Pseudomonas fermentation biological agent based on stevia polysaccharide as well as preparation method and application of pseudomonas fermentation biological agent

Abstract

The invention relates to the technical field of microorganisms, in particular to a stevia polysaccharide-based pseudomonas fermentation biological agent as well as a preparation method and application thereof.According to the stevia polysaccharide-based pseudomonas fermentation biological agent and the preparation method and application thereof, the stevia polysaccharide is secondarily utilized, experiments prove that the stevia polysaccharide can replace a traditional carbon source, and reported in the prior art, a stevia extract has an antibacterial effect on pseudomonas sp. Research finds that the stevia polysaccharide has a very obvious growth promoting effect on Pseudomonas sp. CD1, the effect of the stevia polysaccharide is remarkably better than that of glucose serving as a carbon source, the growth of pseudomonas bacterial colonies is remarkably promoted, the effect of the stevia polysaccharide is better than that of a traditional glucose carbon source, the existing technical barriers are effectively broken through, in addition, the fermented biological preparation can effectively turn waste into wealth, and the cost is reduced. The method reduces environmental pollution and cost, realizes efficient resource utilization of stevia polysaccharide, reduces dependence of agricultural production on synthetic chemical fertilizers, and meets green agricultural requirements.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Pseudomonas fermentation biological agent based on stevia polysaccharide, its preparation method, and its application. Background Technology

[0002] Stevia polysaccharides, a byproduct of stevia processing, are high-molecular-weight polysaccharides that are difficult for ordinary microorganisms to utilize directly. These byproducts are often discarded due to their excessive bitterness, resulting in resource waste and environmental pollution. Current utilization of stevia polysaccharides is insufficient, necessitating the development of new resource-based utilization pathways. On the other hand, Plant Growth-Promoting Rhizobacteria (PGPR), as an environmentally friendly bio-fertilizer technology, is widely researched and applied in agricultural production. PGPR can promote crop growth through multiple pathways, including improving nutrient utilization, producing plant hormones, and enhancing plant resistance. Its application can effectively reduce the use of chemical fertilizers and pesticides, improving crop yield and quality. Therefore, utilizing microbial fermentation to convert stevia polysaccharides into plant growth promoters not only holds promise for solving waste problems but also for developing high-value agricultural application products.

[0003] Existing technologies report that stevia extracts have broad antibacterial activity. For example, the article "Research Progress on the Biological Functions of Steviosides and Their Application in Livestock and Poultry Production" by He Zhentao mentions that stevia extracts are effective against Pseudomonas aeruginosa (…). Pseudomonas sp. Stevia has antibacterial effects; existing technology, in the article "Research Progress on Chemical Constituents and Pharmacological Activities of Stevia" by Liu Qiong, mentions that water, methanol, ethyl acetate and hexane extracts of stevia have antibacterial effects against Pseudomonas aeruginosa (…). Pseudomonas sp. Stevia has antibacterial properties, so it would be a major breakthrough if Pseudomonas could utilize stevia polysaccharide as a single carbon source.

[0004] Stevia is widely used in food additives, sweeteners, and the pharmaceutical industry. However, the byproducts of stevia processing contain a large amount of stevia polysaccharides. If these are not properly utilized, they not only have adverse environmental impacts but also waste significant nutrient resources. To address these problems, this invention proposes using stevia polysaccharides as a high-quality carbon source for Pseudomonas liquid fermentation, optimizing Pseudomonas culture efficiency, achieving high-value utilization of stevia polysaccharides, and overcoming the technical barriers to the antibacterial effect of stevia leaf extract on Pseudomonas. Summary of the Invention

[0005] In view of the above, it is necessary to utilize stevia polysaccharides, explore their potential in Pseudomonas fermentation, break through the technical barriers of the antibacterial effect of stevia leaf extract on Pseudomonas, effectively utilize stevia polysaccharides at high value, and reduce environmental pollution.

