Biological disinfectant preparation for pathogenic microorganisms in closed and semi-closed space, preparation method and application method

Abstract

The application discloses a biological disinfection preparation for pathogenic microorganisms in airtight and semi-hermetic space, a preparation method and an application method. The biological disinfection preparation comprises a CO2 concentration response type composite biological polysaccharide carrier and a composite active component coated by the carrier; wherein the carrier is compounded by sodium alginate, xanthan gum and hyaluronic acid to form a microcapsule system with a porous network structure; and the composite active component comprises specific deep-sea pressure-resistant bacterial lyase, volcanic thermophilic protease, triterpenoid glycyrrhizinic acid and phenolic gallic acid. In addition, the application also comprises an application method for airtight space atomization by generating super-fine mist droplets with a particle size of less than 5 microns through a piezoelectric microporous atomizer. The application can efficiently degrade pathogens by the synergistic effect of extreme environment enzymes and plant extracts, and can realize on-demand triggering release by sensing CO2 concentration changes through the polysaccharide carrier, so as to avoid chemical residues in traditional disinfection and realize long-acting dynamic disinfection for human-machine coexistence.

Description

Technical Field

[0001] This invention relates to biological disinfectant preparations, preparation methods, and application methods, and particularly to a biological disinfectant preparation, preparation method, and application method for pathogenic microorganisms in enclosed and semi-enclosed spaces. Background Technology

[0002] Enclosed spaces (such as underground fortifications, laboratories, and passenger cabins) often have poor air circulation, low air exchange rates, and high humidity and temperature, making them ideal environments for the growth of pathogenic microorganisms such as bacteria, fungi, and viruses, posing a continuous threat to the health of people inside. Traditional disinfection methods mainly rely on chemical agents or physical means. Common chemical disinfection methods include the use of chlorine-containing disinfectants, alcohol, or hydrogen peroxide, while physical methods primarily involve ultraviolet light irradiation. In recent years, with the rise of green and environmentally friendly concepts, enzyme-based disinfection technology has gradually been applied as a new green alternative. This technology currently mainly relies on enzyme proteins derived from common microorganisms, such as lysozyme and protease, which destroy the structure of microorganisms through biodegradation to achieve sterilization.

[0003] Existing technologies still have many limitations in practical applications, specifically as follows: (1) Safety and side effects: Although traditional chemical disinfectants have a certain bactericidal effect, they generally have problems such as drug residue, strong corrosiveness, and great irritation to human skin and mucous membranes.

[0004] (2) Limitations of physical disinfection: Methods such as ultraviolet lamp irradiation have obvious blind spots, relatively low disinfection efficiency, and it is difficult to achieve dynamic and continuous disinfection under the condition of human-machine coexistence.

[0005] (3) Disruption of microecological balance: Although existing conventional enzyme preparations can degrade pathogens, they also have a strong inhibitory effect on the normal flora that are beneficial to the human body, which can easily disrupt the microecological balance of the human body or the environment.

[0006] (4) Lack of intelligent response mechanism: Existing enzyme preparation technologies often lack the ability to sense and respond to environmental factors (such as changes in humidity, temperature or microbial concentration), making it difficult to achieve on-demand, scenario-based release. Due to their inability to adapt to complex and ever-changing actual environments, existing enzyme technologies are unable to meet the dynamic requirements of continuous, adaptive and highly safe disinfection in enclosed spaces. Summary of the Invention

[0007] The purpose of this invention is to provide a biological disinfectant preparation, preparation method, and application method for pathogenic microorganisms in enclosed and semi-enclosed spaces. It can efficiently degrade pathogens by utilizing the synergistic effect of enzymes in extreme environments and plant extracts, and achieve on-demand release based on scene-bound changes in CO2 concentration (>1000ppm) through a polysaccharide carrier. This effectively avoids the chemical residues and microecological damage of traditional disinfection, and safely achieves long-term dynamic disinfection that allows for coexistence between humans and machines.

