Biological pollutant in-situ reduction method based on amoeba intelligent perception

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a biological pollutant in-situ reduction method based on amoeba intelligent perception. It is found for the first time that the Hammanamoeba has an excellent removal effect on biological pollutants on the surface of a treatment medium, and a biological preparation is further prepared by taking the Hammanamoeba spores as active ingredients. The biological agent can efficiently remove pathogenic microorganisms such as klebsiella pneumoniae and biological membranes of the pathogenic microorganisms on the surface of an object, has the advantages of lasting effect, ecological safety, no secondary pollution, difficulty in inducing drug resistance and the like, and is superior to a traditional chemical disinfection and physical treatment mode. Moreover, the biological agent has good storage stability, the survival rate of spores is still kept at 85% or above after the biological agent is stored for 2 months under the low-temperature and dark condition, the requirements of commercialized storage and transportation are completely met, and the biological agent has wide application value in biological pollution treatment in various scenes such as medical environments and public facilities.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology. More specifically, it relates to an in-situ reduction method of biological pollutants based on ameba intelligent perception. BACKGROUND

[0002] Surface bio-contamination is one of the major challenges in the field of global public health. The colonization, enrichment and spread of microorganisms such as bacteria, fungi, viruses and algae, as well as their metabolites and genetic material (such as antibiotic resistance genes, ARGs) on various surfaces (including medical devices, building walls and food processing equipment) continue to threaten human health and ecological safety. With the intensification of environmental pollution and the continuous evolution of microbial drug resistance, research shows that biofilms and their embedded drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), not only can survive for a long time, but also can release ARGs. In addition, the physical barrier formed by extracellular polymeric substances (EPS) can hinder the penetration of disinfectants, protect the bacterial population in the film, and increase the risk of cross-infection and horizontal gene transfer (HGT).

[0003] Current commonly used treatment technologies still have obvious limitations. Broad-spectrum chemical disinfectants (such as hypochlorite and quaternary ammonium salt) can kill free surface microorganisms, but it is difficult to effectively penetrate the biofilm, and the removal effect of free ARGs and dormant pathogens in the film is limited, reflecting the limitations of chemical methods in dealing with biological pollution. Mechanical methods such as high-pressure water jet and ultrasonic cleaning can strip part of the biofilm, but they can easily damage the surface structure of the object, and the biofilm fragments after stripping may still carry active ARGs, leading to secondary pollution. Ultraviolet (UV-C) irradiation can damage microbial DNA, but it has weak degradation ability for free ARGs fragments released in the environment, reflecting the limitations of physical technology. Biological control strategies such as bacteriophages and antibacterial peptides have specific bactericidal ability, but they are difficult to deal with mixed biofilms of multiple species (such as bacterial-fungal symbiotic systems) due to single target, and have poor effect on eukaryotic pathogens (such as Aspergillus). The colonization stability of engineered bacteria (such as Bacillus subtilis expressing lysozyme) is low due to the influence of environmental factors such as temperature and pH, and there is an ecological risk of engineered bacteria escape.

[0004] Amoeba, as a kind of protists with broad-spectrum predation activity, is considered as an efficient'microbial predator' in nature. This kind of single-celled eukaryotes plays a key role in the regulation of environmental microbial communities, and can identify and eliminate various microorganisms such as bacteria and fungi through phagocytosis, and even effectively reduce the resistance genes in soil. For example, a model amoeba Dictyostelium discoideum can effectively reduce the resistance genes in soil, which is disclosed in a Chinese patent. However, the pollution control technology based on this organism still faces the practical problem of insufficient removal efficiency, and the production of fruiting bodies during spore reproduction will cause obvious trace residues on smooth surfaces.

[0005] Therefore, it is urgent to develop a new governance strategy with high-efficiency microbial removal efficiency, long-term ecological safety and no secondary pollution risk, so as to effectively block the transmission route of pathogenic microorganisms in the environment. SUMMARY

[0006] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the prior art that lack a method with high-efficiency microbial removal efficiency, long-term ecological safety and no secondary pollution risk. The primary object is to provide the application of Vermamoeba vermiformis in treating biological pollutants on the surface of a medium.

