Preparation method of a suspended microbial seed coating agent and application thereof in improving seed vigor

Abstract

This invention relates to the field of seed coating technology, and in particular to a method for preparing a suspended microbial seed coating agent and its application in improving seed vigor. This invention creatively improves the preservation of microbial spore activity, reduces the number of inactivated spores, and the preparation process of the suspended microbial seed coating agent is mild, simple, and low-cost. The suspended microbial seed coating agent prepared by the method exhibits good suspension effect, high temperature resistance, and high encapsulation rate. The suspended microbial seed coating agent provided by this invention significantly improves the germination rate and growth of maize seeds under adverse conditions (salt and drought), and has extremely strong application prospects.

Description

Technical Field

[0001] This invention relates to the field of seed coating technology, and in particular to a method for preparing a suspended microbial seed coating agent and its application in improving seed vigor. Background Technology

[0002] In agricultural production, seed reserves become crucial when replanting or reseeding is necessary due to disasters. However, seed viability inevitably declines during storage, making the enhancement of seed viability a key aspect of agricultural production and of immeasurable importance for maintaining basic food production. Seed coating agents, as specialized preparations directly coated on the seed surface, effectively carry active ingredients and form a protective barrier around the seed. While traditional chemical seed coating agents are widely used, long-term use can lead to increased pathogen resistance, pesticide residues, soil microecological imbalance, and environmental pollution. Microbial seed coating agents, characterized by their green, efficient, and safe properties, represent a growing trend in seed coating development. However, the ease of microbial cell inactivation, short shelf life, and susceptibility to environmental influences are major limiting factors. Microencapsulation technology can provide a physical barrier for microorganisms, enhancing their tolerance to adverse external environments and improving their activity. Therefore, applying microencapsulation technology to microbial seed coating agents is a significant development trend. Traditional microencapsulation technologies include spray drying, extrusion, endogenous / exogenous emulsification, and interfacial polymerization. However, these technologies can lead to thermal damage to the active microorganisms inside the microcapsules, poor storage stability, porous wall materials, low production efficiency, and high costs. Therefore, it is crucial to select a mild, highly efficient, simple, and low-cost microencapsulation technology for encapsulating microorganisms.

[0003] Microbial seed coating agents often face limitations in their development due to their short shelf life and stringent storage requirements. Bacillus species, in addition to their high survival rate, abundant secondary metabolites, and strong resistance, also hold a significant position in the field of microbial seed coating agents due to their ability to produce spores and their tolerance to high temperatures. Furthermore, preparing spores into powder form enables microbial seed coating agents to achieve high stability, long shelf life, and convenient application. However, drying can affect the survival of non-spore-forming bacteria; therefore, it is necessary to find an osmotic regulator to maintain the cellular integrity of spores during the drying process.

[0004] Electrostatic attraction is a technique for preparing microcapsules by the attraction between positive and negative charges. It is used in production due to its advantages such as mildness, safety, good biocompatibility, and high survival rate. However, it has disadvantages such as pH sensitivity and complex processes, and the technology urgently needs further improvement.

[0005] In summary, there are currently no microbial microcapsules that offer good stability, can easily and effectively improve the microbial encapsulation rate, have low production costs, and are suitable for large-scale preparation. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for preparing a suspended microbial seed coating agent and its application in improving seed vigor. This invention improves the retention of microbial spore activity, reduces the number of inactivated spores, and the preparation process of the suspended microbial seed coating agent is mild, simple, and low-cost. The resulting suspended microbial seed coating agent exhibits good suspension performance, high temperature resistance, and high encapsulation rate.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing a suspended microbial seed coating agent, comprising the following steps:

[0009] Bacillus subtilis was activated and cultured, and the precipitate was collected by centrifugation.

[0010] The precipitate is resuspended using a activating agent solution and dried to obtain the core material; the activating agent includes one or more of proline, betaine, and mannitol;

[0011] The wall material A solution and the core material are first mixed to obtain a first mixed solution; the wall material A includes one or more of gelatin, pectin, cationic etherified starch, cationic cellulose and chitosan.