[0006] To achieve the above objectives, the technical solution adopted by this invention is: a Pseudomonas fermentation biological agent based on stevia polysaccharides, wherein the Pseudomonas fermentation biological agent is composed of Pseudomonas species. Pseudomonas It was obtained by fermentation of strain CD1, stevia polysaccharide solution, and MSM medium; the aforementioned *Pseudomonas* spp. Pseudomonas The preservation information for strain CD1 is: Pseudomonas spp. CD1 Pseudomonas sp. CD1, its classification name is: Pseudomonas sp. CD1, classified in Chinese as Pseudomonas spp. CD1, with accession number CCTCC NO: M20231300, is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on July 17, 2023. This strain has been published in patent application 202311758216.X.

[0007] Furthermore, the stevia polysaccharide solution is prepared by diluting it 50-100 times.

[0008] Furthermore, the MSM culture medium obtained by fermentation consists of the following components: 2.0 g / L of (NH4)2SO4, 0.2 g / L of MgSO4·7H2O, 0.01 g / L of CaCl2·2H2O, 0.001 g / L of FeSO4·7H2O, 1.5 g / L of Na2HPO4·12H2O, and 41.5 g / L of KH2PO4.

[0009] The present invention also includes a method for preparing the Pseudomonas fermentation biological agent, the method comprising: using Pseudomonas spp. Pseudomonas strain CD1 was inoculated into MSM medium with stevia polysaccharide as the sole carbon source and cultured at 22–30°C for 48–72 hours to obtain a Pseudomonas fermentation biological agent; the Pseudomonas spp. Pseudomonas The preservation number of sp. strain CD1 is CCTCC NO: M20231300.

[0010] Furthermore, the stevia polysaccharide is diluted to a concentration of 50-100 times.

[0011] The present invention also includes the application of the Pseudomonas fermentation biological agent or the Pseudomonas fermentation biological agent prepared by the method to promote cucumber growth.

[0012] The present invention also includes the aforementioned *Pseudomonas* genus. Pseudomonas A compound system of sp. strain CD1, wherein the compound system also contains stevia polysaccharide.

[0013] The present invention also includes a method for improving the quality and efficiency of cucumber cultivation by applying the aforementioned promoter, the specific method of which is as follows.

[0014] (1) Pick the aforementioned Pseudomonas spp. Pseudomonas A single colony of strain CD1 was inoculated into TSB liquid medium and cultured in a constant temperature shaker at 25-30℃ and 170-200 rpm for 10-15 hours to obtain a seed culture. The seed culture was then inoculated into TSB liquid medium at a volume ratio of 1:100 and cultured in large quantities at 25-30℃ and 170-200 rpm for 8-12 hours to obtain *Pseudomonas* spp. Pseudomonas Fermentation broth of strain CD1.

[0015] (2) Pseudomonas spp. Pseudomonas The viable cell count in the fermentation broth of strain CD1 was determined by plate counting. The broth was then centrifuged to remove the precipitate, yielding a supernatant. This supernatant was diluted with sterile water to a viable cell concentration of 10⁻⁶. 7 The fermentation solution of CFU / mL was diluted and poured into the soil around the roots of cucumber plants.

[0016] (3) Dilute stevia polysaccharide and sterile water at a volume ratio of 1:100, and then pour the diluted solution into the cucumber soil after inoculation in step (2).

[0017] This invention has the following beneficial effects: 1. This invention utilizes stevia polysaccharides in a secondary manner. Experiments have shown that stevia polysaccharides can replace traditional carbon sources. Existing technologies report that stevia extracts have an effect on Pseudomonas aeruginosa (…). Pseudomonas sp. Stevia polysaccharides have antibacterial effects; in this application, the research group found that stevia polysaccharides do not have inhibitory effects on all microorganisms. Even among Pseudomonas strains, the antibacterial effects vary depending on the strain, especially against the Pseudomonas species mentioned in this application. Pseudomonas sp. CD1 has a very obvious growth-promoting effect, which is significantly better than glucose as a carbon source in promoting the growth of Pseudomonas colonies and is superior to traditional glucose as a carbon source.