[0007] The second object of the present application is a method for in-situ reduction of biological pollutants using the Vermamoeba vermiformis, which comprises the step of contacting the surface of the medium contaminated by the biological pollutants with the Vermamoeba vermiformis.

[0008] The third object of the present application is to provide a biological agent.

[0009] The fourth object of the present application is to provide a preparation method of the biological agent.

[0010] The fifth object of the present application is to provide the application of the biological agent in treating biological pollutants on the surface of a medium.

[0011] The sixth object of the present application is to provide a method for in-situ reduction of biological pollutants using the biological agent.

[0012] The above objects of the present application are achieved by the following technical solutions:

[0013] The present application protects the application of Vermamoeba vermiformis in treating biological pollutants on the surface of a medium, wherein the Vermamoeba vermiformis is DW8S, which has been deposited in the China Center for Type Culture Collection on July 2, 2024, and the deposit number is CCTCC NO: C2024147.

[0014] The present application protects a method for reducing biological pollutants in situ, which comprises the step of contacting a medium surface contaminated by biological pollutants with Vermamoeba vermiformis DW8S.

[0015] The Vermamoeba vermiformis DW8S has been deposited with the China Center for Type Culture Collection on July 2, 2024, and has a deposit number of CCTCC NO: C2024147.

[0016] The present application first discovered that the above-mentioned Vermamoeba vermiformis has excellent cleaning effect in treating biological pollutants on the surface of the medium, which is significantly higher than the existing model Amoeba Dictyostelium discoideum, and the Amoeba naturally hibernates after the treatment is completed, without leaving obvious traces and without the risk of secondary pollution.

[0017] Further, the Vermamoeba is Vermamoeba spore.

[0018] Preferably, the concentration of the Vermamoeba spore is 1×10 6 ~ 1×10 8 / mL.

[0019] More preferably, the concentration of the Vermamoeba spore in the biological agent is 1×10 7 / mL.

[0020] Preferably, the medium is a solid or semi-solid medium; more preferably, the medium comprises one or more of a solid culture medium, plastic, glass, metal, building wall surface, and soil.

[0021] Preferably, the solid culture medium is a non-nutrient medium.

[0022] Preferably, the biological pollutants include any one or more of pathogenic microorganisms and their metabolites, pathogenic microorganism biofilms, and resistance genes carried by pathogenic microorganisms.

[0023] The present application protects a biological agent comprising Vermamoeba spores, wherein the Vermamoeba is Vermamoeba vermiformis DW8S, which has been deposited with the China Center for Type Culture Collection on July 2, 2024, and has a deposit number of CCTCC NO: C2024147.

[0024] The aforementioned biopharmaceutical based on *Entamoeba harmanii* exhibits significantly higher removal efficiency than the existing model amoeba *Dictyostelium discoideum*. Furthermore, the entire process targets only the specific contaminant, without damaging surface materials, producing no harmful residues, or inducing resistance. After treatment, the amoebas naturally enter dormancy, leaving no obvious traces and posing no risk of secondary contamination, thus effectively blocking the transmission pathways of pathogenic microorganisms in the environment. Moreover, this biopharmaceutical demonstrates good storage stability; after two months of storage under low-temperature, light-protected conditions, the spore survival rate remains above 85%, fully meeting the requirements for commercial storage and transportation. Therefore, this biopharmaceutical combines high microbial removal efficiency, long-term ecological safety, no risk of secondary contamination, and good storage stability.

[0025] Preferably, the concentration of *Amoeba harmanii* spores in the biological agent is 1 × 10⁻⁶. 6 ~1×10 8 per mL.

[0026] More preferably, the concentration of Acanthamoeba harmanii spores in the biological agent is 1×10⁻⁶. 7 per mL.

[0027] Furthermore, the biological agent further includes an antifreeze, a dispersant, and a neutral buffer solution; preferably, the biological agent further includes 18 vol% to 22 vol% of antifreeze, 0.01 vol% to 0.1 vol% of dispersant, and the balance being a neutral buffer solution.

[0028] More preferably, the biological agent further comprises 19 vol% to 21 vol% of antifreeze, 0.04 vol% to 0.06 vol% of dispersant, and the balance being a neutral buffer solution.