[0012] The wall material B solution and the first mixed solution are mixed a second time to obtain a second mixed solution; the wall material B includes one or more of whey protein, soy protein, corn protein and silk fibroin.

[0013] The wall material C solution and the second mixed solution are mixed in a third step to obtain the suspended microbial seed coating agent; the wall material C includes one or more of seaweed polysaccharides, polyvinyl alcohol and hydroxypropyl methylcellulose.

[0014] Preferably, in the suspended microbial seed coating agent, the content of wall material A is 5 g / L-20 g / L, the content of wall material B is 5 g / L-20 g / L, and the content of wall material C is 2 g / L-10 g / L; the ratio of the number of viable bacteria in the core material to the total mass of wall materials A, B, and C in the suspended microbial seed coating agent is 10. 8 -10 10 CFU: 1g.

[0015] Preferably, the Bacillus subtilis is Bacillus subtilis. Bacillus subtilis KC-1, with accession number CGMCC NO.35237.

[0016] Preferably, the culture medium used for the culture comprises the following components at the following concentrations: peptone 10.0 g / L, beef extract 10.0 g / L, KCl 2.0 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 1 g / L and MnSO4 1 g / L; the pH of the culture medium is 7.0-7.2.

[0017] Preferably, the mass ratio of the precipitate to the activator is 1:50; and the concentration of the activator in the activator solution is 20-50 g / L.

[0018] Preferably, the wall material A is dissolved in water at 50℃-80℃ to obtain a solution of the wall material A; and the wall material C is dissolved in water at 50℃-80℃ to obtain a solution of the wall material C.

[0019] Preferably, the rotation speed of the second mixing is 400 rpm to 1000 rpm; the rotation speed of the third mixing is 400 rpm to 1000 rpm, and the temperature is 50℃ to 80℃.

[0020] This invention provides the application of the suspended microbial seed coating agent prepared by the preparation method described above in improving seed vigor and / or improving seed stress resistance.

[0021] Preferably, improving seed stress resistance includes increasing seed germination rate and / or promoting growth under adverse conditions; the adverse conditions include salt and / or drought stress.

[0022] Preferably, the seeds include reserve seeds.

[0023] Beneficial effects:

[0024] (1) The preparation method provided by the present invention is simple and can significantly improve the heat resistance of spores. Using spores as core material, the spore survival rate is ≥96% after heat treatment at 70℃ for 30 min; and the spore survival rate is ≥60% after heat treatment at 90℃ for 10 min. This is beneficial for the spores to provide processing stability for subsequent preparation of microcapsules.

[0025] (2) The suspended microbial seed coating agent prepared by the method provided by the present invention can improve the storage stability of spores. Compared with not adding any one of the wall materials A, B and C described in the present invention, the seed coating agent obtained by adding the wall materials A, B and C provided by the present invention has better storage stability and avoids the reduction of microbial activity in the suspended seed coating agent.

[0026] (3) The preparation method provided by the present invention is mild, simple and low cost. The prepared suspended microbial seed coating agent has good suspension effect, high temperature resistance and high encapsulation rate.

[0027] (4) The suspended microbial seed coating agent prepared by the method provided in this invention can improve the germination and growth of maize seeds under drought and salt stress.

[0028] Biological Preservation Instructions

[0029] Bacillus subtilis KC-1, classified as Bacillus subtilis Bacillus subtilis It was deposited on July 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.35237. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0031] Figure 1 Schematic diagram of suspending agents for different ratios of wall material A and wall material B;

[0032] Figure 2 Comparison of uncoated and microbially coated corn seeds in sterile water after 7 days;

[0033] Figure 3 A comparison of uncoated and suspended microbial-coated maize seeds after 7 days under salt stress;

[0034] Figure 4 Comparison of uncoated and suspended microbial-coated maize seeds after 7 days in PEG6000 (drought) treatment;

[0035] Figure 5 Comparison of maize seeds after 7 days under salt stress and PEG6000 (drought) treatment: uncoated and suspended microbial coating.