[0018] 2. This application utilizes stevia polysaccharides and Pseudomonas ( ) Pseudomonas sp.Processing CD1 into a microbial agent effectively turns waste into treasure, reducing environmental pollution and lowering costs. This component has a particularly significant growth-promoting effect on strain CD1. Combined with the growth-promoting effect of this strain, it achieves efficient resource utilization of stevia polysaccharides, reducing agricultural production's dependence on synthetic fertilizers and meeting the needs of green agriculture. Pot experiments have demonstrated that the fermented biological agent can significantly increase seedling biomass, improve root development, and promote plant growth. The fermented biological agent prepared in this invention optimizes the Pseudomonas liquid fermentation process. By adjusting the stevia polysaccharide concentration, pH, and culture conditions, production efficiency is improved, and the liquid fermentation colony count can reach 10^6. 9 The output of active ingredients is stable at 1 / mL. Attached Figure Description

[0019] Figure 1 The diagram shows the fermentation process of strain CD1; where CK is carbon-free basal medium (MSM), A is a fermentation diagram with glucose as the carbon source, B is a fermentation diagram with 50 times dilution of stevia polysaccharide, and C is a fermentation diagram with 100 times dilution of stevia polysaccharide.

[0020] Figure 2 A graph showing the enrichment analysis of differentially expressed metabolic pathways under different treatments.

[0021] Figure 3 A KEGG-annotated pathway diagram for the synthesis of amino acids from differentially metabolized metabolites.

[0022] Figure 4 This is a diagram showing the results of a pot experiment on cucumber growth using stevia polysaccharides and the CD1 strain.

[0023] Figure 5 The figure shows the experimental results of stevia polysaccharide and CD1 strain on the fresh weight of the underground parts of cucumber.

[0024] Figure 6 The figure shows the experimental results of stevia polysaccharide and CD1 strain on the fresh weight of the aboveground parts of cucumber.

[0025] Figure 7 The figure shows the experimental results of the effect of stevia polysaccharide and CD1 strain on the total fresh weight of cucumber growth.

[0026] Figure 8 This is a diagram showing the results of a pot experiment on cucumber growth using the supernatant from the fermentation of *Steviolus polysaccharide*.

[0027] Figure 9 This is a diagram showing the experimental results of the fermentation supernatant of Stevia polysaccharide on the underground parts of cucumber growth.

[0028] Figure 10 This is a diagram showing the experimental results of the fermentation supernatant of Stevia polysaccharide on the aboveground parts of cucumber.

[0029] Figure 11The figure shows the experimental results of the fermentation supernatant of Stevia polysaccharide on the total dry weight of cucumber growth.

[0030] Figure 12 The figure shows the experimental results of the stevia polysaccharide-based Pseudomonas aeruginosa fermentation system and the effect of non-fermentation on the underground growth of cucumber.

[0031] Figure 13 The figure shows the experimental results of the stevia polysaccharide-based Pseudomonas aeruginosa fermentation system and the non-fermentation system on the aboveground parts of cucumber growth.

[0032] Figure 14 The figure shows the experimental results of the stevia polysaccharide-Pseudomonas aeruginosa fermentation system and the effect of non-fermentation on the total dry weight of cucumber growth. Information on the preservation of biological materials

[0033] The preservation information for strain CD1 in this application is as follows: *Pseudomonas* spp. Pseudomonas sp. CD1, its classification name is: Pseudomonas sp. CD1, classified in Chinese as Pseudomonas spp. CD1, with accession number CCTCC NO: M20231300, is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on July 17, 2023. This strain has been published in patent application 202311758216.X. Detailed Implementation

[0034] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0035] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features. Example 1

[0036] This embodiment studies the effects of stevia polysaccharides on the growth of various microorganisms, as detailed below.

[0037] 1. The stevia polysaccharides used in the embodiments of this application were all purchased from Dongtai Haorui Biotechnology Co., Ltd., and their main indicators and properties are shown in Table 1.