[0029] Furthermore, the biological agent satisfies one or more of the conditions in (1) to (4):

[0030] (1) The antifreeze agent includes one or two of glycerin and trehalose;

[0031] (2) The dispersant includes one or both of ethyl phenyl polyethylene glycol (NP-40) and Tween 80;

[0032] (3) The pH of the neutral buffer solution is 6.8 to 7.4;

[0033] (4) The neutral buffer solution includes phosphate buffer solution or HEPES buffer solution.

[0034] Using the above-mentioned antifreeze as a cryoprotectant for amoebae, all of the above reagents can play a role in cryoprotection. More preferably, the antifreeze is glycerol.

[0035] The above-mentioned dispersants do not have a significant impact on spore activity and stability.

[0036] Preferably, the phosphate buffer solution includes any one of potassium phosphate buffer, standard phosphate buffer saline, and Durham phosphate buffer saline, more preferably potassium phosphate buffer. All of the above buffer solutions have good pH stability and a good effect on maintaining spore dormancy.

[0037] Furthermore, the potassium phosphate buffer is a KK2 buffer. As a commonly used formulation, the KK2 buffer comprises the following components: 2.2 g / L potassium dihydrogen phosphate, 0.7 g / L dipotassium hydrogen phosphate, and water to a final volume of 1 L.

[0038] Furthermore, the dosage form of the biological agent includes a spray.

[0039] The present invention protects a method for preparing the biological agent, comprising the following steps: mixing the components according to the specified ratio to obtain the biological agent.

[0040] As a preferred embodiment, the preparation method of the biological agent includes the following steps: incubating amoebae, collecting its spores in a neutral buffer solution to prepare an amoebae spore suspension, adding other components to the obtained amoebae spore suspension in a certain proportion, mixing well, and obtaining the biological agent.

[0041] Preferably, the spores collected are second-generation spores.

[0042] Furthermore, the incubation conditions are as follows: culture under light at 20–30°C (preferably 21°C) for 5–9 days (preferably 7 days). After the spores are revived, they are incubated under the above conditions to obtain the first generation of spores. A second incubation is then performed to obtain the second generation of spores.

[0043] Furthermore, the culture medium used for resuscitation is a nutrient agar plate, and the culture medium used for incubation is a non-nutrient agar plate.

[0044] Furthermore, the formulation of the nutrient agar plate is as follows: glucose 2.0 g / L, peptone 2.0 g / L, yeast extract 2.0 g / L, magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.9 g / L, dipotassium hydrogen phosphate 1.0 g / L, agar 15.0 g / L, and water to a final volume of 1 L. Before use, the components are mixed in the specified proportions, autoclaved at 121°C for 20 minutes, and then cooled and solidified to obtain the final product.

[0045] Furthermore, the formula for the non-nutrient agar plate is as follows: potassium dihydrogen phosphate 2.2 g / L, dipotassium hydrogen phosphate 0.7 g / L, agar 15.0 g / L, and water to a final volume of 1 L. Before use, the components are mixed in the specified proportions, autoclaved at 121°C for 20 minutes, and allowed to cool and solidify to obtain the final product.

[0046] This invention also protects the use of the biological agent in treating biological contaminants on the surface of a medium;

[0047] Preferably, the medium is a solid or semi-solid medium; more preferably, the medium includes one or more of solid culture media, plastics, glass, metals, building walls, and soil.

[0048] Preferably, the solid culture medium is a non-nutritive culture medium.

[0049] Preferably, the biological pollutant includes any one or more of the following: pathogenic microorganisms and their metabolites, pathogenic microbial biofilms, and resistance genes carried by pathogenic microorganisms.

[0050] The present invention also protects a method for in-situ reduction of biological contaminants, the method comprising the step of contacting the surface of a medium contaminated by the biological contaminant with the biological agent.

[0051] Furthermore, when the biological agent is in the form of a spray, it is sprayed onto the surface of the medium contaminated by biological pollutants, and the spraying amount of the biological agent is (1~10)×10⁻⁶. 4 Spores per square centimeter.