[0036] Figure 6 This is a flowchart illustrating the preparation process of suspended microbial seed coating agents. Detailed Implementation

[0037] This invention provides a method for preparing a suspended microbial seed coating agent, comprising the following steps:

[0038] Bacillus subtilis was activated and cultured, and the precipitate was collected by centrifugation.

[0039] The precipitate is resuspended using a activating agent solution and dried to obtain the core material; the activating agent includes one or more of proline, betaine, and mannitol;

[0040] The wall material A solution and the core material are first mixed to obtain a first mixed solution; the wall material A includes one or more of gelatin, pectin, cationic etherified starch, cationic cellulose and chitosan.

[0041] The wall material B solution and the first mixed solution are mixed a second time to obtain a second mixed solution; the wall material B includes one or more of whey protein, soy protein, corn protein and silk fibroin.

[0042] The wall material C solution and the second mixed solution are mixed in a third step to obtain the suspended microbial seed coating agent; the wall material C includes one or more of seaweed polysaccharides, polyvinyl alcohol and hydroxypropyl methylcellulose.

[0043] This invention involves activating and culturing Bacillus subtilis, then collecting the precipitate by centrifugation. As one embodiment, the Bacillus subtilis is specifically Bacillus subtilis. Bacillus subtilis KC-1, with accession number CGMCC NO.35237. In one embodiment, the present invention first obtains single colonies of *Bacillus subtilis* through LB solid-state activation culture, then inoculates these single colonies into LB liquid culture to obtain a seed culture, and finally inoculates the seed culture into a spore-inducing medium for culture. The resulting culture is then collected by centrifugation. In one embodiment, the spore-inducing medium comprises the following components at the following concentrations: peptone 10.0 g / L, beef extract 10.0 g / L, KCl 2.0 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 1 g / L, and MnSO4 1 g / L; the pH of the medium is 7.0-7.2. In one embodiment, the culture temperature is 28-30℃; the centrifugation speed is 8000 rpm for 5 min.

[0044] After obtaining the precipitate, the present invention resuspends the precipitate using a surviving agent solution and dries it to obtain the core material. In one embodiment, the surviving agent is proline. In another embodiment, the mass ratio of the precipitate to the surviving agent is 1:50; the concentration of the surviving agent in the surviving agent solution is 20-50 g / L. In another embodiment, the drying method is freeze-drying. The present invention, through the cultivation, isolation, preservation, and drying of spores, greatly improves the survival rate and storage stability of spores.

[0045] In one embodiment, the suspended microbial seed coating agent contains wall material A at a content of 5 g / L-20 g / L, wall material B at a content of 5 g / L-20 g / L, and wall material C at a content of 2 g / L-10 g / L; the ratio of the number of viable bacteria in the core material to the total mass of wall materials A, B, and C in the suspended microbial seed coating agent is 10. 8 -10 10CFU: 1g. In one embodiment, the suspended microbial seed coating agent contains wall material A at 10g / L-15g / L, wall material B at 15g / L-20g / L, and wall material C at 3g / L-5g / L; the ratio of the viable bacteria count in the core material to the total mass of wall materials A, B, and C in the suspended microbial seed coating agent is 10:1. 8 CFU: 1g. In one embodiment, the mass ratio of wall material A to wall material B in the suspended microbial seed coating agent is 1:2.

[0046] In one embodiment, wall material A is dissolved in water at 50°C-80°C to obtain a solution of wall material A; wall material C is dissolved in water at 50°C-80°C to obtain a solution of wall material C.

[0047] The wall material A, operating at a temperature between 65-75℃, maintains a suitable solution viscosity, which facilitates the formation of uniform, fine droplets and subsequent gelation and solidification of the microcapsule structure during cooling. The positively charged wall material A interacts more readily with the negatively charged spore surface via electrostatic forces, resulting in a denser and more stable composite interfacial film, improving encapsulation efficiency and the mechanical strength of the microcapsules. This temperature range is also significantly lower than the lethal temperature of the spores, maximizing the protection of core biological activity. Rotational speed optimizes particle size and uniformity, promoting homogeneous multi-component compounding. Stepwise mixing follows the electrostatic layer-by-layer assembly principle of "negative-positive-negative-positive," maximizing charge interaction at each layer to form a well-structured, dense microcapsule wall.