[0038]

[0039] 2. Experimental Method: Thirteen existing growth-promoting bacteria strains were inoculated into MSM inorganic salt medium (pH adjusted to 7.0) with stevia polysaccharide as the sole carbon source, and incubated at 30℃ and 170 r·min. -1 After shaking for 48 hours, the OD of the bacterial culture was measured using an ELISA reader. 600The growth status of the strains was measured over 24 hours. MSM medium supplemented with glucose (a common carbon source) was used as a positive control (Glu) to verify the growth ability of the strains, while MSM medium without stevia polysaccharide was used as a negative control (CK). The growth status of each strain is shown in Table 2.

[0040]

[0041] As shown in Table 2: In MSM medium with stevia polysaccharide as the sole carbon source, the growth abilities of the 13 identified strains differed significantly: The 24hOD of the Pseudomonas CD1 strain was significantly different. 600 The OD value reached 1.08 (50-fold dilution) and remained at 0.58 at 100-fold dilution, significantly higher than the negative control group, demonstrating its efficient degradation and utilization of stevia polysaccharides; while the OD values ​​of 50-fold and 100-fold dilutions of the related Pseudomonas LB4 were significantly higher. 600 The OD value was approximately 0.09, slightly lower than the negative control group (CK), but the difference was not significant. The reason for these experimental results may be that stevia polysaccharide has a certain inhibitory effect on the LB4 strain of the same genus, or it can be considered that the LB4 strain of the same genus has difficulty utilizing stevia polysaccharide as its sole carbon source. Besides strain LB4, Table 2 shows the OD values ​​of 11 other strains (Neosphospira LB9, Burkholderia BW53 / BW419 / BW28 / BW711, etc., Bacillus SQR9 / B34 / BW82 / B18 / B7, etc.). 600 The values ​​were all below 0.65, showing no significant difference from the negative control (CK), indicating that these strains could not utilize stevia polysaccharide as their sole carbon source. Meanwhile, all strains showed significant proliferation in the "glucose-MSM" positive control (Glu), ruling out the possibility of insufficient inoculation activity. Therefore, CD1 exhibits a unique and significant advantage in utilizing stevia polysaccharide, enabling rapid growth with stevia polysaccharide as the sole carbon source. Example 2

[0042] This embodiment studies the effect of stevia polysaccharide on Pseudomonas fermentation biological agents, as detailed below.

[0043] 1. Preparation of CD1 strain stevia polysaccharide fermentation inoculum: *Pseudomonas CD1* was inoculated into MSM medium with stevia polysaccharide as the sole carbon source (stevia polysaccharide diluted 50-fold and 100-fold respectively), and cultured at 22–30℃ under aerobic conditions for 48–72 hours to obtain the CD1 stevia polysaccharide fermentation inoculum. During this process, the color of the culture system gradually changed from an initial yellow to a dark green, such as… Figure 1As shown in the figure, this is a schematic diagram of the fermentation of strain CD1; where CK is the passive carbon basal medium (MSM), A is a schematic diagram of fermentation with glucose as the carbon source, B is a schematic diagram of fermentation with 50 times the concentration of stevia polysaccharide, and C is a schematic diagram of fermentation with 100 times the concentration of stevia polysaccharide. This indicates that the strain produced dark metabolites during the metabolism of stevia polysaccharide.

[0044] 2. Material analysis of the CD1 strain stevia polysaccharide fermentation agent: Metabolomics analysis of the CD1 strain under stevia polysaccharide induction was performed using GC-MS. The results showed that the CD1 strain upregulated metabolic pathways related to indoleacetic acid (IAA) synthesis and accumulated a large amount of aromatic amino acids (precursors to IAA), such as tryptophan. Figure 2 and Figure 3 As shown; where, Figure 2 A graph showing the enrichment analysis of differentially expressed metabolic pathways under different treatments. Figure 3 A KEGG-annotated pathway diagram for the synthesis of amino acids from differentially metabolized metabolites. Example 3

[0045] According to the experiment in Example 2, a pot experiment was conducted on the combination of growth-promoting Pseudomonas strain CD1 and stevia polysaccharide. The details are as follows: Test strain: CD1 stevia polysaccharide fermentation agent prepared in Example 2 at 100 times concentration; Test crop: cucumber.