[0052] Furthermore, the in-situ reduction mechanism is as follows: (1) Intelligent sensing predation and reduction process: Spores are sensitive to pathogenic microorganisms and their metabolites, pathogenic microbial biofilms, and ARGs that may be carried. They germinate when contamination is present, especially under conditions of 20-30℃. After the spore preparation is uniformly delivered to the contaminated surface through a spray device, the spores of *Entamoeba hammannii* germinate into trophozoites in a short time. The trophozoites prey on and digest biological pollutants, achieving the primary removal of biological pollutants; (2) Cyclic continuous action: After the pollutants are removed, *Entamoeba hammannii* forms new spores and waits on the surface of the medium. When new contamination is encountered, the spores germinate again, achieving cyclic reduction without repeated application. (3) Deep removal: The trophozoites can directly engulf and degrade pathogens and ARGs in situ, achieving root-cause removal. The entire process targets only the target pollutants, does not damage the surface material, does not produce harmful residues, and does not induce resistance. After treatment, the amoeba naturally goes dormant, with no risk of secondary pollution.

[0053] Therefore, the in-situ biological reduction method based on amoeba intelligent sensing and predation function described in this invention has intelligent sensing, high stability of recycling and excellent deep clearance ability. This method is based on Harman amoeba spore biological agents.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] This invention marks the first discovery of the excellent removal effect of *Entamoeba hemangiosum* on biological contaminants on surfaces. Furthermore, a biological agent was prepared using *Entamoeba hemangiosum* spores as the active ingredient. This biological agent can efficiently remove pathogenic microorganisms such as *Klebsiella pneumoniae* and their biofilms from object surfaces, offering advantages such as long-lasting action, ecological safety, no secondary pollution, and low likelihood of inducing drug resistance, surpassing traditional chemical disinfection and physical treatment methods. Moreover, this biological agent exhibits good storage stability; after two months of storage under low-temperature, light-protected conditions, the spore survival rate remains above 85%, fully meeting the requirements for commercial storage and transportation. It has broad application value in the treatment of biological contamination in various scenarios such as medical environments and public facilities. Attached Figure Description

[0056] Figure 1 Morphological images of two morphologically intact amoebae spores (incubated on non-nutrient medium) after isolation and purification. (a) is an optical microscope image of *Vermamoeba vermiformis* DW8S spores; (b) is an optical microscope image of *Dictyostelium discoideum* QS9 spores; (c) is a side view of *Vermamoeba vermiformis* DW8S spores under normal growth conditions; and (d) is a side view of *Dictyostelium discoideum* QS9 spores under normal growth conditions.

[0057] Figure 2 The graph shows the 24-hour reduction effect of the amoeba spray formulation on Klebsiella pneumoniae on the surface of various media.

[0058] Figure 3 Statistical chart showing the reduction effect and response time (short-term 0-8h response) of amoeba spray formulation.

[0059] Figure 4 A statistical chart showing the reduction effect of amoeba spray formulation on surface microorganisms (three applications of Klebsiella pneumoniae).

[0060] Figure 5 A statistical chart showing the reduction rate of surface microorganisms by amoeba spray formulations (three applications of Klebsiella pneumoniae). Detailed Implementation

[0061] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0062] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0063] Figure 1 (a) indicates Figure 1 Figure (a) in the diagram is used as an example; the other figures are named similarly.

[0064] Example 1: Cultivation, purification, and formulation preparation of amoebae spores

[0065] 1.1 Materials and Methods

[0066] Amoebic strain: The amoeba in the preparation is *Vermamoeba vermiformis* DW8S, which was deposited at the China Center for Type Culture Collection on July 2, 2024, with accession number CCTCC NO: C2024147. The experimental data also used *Dictyostelium discoideum* QS9 (disclosed in CN117600218A) as a supplementary control. Furthermore, it should be noted that *V. vermiformis* itself has extremely weak pathogenicity and does not cause deep tissue infection or systemic disease (Delafont, V., et al., *Vermamoeba vermiformis: a Free-Living Amoeba of Interest*. *Microbial Ecology*, 2018. 76(4): p. 991-1001.). Therefore, the *Vermamoeba vermiformis* DW8S of this invention has ecological safety.

[0067] Culture medium for resuscitation: Nutrient agar plates. Formula: glucose 2.0 g / L, peptone 2.0 g / L, yeast extract 2.0 g / L, magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.9 g / L, dipotassium hydrogen phosphate 1.0 g / L, agar 15.0 g / L, deionized water to a final volume of 1 L, autoclave at 121°C for 20 minutes.