[0048] In one embodiment, the rotation speed of the second mixing is 400 rpm to 1000 rpm; the rotation speed of the third mixing is 400 rpm to 1000 rpm, and the temperature is 50℃ to 80℃.

[0049] The preparation method provided by this invention improves the protective effect of microcapsules on microorganisms through effective encapsulation. The suspended microbial seed coating agent prepared by this invention can promote seed germination and growth, as well as improve seed resistance to stress (salt stress, drought stress).

[0050] Based on the above advantages, the present invention provides the application of the suspended microbial seed coating agent prepared by the preparation method described in the above technical solution in improving seed vigor and / or improving seed stress resistance.

[0051] In one implementation, improving seed stress resistance includes increasing seed germination rate and / or promoting growth under adverse conditions; the adverse conditions include salt and / or drought stress.

[0052] In one embodiment, the seeds include reserve seeds. In another embodiment, the reserve seeds include reserve corn seeds. In one embodiment, the weight ratio of the suspended microbial seed coating agent to the corn seeds is 1:(25-30).

[0053] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes a method for preparing a suspended microbial seed coating agent provided by the present invention and its application in improving seed vigor, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0054] Preparation Example

[0055] The formula for LB solid medium is: 10 g / L tryptone, 5 g / L beef extract, 10 g / L sodium chloride, 15 g / L agar, and pH 7.0-7.2.

[0056] The LB liquid medium formula is: 10 g / L tryptone, 5 g / L beef extract, 10 g / L sodium chloride, pH 7.0-7.2.

[0057] The induction medium was Schaeffer's spore medium, prepared as follows: 10.0 g peptone, 10.0 g beef extract, 2.0 g KCl, 0.5 g MgSO4·7H2O, and deionized water were added to a final volume of 1 L, pH 7.0-7.2. After sterilization and cooling to 50°C, 1 mL of CaCl2 solution and 1 mL of MnSO4 solution were added; the concentration of the CaCl2 solution was 1 g / L, and the concentration of the MnSO4 solution was 1 g / L.

[0058] Example 1

[0059] Isolated Bacillus subtilis KC-1 was picked and inoculated into LB solid medium and activated at 28-30℃. After colonies grew, the colonies on the plate were inoculated into LB liquid medium for culture with shaking (300 rpm) at 30℃ for 18-20 hours, until the bacterial culture became turbid (OD). 600 ≈1.0), to obtain seed culture. The seed culture was inoculated into induction medium and cultured at 28-30℃ for 48 h (OD ≈1.0). 600 After setting the concentration to 0.9-1.2, the culture was centrifuged at 8000 rpm for 5 min, and the spore precipitate was collected. The collected spore precipitate and the surviving agent (proline) suspension were stirred for 30 min to ensure that the surviving agent and spores were fully penetrated and bound, resulting in a bacterial solution containing the surviving agent; the mass ratio of the spore precipitate to the surviving agent was 1:50; and the concentration of the surviving agent suspension was 4% (w / v).

[0060] Example 2

[0061] The method is similar to that in Example 1, except that the activator is betaine.

[0062] Example 3

[0063] The method is similar to that in Example 1, except that the activator is mannitol.

[0064] Test Example 1

[0065] OD of spore precipitate and viability agent suspension after 7 days in Examples 1-3 was tested. 600 The values ​​were set as a control group without the mixed activator, and the results are shown in Table 1.

[0066] Table 1. Effects of different surviving agents on spore growth and viability.

[0067]

[0068] The results showed that, compared with the control group without any preservatives, the treatment groups with proline, betaine, and mannitol all increased the OD of the spore-forming bacterial culture. 600 The values ​​indicate that these surviving agents all improved spore survival. Among the different surviving agents, proline showed the best spore survival effect. Therefore, proline was chosen as the spore surviving agent in subsequent selections.

[0069] Example 4

[0070] 1. Core material preparation (see flowchart) Figure 6 (1. Preparation of spores)

[0071] The bacterial solution containing the viability preservative was prepared using the method in Example 1, and then freeze-dried into spore powder to obtain the core material.