[0008] To achieve the above objectives, a first aspect of the present invention provides a biological disinfectant for pathogenic microorganisms in enclosed and semi-enclosed spaces, comprising: CO2 concentration-responsive composite biopolysaccharide carrier, wherein the carrier constitutes a microcapsule system with a porous network structure, and a composite active component is encapsulated by the carrier; The composite active components include microbial lysins, proteases, triterpenoids, and phenolic compounds, which are used to produce a synergistic bactericidal effect against pathogenic microorganisms.

[0009] In one embodiment of the present invention, the composite biopolysaccharide carrier is composed of sodium alginate, natural polysaccharides and hyaluronic acid.

[0010] In one embodiment of the present invention, the natural polysaccharide is xanthan gum; The composite biopolysaccharide carrier is composed of sodium alginate, xanthan gum, and hyaluronic acid in a mass ratio of (2.5-3.5):(0.8-1.2):(1.8-2.2).

[0011] In one embodiment of the present invention, the microbial lysin is a deep-sea pressure-resistant bacterial lysin; the protease is a volcanic thermophilic bacterial protease; the triterpenoid compound is a triterpenoid glycyrrhetinic acid; and the phenolic compound is a phenolic gallic acid.

[0012] In one embodiment of the present invention, the amino acid sequence of the volcanic thermophilic protease is as shown in SEQ ID NO: 2; or it is a derived polypeptide that has more than 90% homology with SEQ ID NO: 2 and has the same cleavage activity.

[0013] The amino acid sequence of the deep-sea pressure-resistant bacterial lysin is shown in SEQ ID NO: 1; or it is a derived polypeptide with more than 90% homology to SEQ ID NO: 1 and with the same cleavage activity.

[0014] The mass ratio of the deep-sea pressure-resistant bacterial lyase, the volcanic thermophilic bacterial protease, the triterpenoid glycyrrhetinic acid, and the phenolic gallic acid is (1.8-2.2):(0.9-1.1):(1.3-1.7):(0.9-1.1).

[0015] In a second aspect of the present invention, a method for preparing a biological disinfectant agent for pathogenic microorganisms in enclosed and semi-enclosed spaces as described above is provided, comprising the following steps: S1. Preparation of composite biological polysaccharide carrier; S2. Microbial lysin separation and purification; S3. Protease isolation and purification; S4. Mixing of composite active components: Weigh the microbial lysin, protease, triterpenoid compound and phenolic compound according to the proportion, and mix them evenly; S5. Formulation Mixing: The composite active components are uniformly dispersed in a composite biological polysaccharide carrier colloidal solution, and a pH adjuster is added to adjust the pH of the system to 6.5-7.5. The mixture is then stirred to obtain the final product.

[0016] In one embodiment of the present invention, in step S1, sodium alginate, natural polysaccharide, and hyaluronic acid are weighed in a solvent at a mass ratio of (2.5-3.5):(0.8-1.2):(1.8-2.2). A crosslinking agent is then added to the mixed solution to form a colloid through intermolecular crosslinking. The solution is then homogenized to obtain a colloidal solution with a stable three-dimensional network structure. Preferably, in step S1, a crosslinking agent is added to the mixed solution to form a colloid with a stable three-dimensional network structure; high-speed homogenization further refines the colloidal particles and improves the uniformity of the system, ultimately obtaining a colloidal solution with a fine texture and stable performance.

[0017] In step S2, the microbial lysin includes a deep-sea pressure-resistant bacterial lysin. In step S3, the protease includes thermophilic volcano protease; In step S4, the deep-sea pressure-resistant bacterial lyase, volcanic thermophilic bacterial protease, triterpenoid glycyrrhetinic acid and phenolic gallic acid are weighed in a mass ratio of (1.8-2.2):(0.9-1.1):(1.3-1.7):(0.9-1.1) and mixed evenly.