[0068] Culture medium used for cultivation: non-nutrient agar plates. Formula: potassium dihydrogen phosphate 2.2 g / L, dipotassium hydrogen phosphate 0.7 g / L, agar 15.0 g / L, deionized water to a final volume of 1 L, autoclave at 121°C for 20 minutes.

[0069] Spore purification: The spores were eluted by centrifugation using KK2 buffer solution (potassium dihydrogen phosphate 2.2 g / L, dipotassium hydrogen phosphate 0.7 g / L, and deionized water to a final volume of 1 L).

[0070] Formulation: KK2 phosphate buffer (pH 7.0) was used as the base solvent, with 20% (v / v) glycerol (Sinopharm Chemical Reagent Co., Ltd.) added as a protective agent and 0.05% (v / v) NP-40 (ethyl phenyl polyethylene glycol) added as a dispersant.

[0071] 1.2 Experimental Procedure

[0072] Spore thawing: Remove the frozen *Amoebae hammannii* spores from the preservation solution and thaw rapidly at room temperature. Take 20 μL of the spore suspension and react it with OD... 600 A food bacteria suspension of 10.0 was spread onto nutrient agar plates and incubated at 21°C under light for 7 days.

[0073] First generation culture: Scrape the successfully revived amoebae spores with a sterile inoculation loop, streak them onto a non-nutrient agar plate, and incubate at 21°C under light for 7 days to obtain the first generation of spores, effectively reducing the number of food bacteria on the culture interface.

[0074] Second generation culture: Spores were scraped from the surface of the first generation culture with a sterile inoculation loop and re-streaked onto a new non-nutrient agar plate. The plate was then cultured at 21°C under light for 7 days to obtain a large number of second generation spores.

[0075] Spore collection and purification: Add 5 mL of pre-chilled KK2 buffer solution to a culture plate, and gently scrape off the spores with a clean inoculation loop. Transfer the suspension to a 15 mL centrifuge tube, centrifuge at 500 × g for 5 minutes at 4 °C, and discard the supernatant to remove any culture medium residue. Repeat the washing and centrifugation twice.

[0076] Formulation preparation: The purified spores were resuspended in KK2 phosphate buffer and counted under an optical microscope using a hemocytometer. The spore concentration was adjusted to 1×10⁻⁶. 8 The spore concentration was used as the stock solution. The final spray formulation was prepared according to the following ratio: 10 mL spore stock solution + 20 mL glycerol + 50 μL 10 vol% NP-40 stock solution + KK2 buffer to a final volume of 100 mL. The final formulation contained 1 × 10⁻⁶ spores. 7 per mL. Aliquot the formulation into brown spray bottles and store at 4°C protected from light.

[0077] The same concentration of amoebae spore preparation (Dictyostelium discoideum QS9) was prepared using the same method, and the other components and their concentrations in the preparation were the same.

[0078] 1.3 Results

[0079] This invention successfully obtained a high-purity suspension of second-generation *Entamoeba hammannii* spores and prepared a designed amoebae spore spray formulation. Microscopic observation revealed the morphology of *Entamoeba hammannii* as follows: Figure 1 As shown in (a), the spores are morphologically intact, without significant bacterial contamination, and do not produce fruiting bodies during normal growth. The surface of the medium ( Figure 1 (c) The grayish-yellow area in the image represents the medium. No traces were found. Figure 1 (c)), while the fruiting body ( ) during the reproduction of spores of *Discocephalus spp.* Figure 1 (b) will result in noticeable traces remaining on smooth surfaces. Figure 1 (d)).

[0080] As can be seen, the Harman Amoeba of the present invention does not damage the surface material, does not produce any residue, and poses no risk of secondary pollution.

[0081] Example 2: Evaluation of the reduction effect of amoeba spray on Klebsiella pneumoniae on different media surfaces

[0082] 2.1 Materials and Methods

[0083] Bacterial strain: Klebsiella pneumoniae isolate.

[0084] Media materials: sterile non-nutrient culture medium, sterile plastic slides, sterile glass slides, sterile stainless steel sheets, sterile red brick pieces (simulating building walls), and sterile soil.