[0046] Disinfection and germination of cucumber seeds: Soak cucumber seeds in sterile deionized water at 50℃ for 30 minutes, then immediately transfer them to sterile water at 30℃ for 1 hour. After draining the water, soak the seeds in 70% ethanol for 3-4 minutes, shaking continuously during the soaking process. Rinse three times with sterile water, then soak in 2% sodium hypochlorite for 3-4 minutes, again shaking continuously. Finally, rinse repeatedly with sterile deionized water until the sodium hypochlorite and the outer coating of the cucumber seeds are completely removed, thus completing the disinfection of the cucumber seeds. Place the disinfected cucumber seeds, which are of similar plumpness, evenly in a 90mm petri dish lined with sterile filter paper. Moisten each filter paper with 3ml of the liquid prepared in the above steps, keeping the filter paper moist. Incubate in the dark at 30℃ for 3-5 days until the cucumber seeds show signs of germination.

[0047] Cucumber seedling cultivation: Select uniform and plump cucumber seeds with white tips and sow them into the seedling substrate. When the cucumbers in the substrate have grown to two true leaves, select seedlings with uniform growth and transplant them for subsequent experiments.

[0048] Inoculation: One week later, inoculate the fermentation broth of Pseudomonas spp. strain CD1 (final concentration 10). 7Stevia polysaccharide (STE) was prepared at a concentration of CFU / g (soil). Stevia polysaccharide fermentation broth from *Pseudomonas* strain CD1 was diluted 100 times (STE+CD1). A water control was included; no bacteria were inoculated (CK). Culture conditions were consistent with other inoculation treatments. Each treatment was replicated 12 times. All plants were grown in a greenhouse with a natural temperature range of 25–30°C and a humidity range of 50–70% RH. Agronomic traits were measured 30 days after transplanting.

[0049] The results are as follows Figures 4-7 As shown: Figure 4 The following figures show the results of the potted plant experiment for the four treatments. Figures 5-7 The effects of stevia polysaccharide (STE) and strain CD1 on cucumber seedling growth were investigated. Figure 7 The influence of the dry weight of the aboveground part Figure 6 For the influence of root weight, Figure 7 The effect on total dry weight. As shown in the figure, after different treatments, the total dry weight of the STE+CD1 group was significantly increased compared with the STE group alone and the CD1 group alone; while the STE group alone and the CD1 group alone were not significantly different from the control CK, indicating that the use of strain CD1 alone and the use of stevia polysaccharide (STE) alone had no significant growth-promoting effect on cucumber seedlings. Example 4

[0050] This embodiment studies the effect of fermentation supernatant of "stevia polysaccharide-CD1 strain" on cucumber growth promotion, as detailed below.

[0051] 1. Prepare the supernatant of the fermentation broth according to the following method.

[0052] ①CK treatment group: Prepare carbon-free basal medium (MSM), add stevia polysaccharide to prepare a 100-fold dilution concentration, and adjust the pH to 7.

[0053] ②SC treatment group: The purchased stevia polysaccharide was diluted 100 times with water, the pH was adjusted to 7, and then CD1 bacterial suspension was inoculated at a volume percentage of 1% and cultured at 30°C and 170 rpm for 72 hours. The precipitate was removed by centrifugation, and the supernatant was collected. The CD1 bacterial suspension was prepared by: inoculating Pseudomonas CD1 (… Pseudomonas The sp. strain was activated, inoculated into TSB medium, and cultured for 24 hours at a temperature of 22-30℃ under aerobic conditions to obtain the corresponding bacterial solution.

[0054] 2. The effects of fermented biological agents on cucumber growth were studied, as detailed below.

[0055] (1) Disinfection and germination of cucumber seeds: Soak cucumber seeds in sterile deionized water at 50℃ for 30 min, then immediately transfer them to sterile water at 30℃ for 1 h. After draining the water, soak the seeds in 70% ethanol for 3-4 min, shaking continuously during the soaking process. Rinse three times with sterile water, then soak in 2% sodium hypochlorite for 3-4 min, shaking continuously as well. Finally, rinse multiple times with sterile deionized water until the sodium hypochlorite and the outer coating of the cucumber seeds are completely removed, thus completing the disinfection of the cucumber seeds. Place the disinfected cucumber seeds with a similar degree of plumpness evenly in a 90 mm petri dish lined with sterile filter paper. Moisten each filter paper with 3 ml of the liquid prepared in the above steps, keeping the filter paper moist. Incubate in the dark at 30℃ for 3-5 days until the cucumber seeds show signs of germination.