[0018] In one embodiment of the present invention, in step S2, the enzyme-producing strain is subjected to high-pressure fermentation culture at 50 MPa to 55 MPa and 4°C to 6°C; the fermentation supernatant is homogenized at low temperature and high pressure at 4°C to 10°C or subjected to ultrasonic disruption, followed by affinity chromatography capture. Preferably, in this embodiment, the affinity chromatography capture step includes: first, designing biomimetic affinity ligands or introducing recombinant protein tags targeting the specific amino acid sequence of the deep-sea pressure-resistant bacterial lysin, and achieving efficient capture of the target enzyme through one or two steps of affinity chromatography. Next, preliminary purification is performed using low-temperature gradient salting-out, followed by concentration and desalting using an ultrafiltration membrane, simultaneously removing small molecule impurities. Then, final purification is performed using reversed-phase or ion-exchange high-performance liquid chromatography (HPLC) to further improve enzyme purity. Finally, a composite protective agent is added to the purified enzyme solution, and the solution is freeze-dried under vacuum to obtain deep-sea pressure-resistant bacterial lysin powder.

[0019] In step S3, the purification process includes pre-treatment of the fermentation supernatant by incubating it at 60°C to 70°C for 30 to 40 minutes for heat denaturation, followed by centrifugation and filtration to remove heat-sensitive contaminating proteins. Preferably, in this embodiment, Sephadex or Superdex series gel filtration columns are used during gel filtration chromatography to achieve a purity of over 95% for the thermophilic volcano protease, maintaining an activity above 20,000 U / mg.

[0020] A third aspect of the present invention provides a method for applying the above-described biological disinfectant agent targeting pathogenic microorganisms in enclosed and semi-enclosed spaces, comprising the following steps: Step 1: Add the biological disinfectant to the piezoelectric microporous atomizer; Step 2: The application rate is 0.5 mL to 1.2 mL per cubic meter, which is atomized by generating droplets with a particle size ≤ 5 μm through the piezoelectric microporous atomizer; Step 3: After atomization, keep the space sealed for 30 to 60 minutes.

[0021] In one embodiment of the present invention, the application is adapted to closed or semi-closed spaces where the CO2 concentration can easily rise to >1000ppm. When the CO2 concentration in the space is >1000ppm, the carrier structure changes, triggering a secondary release of the encapsulated composite active components.

[0022] The present invention has the following beneficial effects: First, this invention utilizes microbial enzymes isolated from extreme environments (such as high-temperature, high-salt, or high-pressure habitats) combined with triterpenoids and phenolic compounds as core bactericidal components. These components have unique origins and high specificity and stability, enabling them to degrade the structural and functional proteins of pathogens. This ensures that the disinfectant maintains its activity in complex environments. While effectively avoiding the problems of drug residues, strong corrosion, and mucosal irritation caused by traditional chemical disinfectants, it significantly reduces the broad-spectrum inhibition of beneficial normal flora in the human body and overcomes the defect of conventional enzyme preparations that easily disrupt the microecological balance.

[0023] Secondly, this invention constructs a CO2 concentration-responsive composite biopolysaccharide carrier composed of sodium alginate, xanthan gum, and hyaluronic acid, enabling disinfectant preparations to intelligently sense and respond to environmental changes. When the risk of pollution increases due to poor air circulation in a confined space, the carrier structure undergoes microscopic swelling or pH response, thereby triggering the secondary release of the encapsulated components. This solves the problem of existing technologies lacking a scenario binding mechanism and making it difficult to achieve on-demand release.

[0024] Furthermore, the porous microcapsule encapsulation structure of the biopolysaccharide carrier, combined with the antioxidant and free radical scavenging effects of phenolic gallic acid in the composite active components, greatly enhances the physicochemical stability of the extreme enzyme protein in complex temperature and humidity environments, achieving scientific sustained release of active ingredients and multi-target synergistic sterilization, and can maintain dynamic disinfection balance for a relatively long time in a closed space.

[0025] Finally, this invention optimizes the distribution and control of disinfectants by working in conjunction with ventilation systems and environmental monitoring equipment. Specifically, relying on the excellent film-forming and air-suspending properties of the composite carrier, this invention enables disinfectant particles to penetrate deep into every corner of enclosed spaces, effectively compensating for the shortcomings of traditional physical methods such as ultraviolet irradiation, which have blind spots. While reducing ineffective consumption in non-target areas and the overall ecological burden, it safely and efficiently achieves three-dimensional dynamic disinfection in a human-machine coexistence state. Attached Figure Description