[0085] Experimental Groups:

[0086] Experimental group: Klebsiella pneumoniae + spraying with an amoeboid spore preparation (Amoeba Hamman) prepared according to the method in Example 1.

[0087] Control group 1: Klebsiella pneumoniae + sprayed blank preparation without spores (containing only 20% glycerol (v / v) + 0.05% (v / v) NP-40 KK2 buffer).

[0088] Control group 2: Klebsiella pneumoniae + sprayed KK2 buffer

[0089] Control group 3: Klebsiella pneumoniae + spraying with the amoebae spore preparation (Dendrobium distichum) prepared according to the method in Example 1.

[0090] (Each group has 3 repetitions at each time point)

[0091] Detection time points after a single application: 0h (baseline), 8h, 16h, and 24h after treatment.

[0092] Detection time points after multiple applications: 0h (baseline value), 24h, 48h, and 72h after treatment.

[0093] Comparison time points for different amoeba preparation application groups: 2h, 4h, 6h, and 8h after treatment.

[0094] 2.2 Experimental Procedure

[0095] Construction of surface bacterial contamination model: Klebsiella pneumoniae strain was evenly spread on nutrient medium and incubated at room temperature for 48 h. A sample was then picked up with a sterile inoculating loop and dissolved in KK2 buffer, adjusting the concentration to 1 × 10⁻⁶. 9 CFU / mL. Add 10 μL of bacterial suspension evenly to the surface of each medium, and air dry in a clean bench for 15 minutes to ensure an initial inoculum of approximately 1 × 10⁻⁶ CFU / mL on each medium surface. 7 CFU.

[0096] Spray treatment: For the experimental group media, 20 μL of amoebae spore preparation (containing approximately 2 × 10⁻⁶ spores) was evenly sprayed using a metered sprayer. 5 (Spores). The control group was sprayed with an equal volume of the corresponding liquid.

[0097] Incubation: Place all the treated media slides in a petri dish lined with sterile filter paper (maintain light and incubate at 21°C).

[0098] Sampling and CFU Counting: At each time point, the media slides were transferred to centrifuge tubes containing 5 mL of elution buffer (KK2 + 0.1% NP-40), and vortexed for 2 minutes to fully elute the amoebae spores. The elution buffer was serially diluted 10-fold, and 100 μL of the appropriately diluted bacterial suspension was inoculated onto SM / 5 agar plates using the spread plate method. After incubation at room temperature upside down for 24 hours, the colonies (CFU) were counted.

[0099] Data analysis: Calculate the number of colonies recovered per square centimeter of media surface (CFU / cm²). 2 ), and calculate the reduction rate.

[0100] 2.3 Results

[0101] Results of a single application of Klebsiella pneumoniae: Figure 2As shown, the amoebae spore spray formulation of this invention exhibits extremely high reduction efficiency against Klebsiella pneumoniae on various media surfaces within a short period of time. Compared with the control group, the spray formulation containing Amoebae hammannii spores effectively eliminated surface microorganisms at all time points (8h, 16h, 24h), with a significantly higher elimination efficiency than the spray formulation containing Dictyophora distichum spores. A elimination efficiency of >95% was achieved (24h) on all smooth surfaces.

[0102] at the same time, Figure 3 The predation patch assay supplemented the evidence for determining the surface microbial clearance behavior of amoebae. The spray formulation containing Entamoeba harmanii spores showed a significantly preferential response to the clearance of surface pathogens, with shorter response time and higher predation efficiency, and the application effect was better than that of the spray formulation containing Diplostomum discoidum spores.

[0103] Results of repeated administration of Klebsiella pneumoniae Figure 4 As shown, after only one application, the Harman amoeba spore spray formulation of this invention can still continuously and efficiently eliminate surface microorganisms even after multiple in-situ applications of Klebsiella pneumoniae, demonstrating good resistance to re-proliferation, intelligent sensing and active removal capabilities, and repeatable disinfection effects. Figure 5 As shown, after multiple in-situ applications of Klebsiella pneumoniae, the Harman amoeba spore spray formulation of the present invention only requires one application and can still maintain a reduction rate of over 90% against Klebsiella pneumoniae for the next 3 days.