[0072] 2. Preparation method of suspended microbial seed coating agent (see flowchart) Figure 6 2. Preparation of suspended microbial seed coating agents

[0073] Dissolve 1g of gelatin (wall material A) in water at 70℃ to obtain 100mL of wall material A solution;

[0074] Add 1g of the core material to the wall material A solution and stir to obtain mixed solution 1;

[0075] Dissolve 1g of whey protein (wall material B) in water to obtain 100mL of wall material B solution. Add mixed solution 1 to wall material B solution and stir at 70℃ and 800rpm for 10min to obtain mixed solution 2.

[0076] Dissolve 0.3g of seaweed polysaccharide (wall material C) in water at 70℃ to obtain 100mL of wall material C solution. Add mixed solution 2 to wall material C solution and stir at 70℃ and 800rpm for 10min to obtain suspended microbial seed coating agent.

[0077] Example 5

[0078] The suspended microbial seed coating agent is similar to that in Example 4, except that 1g of whey protein is replaced with 3g of whey protein (i.e., wall material A: wall material B = 1:3).

[0079] Example 6

[0080] The suspended microbial seed coating agent is similar to that in Example 4, except that 1g of whey protein is replaced with 2g of whey protein (i.e., wall material A: wall material B = 1:2).

[0081] Example 7

[0082] The suspended microbial seed coating agent is similar to that in Example 4, except that 1g of gelatin is replaced with 2g of gelatin (i.e., wall material A: wall material B = 2:1).

[0083] Example 8

[0084] The suspended microbial seed coating agent is similar to that in Example 4, except that 1g of gelatin is replaced with 3g of gelatin (i.e., wall material A: wall material B = 3:1).

[0085] Test Example 2

[0086] The turbidity (OD) of the suspended microbial seed coating agents in Examples 4-8 was detected and observed. 400 Value, zeta potential, and encapsulation efficiency were measured. Zeta potential was determined using a zeta potential analyzer (Beckman Coulter, UK). Encapsulation efficiency was determined according to the method described in the reference: [Gong M, He J, Kong M, Huo Q, Jiang Y, Song J, Han W and Lv G (2023) A microencapsulation approach to design microbial seed coatings to boost wheatseed germination and seedling growth under salt stress. Front. Plant Sci. 14:1283590. doi: 10.3389 / fpls.2023.1283590]. Results are shown in […]. Figure 1 And Table 2.

[0087] Table 2 Detection results of different suspended microbial seed coating agents

[0088]

[0089] The results show that as the ratio of wall material A to wall material B gradually increases, OD 400nm The zeta potential gradually increases and then decreases, and the embedding efficiency also shows an initial increase followed by a decrease. The embedding efficiency is highest at a ratio of 1:2 for wall material A to wall material B, reaching 90.1%.

[0090] Example 9

[0091] The suspended microbial seed coating agent prepared in Example 6 was used to coat seeds (Zhengdan 958) at a weight ratio of 1:30. The seeds were corn seeds (Zhengdan 958) that had been stored for one and a half years and aged. After coating, the seeds were air-dried naturally and placed in sterile water-moistened petri dishes. Seeds without coating were used as a control group (CK). Germination potential was measured after 3 days, and the number of germinated seeds was counted daily. After 7 days, the germination rate and root length were measured. The results are shown in Table 3. Figure 2 .

[0092] Table 3 Germination potential, germination rate, and root length of different treatment groups in sterile water.

[0093]

[0094] The results showed that, compared with the control (CK), the germination potential, germination rate and root length of the coating treatment increased significantly by 21.19%, 22.56% and 46.46%, respectively.

[0095] Example 10

[0096] The experimental method was similar to that of Example 9, except that the coated seeds were placed in a petri dish moistened with sterile saline solution at a concentration of 6%. The results are shown in Table 4 and [Table data would be inserted here]. Figure 3 .

[0097] Table 4 Germination potential, germination rate, and root length of different treatment groups in sterile saline solution

[0098]

[0099] The results showed that, compared with the control (CK), the germination potential, germination rate, and root length of the coating treatment increased significantly by 19.41%, 28.61%, and 27.57%, respectively.