[0026] Figure 1 The flowchart of a method for preparing a biological disinfectant for pathogenic microorganisms in enclosed and semi-enclosed spaces, according to an embodiment of the present invention, is disclosed. Figure 2 The diagram illustrates the antibacterial effect of a biological disinfectant agent against pathogenic microorganisms in enclosed and semi-enclosed spaces, according to an embodiment of the present invention; wherein, Figure 2 A shows a comparison of the antibacterial effects of a biological disinfectant agent targeting pathogenic microorganisms in enclosed and semi-enclosed spaces against Escherichia coli according to an embodiment of the present invention. Figure 2B illustrates the inhibition zone of Escherichia coli produced by a biological disinfectant agent targeting pathogenic microorganisms in enclosed and semi-enclosed spaces, according to an embodiment of the present invention. Figure 2 C reveals a comparative diagram showing the antibacterial effect of a biological disinfectant agent targeting pathogenic microorganisms in enclosed and semi-enclosed spaces against Staphylococcus aureus according to an embodiment of the present invention. Figure 2 D illustrates the inhibition zone of Staphylococcus aureus produced by a biological disinfectant agent targeting pathogenic microorganisms in enclosed and semi-enclosed spaces, according to an embodiment of the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0028] The first type of embodiment is the formulation and experimental verification of the present invention.

[0029] The first embodiment of the present invention is the formulation and preparation process of the present invention, combined with... Figure 1 The first embodiment is described below. This embodiment aims to obtain a biological disinfectant agent that targets pathogenic microorganisms in enclosed and semi-enclosed spaces. It includes: a CO2 concentration-responsive composite biopolysaccharide carrier, the carrier forming a microcapsule system with a porous network structure, and a composite active component encapsulated by the carrier; wherein, the composite active component contains microbial lysin, protease, triterpenoids and phenolic compounds, which are used to produce a synergistic bactericidal effect against pathogenic microorganisms.

[0030] In this embodiment, the composite biological polysaccharide carrier is composed of sodium alginate, natural polysaccharides, and hyaluronic acid.

[0031] In this embodiment, the natural polysaccharide is xanthan gum; the composite biological polysaccharide carrier is composed of sodium alginate, xanthan gum, and hyaluronic acid in a mass ratio of (2.5-3.5):(0.8-1.2):(1.8-2.2).

[0032] In this embodiment, the microbial lysin is a deep-sea pressure-resistant bacterial lysin; the protease is a volcanic thermophilic bacterial protease; the triterpenoid compound is glycyrrhetinic acid; and the phenolic compound is gallic acid.

[0033] In this embodiment, the amino acid sequence of the volcanic thermophilic protease is shown in SEQ ID NO: 2; or it is a derived polypeptide with more than 90% homology to SEQ ID NO: 2 and the same cleavage activity. The amino acid sequence of the deep-sea pressure-resistant bacterial lyase is shown in SEQ ID NO: 1; or it is a derived polypeptide with more than 90% homology to SEQ ID NO: 1 and the same cleavage activity. The mass ratio of the deep-sea pressure-resistant bacterial lyase, the volcanic thermophilic protease, the triterpenoid glycyrrhetinic acid, and the phenolic gallic acid is (1.8-2.2):(0.9-1.1):(1.3-1.7):(0.9-1.1).

[0034] In this embodiment, a method for preparing a biological disinfectant agent targeting pathogenic microorganisms in enclosed and semi-enclosed spaces includes, S1. Preparation of composite biological polysaccharide carrier: S11. Weigh sodium alginate, xanthan gum, and hyaluronic acid as raw materials for the composite biological polysaccharide carrier according to a mass ratio of (2.5-3.5):(0.8-1.2):(1.8-2.2). Stir at 30 rpm / min for 30 min at 80°C to fully dissolve them in the solvent to obtain a mixed solution. In this embodiment, in the lower limit verification group, the mass ratio of sodium alginate, xanthan gum, and hyaluronic acid in the composite biological polysaccharide carrier was adjusted to 2.5:0.8:1.8. Experiments show that this lower limit formulation is suitable for environments with small space volume, low CO2 concentration, and basic sterilization requirements. In the upper limit verification group, the mass ratio of sodium alginate, xanthan gum, and hyaluronic acid in the composite biological polysaccharide carrier was adjusted to 3.5:1.2:2.2. Experiments show that this upper limit formulation is suitable for environments with large space volume, controllable CO2 concentration, and higher sterilization requirements.