[0104] Example 3: Stability test of amoeba spore formulation

[0105] 3.1 Method

[0106] The amoebae spore preparation prepared in Example 1 was stored at 4°C in the dark. Samples were taken at 0, 1, and 2 months after storage. Spore activity was assessed using the predatory spot counting method: the preparation was serially diluted 10-fold, and 10 μL of spore solution from each dilution was added dropwise to a non-nutrient agar plate covered with food source bacteria. After the liquid was absorbed, the plate was incubated at 21°C under light for 36 hours. The number of patches (predatory spots) formed around each spot was observed and counted to calculate the concentration of still-active spores.

[0107] Table 1. Changes in the activity of amoebae spore preparations after storage in the dark (based on predatory spot counting method)

[0108]

[0109] *Only representative dilution data are listed for counts within the ideal range (20-200 patches).

[0110] The results are shown in Table 1. The formulation of the present invention can effectively protect the activity of amoebae spores. After being stored at 4°C in the dark for 2 months, the spore survival rate still remained above 85%, which fully meets the requirements for commercial storage and transportation.

[0111] In summary, the above results demonstrate that the present invention has successfully prepared a highly efficient and stable amoeboid spore spray formulation. This formulation can be widely applied to surfaces of various materials and has an excellent and sustained reduction effect on pathogenic bacteria (such as Klebsiella pneumoniae) or the microbial films they form. Furthermore, the formulation exhibits good storage stability at 4°C and has a shelf life of more than 2 months.

[0112] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of Harman amoeba in treating biological contaminants on media surfaces, characterized in that, The Harman amoeba, Vermamoeba vermiformis DW8S, was deposited at the China Center for Type Culture Collection on July 2, 2024, with accession number CCTCC NO: C2024147.

2. A method for in-situ reduction of biological pollutants, characterized in that, The method includes the step of bringing the surface of a medium contaminated with biological contaminants into contact with Amoebae hammannii spores; The Harman amoeba, Vermamoeba vermiformis DW8S, was deposited at the China Center for Type Culture Collection on July 2, 2024, with accession number CCTCC NO: C2024147.

3. A biological agent, characterized in that, The biological agent contains hammamoeba spores, and the hammamoeba is Vermamoeba vermiformis DW8S, which was deposited at the China Center for Type Culture Collection on July 2, 2024, with accession number CCTCC NO: C2024147. Preferably, the concentration of *Amoeba harmanii* spores in the biological agent is 1 × 10⁻⁶. 6 ~1×10 8 per mL.

4. The biological agent according to claim 3, characterized in that, The biological agent also includes an antifreeze, a dispersant, and a neutral buffer solution.

5. The biological agent according to claim 4, characterized in that, The biological agent also contains 18 vol% to 22 vol% of antifreeze, 0.01 vol% to 0.1 vol% of dispersant, and the balance being a neutral buffer solution.

6. The biological agent according to claim 4 or 5, characterized in that, The biological agent satisfies one or more of the conditions in (1) to (4): (1) The antifreeze agent includes one or two of glycerin and trehalose; (2) The dispersant includes one or both of ethyl phenyl polyethylene glycol and Tween 80; (3) The pH of the neutral buffer solution is 6.8 to 7.4; (4) The neutral buffer solution includes phosphate buffer solution or HEPES buffer solution.

7. The biological agent according to claim 3, characterized in that, The dosage form of the biological agent includes a spray.

8. A method for preparing the biological agent according to any one of claims 3 to 7, characterized in that, The process includes the following steps: mixing the components according to the specified ratio to obtain the biological agent.

9. The use of the biological agent according to any one of claims 3 to 7 in treating biological contaminants on the surface of a medium; Preferably, the medium is a solid or semi-solid medium; more preferably, the medium includes one or more of solid culture media, glass, plastic, metal, building walls, and soil. Preferably, the biological pollutant includes one or more of the following: pathogenic microorganisms and their metabolites, pathogenic microbial biofilms, and resistance genes; Preferably, the pathogenic microorganism is Klebsiella pneumoniae.

10. A method for in-situ reduction of biological pollutants, characterized in that, The method includes the step of contacting the surface of a medium contaminated with biological pollutants with the biological agent described in any one of claims 3 to 7.

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