[0056] (2) Cucumber seedling raising: Select uniform and plump cucumber seeds with white tips and sow them into the seedling substrate. When the cucumbers in the substrate have grown to 2 true leaves, select seedlings with uniform growth and transplant them for subsequent experiments.

[0057] (3) Potted plant experimental treatment: The experimental setup was set up according to the two treatments in Table 3, with six replicates. One week after transplanting, the roots were irrigated. After 30 days of cultivation, the cucumber phenotypic traits were measured.

[0058]

[0059] Experimental results are as follows Figures 8-11 As shown: Figure 8 The figures show the results of the potted plant experiments for the two treatments. Figures 9-11 The effect of CD1 stevia polysaccharide fermentation supernatant on cucumber seedling growth was studied. Figure 9 The influence of the dry weight of the underground portion, Figure 10 The influence of the dry weight of the aboveground part Figure 11 The figure shows the effect of total dry weight. As can be seen from the figure, after different treatments, the fermentation supernatant of "stevia polysaccharide-CD1 strain" can significantly promote plant growth compared with CK, which also shows that the fermentation broth of "stevia polysaccharide-CD1 strain" is rich in growth-promoting substances and can significantly promote cucumber growth. Example 5

[0060] This embodiment studies the effect of fermentation of "stevia polysaccharide-CD1 strain" on the growth promotion of cucumber seedlings, as detailed below.

[0061] Test strain: Pseudomonas strain CD1.

[0062] Disinfection and germination of cucumber seeds: Soak cucumber seeds in sterile deionized water at 50℃ for 30 minutes, then immediately transfer them to sterile water at 30℃ for 1 hour. After draining the water, soak the seeds in 70% ethanol for 3-4 minutes, shaking continuously during the soaking process. Rinse three times with sterile water, then soak in 2% sodium hypochlorite for 3-4 minutes, again shaking continuously. Finally, rinse repeatedly with sterile deionized water until the sodium hypochlorite and the outer coating of the cucumber seeds are completely removed, thus completing the disinfection of the cucumber seeds. Place the disinfected cucumber seeds, which are of similar plumpness, evenly in a 90mm petri dish lined with sterile filter paper. Moisten each filter paper with 3ml of the liquid prepared in the above steps, keeping the filter paper moist. Incubate in the dark at 30℃ for 3-5 days until the cucumber seeds show signs of germination.

[0063] Cucumber seedling cultivation: Select uniform and plump cucumber seeds with white tips and sow them into the seedling substrate. When the cucumbers in the substrate have grown to two true leaves, select seedlings with uniform growth and transplant them for subsequent experiments.

[0064] Inoculation: One week later, plants were inoculated with the CD1 strain of stevia polysaccharide fermentation agent prepared in Example 2 (SC fermentation), and with a 1:100 dilution of stevia polysaccharide in the CD1 strain of Pseudomonas spp. fermentation broth (SC without fermentation). A water control treatment was set up: no inoculation agent was used (CK). The culture conditions were the same as other inoculation treatments. Each treatment was replicated 12 times. All plants were grown in a greenhouse with a natural temperature range of 25–30°C and a humidity range of 50–70% RH. Agronomic traits were measured 30 days after transplanting.

[0065] Experimental results are as follows Figures 12-14 As shown: Figures 12-14 The effect of CD1 stevia polysaccharide fermentation supernatant on cucumber seedling growth was studied. Figure 12 The influence of the fresh weight of the underground part Figure 13 The impact of the fresh weight of the above-ground parts Figure 14 The figure shows the effect of total dry weight. As can be seen from the figure, after different treatments, the fermentation of "stevia polysaccharide-CD1 strain" significantly promoted plant growth compared with CK and no fermentation. This also shows that "stevia polysaccharide-CD1 strain" needs to undergo fermentation treatment to produce growth-promoting substances and significantly promote cucumber growth.