[0100] Example 11

[0101] The experimental method was similar to that of Example 9, except that the coated seeds were placed in a petri dish moistened with sterile water containing 15% PEG6000 (simulating a drought environment). The results are shown in Table 5 and... Figure 4 .

[0102] Table 5 Germination potential, germination rate, and root length of different treatment groups under drought conditions.

[0103]

[0104] The results showed that, compared with the control (CK), the germination potential, germination rate and root length of the coating treatment increased significantly by 15.90%, 17.44% and 61.15%, respectively.

[0105] Example 12

[0106] The experimental method was similar to that of Example 9, except that the sterile water was replaced with a mixture of sterile water containing 15% PEG6000 and sterile saline solution. The volume ratio of the sterile water containing 15% PEG6000 to the sterile saline solution was 1:1, and the mass concentration of the sterile saline solution was 6%. The results are shown in Table 6 and [Table data would be inserted here]. Figure 5 .

[0107] Table 6 Germination potential, germination rate, and root length of different treatment groups under drought conditions.

[0108]

[0109] The results showed that, compared with the control (CK), the germination potential, germination rate and root length of the coating treatment increased significantly by 24.01%, 21.08% and 19.20%, respectively.

[0110] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a suspended microbial seed coating agent, characterized in that, Includes the following steps: Bacillus subtilis was activated and cultured, and the precipitate was collected by centrifugation; the Bacillus subtilis was Bacillus subtilis. Bacillus subtilis KC-1, accession number CGMCC NO.35237; The precipitate is resuspended in a activating agent solution and dried to obtain the core material; the activating agent is one or more of proline, betaine, and mannitol; the mass ratio of the precipitate to the activating agent is 1:50; The wall material A solution and the core material are first mixed to obtain a first mixed solution; the wall material A is one or more of gelatin, pectin, cationic etherified starch, cationic cellulose and chitosan. The wall material B solution and the first mixed solution are mixed a second time to obtain a second mixed solution; the wall material B is one or more of whey protein, soy protein, corn protein and silk fibroin. The wall material C solution and the second mixed solution are mixed in a third step to obtain the suspended microbial seed coating agent; the wall material C is one or more of seaweed polysaccharide, polyvinyl alcohol and hydroxypropyl methylcellulose; The concentration of wall material A solution is 10 g / L; the concentration of wall material B solution is 20 g / L; the concentration of wall material C solution is 3 g / L; in the suspended microbial seed coating agent, the mass ratio of wall material A to wall material B is 1:2; the mass ratio of wall material A to wall material C is 1:0.3; the ratio of the number of viable bacteria in the core material to the total mass of wall materials A, B, and C in the suspended microbial seed coating agent is 10. 8 -10 10 CFU: 1g.

2. The preparation method according to claim 1, characterized in that, The culture medium used for the culture comprises the following components at the following concentrations: peptone 10.0 g / L, beef extract 10.0 g / L, KCl 2.0 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 1 g / L and MnSO4 1 g / L; the pH of the culture medium is 7.0-7.

2.

3. The preparation method according to claim 1, characterized in that, The concentration of the activator in the activator solution is 20-50 g / L.

4. The preparation method according to claim 1, characterized in that, The wall material A is dissolved in water at 50℃-80℃ to obtain a solution of wall material A; the wall material C is dissolved in water at 50℃-80℃ to obtain a solution of wall material C.

5. The preparation method according to claim 1, characterized in that, The second mixing speed is 400rpm-1000rpm; the third mixing speed is 400rpm-1000rpm, and the temperature is 50℃-80℃.

6. The application of the suspended microbial seed coating agent prepared by the preparation method according to any one of claims 1 to 5 in improving seed vigor and / or improving seed stress resistance.

7. The application according to claim 6, characterized in that, The improvement of seed stress resistance includes increasing seed germination rate and / or promoting growth under adverse conditions; the adverse conditions include salt and / or drought stress.

8. The application according to claim 6 or 7, characterized in that, The seeds include reserve seeds.

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