[0066] In summary, this invention utilizes stevia polysaccharides in a secondary manner. Experiments have demonstrated that these stevia polysaccharides can replace traditional carbon sources. Existing technologies report that stevia extracts have an effect on *Pseudomonas* (…). Pseudomonas sp. Stevia polysaccharides have antibacterial effects; in this application, the research group discovered through research that: Stevia polysaccharides have antibacterial effects against the Pseudomonas aeruginosa (Pseudomonas aeruginosa) of this application. Pseudomonas sp.CD1 has a very significant growth-promoting effect, which is significantly better than when glucose is used as a carbon source to significantly promote the growth of Pseudomonas colonies and is superior to traditional glucose carbon source. This discovery effectively breaks through the existing technical barriers. In addition, the fermentation biological agent of this application can effectively turn waste into treasure, reduce environmental pollution and lower costs, realize the efficient resource utilization of stevia polysaccharides, reduce the dependence of agricultural production on synthetic fertilizers, and meet the needs of green agriculture.

[0067] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A Pseudomonas fermentation biological agent based on stevia polysaccharides, characterized in that, The Pseudomonas fermentation biological agent is composed of Pseudomonas genus. Pseudomonas It was obtained by fermentation of strain CD1, stevia polysaccharide solution, and MSM medium; the aforementioned *Pseudomonas* spp. Pseudomonas The accession number of sp. strain CD1 is CCTCC NO: M20231300. This strain is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on July 17, 2023. The stevia polysaccharide solution was prepared by diluting it 50-100 times.

2. The Pseudomonas fermentation biological agent according to claim 1, characterized in that, The MSM medium, obtained by fermentation, consists of the following components: 2.0 g / L (NH4)2SO4, 0.2 g / L MgSO4·7H2O, 0.01 g / L CaCl2·2H2O, 0.001 g / L FeSO4·7H2O, 1.5 g / L Na2HPO4·12H2O, and 41.5 g / L KH2PO.

3. A method for preparing the *Pseudomonas* fermentation biological agent as described in any one of claims 1 or 2, characterized in that, The method is as follows: *Pseudomonas* spp. Pseudomonas strain CD1 was inoculated into MSM medium with stevia polysaccharide as the sole carbon source and cultured at 22–30°C for 48–72 hours to obtain the Pseudomonas fermented biological agent; the Pseudomonas spp. Pseudomonas The preservation number of sp. strain CD1 is CCTCC NO: M20231300, and the dilution concentration of the stevia polysaccharide is 50-100 times.

4. The application of the Pseudomonas fermentation biological agent according to any one of claims 1 or 2, or the application of the Pseudomonas fermentation biological agent prepared by the method according to any one of claims 3, in promoting cucumber growth.

5. Containing the *Pseudomonas* genus as described in claim 1 Pseudomonas The compound system of sp. strain CD1 is characterized by, The compound system also contains stevia polysaccharides.

6. A method for improving the quality and efficiency of cucumber cultivation using a Pseudomonas fermentation biological agent prepared according to claim 3, the specific method being as follows: (1) Pick the aforementioned Pseudomonas spp. Pseudomonas A single colony of strain CD1 was inoculated into TSB liquid medium to obtain Pseudomonas spp. Pseudomonas Fermentation broth of strain CD1; (2) Take the Pseudomonas spp. from step (1) Pseudomonas The viable cell count in the fermentation broth of strain CD1 was determined by plate counting. The broth was then centrifuged to remove the precipitate, yielding a supernatant. This supernatant was diluted with sterile water to a viable cell concentration of 10⁻⁶. 7 A CFU / mL fermentation dilution was applied to the soil around the roots of cucumber plants. (3) Dilute stevia polysaccharide and sterile water at a volume ratio of 1:100, and then pour the diluted solution into the cucumber soil after inoculation in step (2).

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