[0035] S12. Add a crosslinking agent to the mixed solution, the amount of which is 0.5 times the standard crosslinking agent dosage (i.e., half the dosage), and then perform intermolecular crosslinking at room temperature and pressure to form a colloid with a stable three-dimensional network structure. In this embodiment, 0.5 times refers to half the standard crosslinking agent dosage used in conventional crosslinking reactions in the art.

[0036] S13. The colloid obtained after S12 is subjected to high-speed homogenization to further refine the colloidal particles and improve the uniformity of the system, ultimately obtaining a colloidal solution with a fine texture and stable performance.

[0037] S2. Preparation of active component 1: Isolation and purification of deep-sea pressure-resistant bacterial lysin (SEQ ID NO: 1).

[0038] S21. Fermentation and collection: Under high pressure fermentation conditions of 50MPa to 55MPa and 4°C to 6°C, the enzyme activity is 2.1 times higher than that under normal pressure fermentation. High pressure fermentation culture of deep-sea pressure-resistant bacteria is carried out. The yield of the target enzyme is increased by optimizing the fermentation conditions. After fermentation, the fermentation supernatant is collected by centrifugation or filtration.

[0039] S22. Low-temperature and high-pressure disruption: This step is a conditional step. If some enzymes are present intracellularly, the fermentation supernatant should be homogenized or ultrasonically disrupted at a low temperature of 4°C to 10°C to minimize enzyme activity loss.

[0040] S23. Affinity Chromatography Capture: This invention utilizes specific biomimetic affinity ligands or recombinant protein tags targeting the specific sequences of deep-sea pressure-resistant bacterial lysins for affinity chromatography capture. This achieves highly efficient capture of the target enzyme through one or two steps of affinity chromatography, significantly reducing the content of extraneous proteins. Preferably, the recombinant protein tags are designed as His-tags (histidine tags) or Strep-tags (streptavidin tags) bound to specific resins.

[0041] S24. Gradient salting out and ultrafiltration: Under conditions of 4°C to 6°C, gradient salting out is performed for preliminary purification, and then ultrafiltration membrane with appropriate molecular weight cutoff is used to complete the concentration and desalting, while removing small molecule impurities simultaneously.

[0042] S25. High-performance liquid chromatography (HPLC) for fine purification: Reversed-phase or ion-exchange HPLC is used for final purification to ensure enzyme purity of over 95% and enzyme activity maintained above 20,000 U / mg. Purity and activity are verified by SDS-PAGE and activity assay. The purification steps for deep-sea pressure-resistant bacterial lysins are the same as those for volcanic thermophilic proteases, namely S34, ion-exchange chromatography, and S35, gel filtration chromatography, as described below.

[0043] S26, Lyophilization with Composite Protectant: A composite protectant consisting of trehalose, mannitol and amino acids is added to the purified enzyme solution, and then the solution is freeze-dried under vacuum to prepare deep-sea pressure-resistant bacterial lysin powder, thereby improving storage stability.

[0044] SEQ ID NO:1 is: MKKVLVATLLLALFASAQASAPADTAPQSIQEAKTAYVADAGKDTVILAGGAFGQTLNTADNGTVFVQKDATDLAVKGVGGTTASISKGDTTVLTPSNNGTAFVSSVADKELTVGAPVLGGTQYITGTSG TPVVTATDGSKVVYDAVGDSGTVLVGSSGSTVVAGADGSTTVVVNAAGDSVVVAHNGTSASVTVSSVGKDVTVQASGDGSTTVVGSTGDATLVVSADGSVTVTASNGTDGTVVVAANGTSVTVTSADGSK S3. Preparation of active component 2: Isolation and purification of thermophilic volcano protease (SEQ ID NO: 2).

[0045] S31. Fermentation and collection: The thermophilic bacteria of volcanoes are fermented at high temperature under conditions of 60°C-80°C. The fermentation broth is collected and the supernatant is separated.

[0046] S32. Heat Denaturation Pretreatment: Utilizing the heat-resistant properties of volcanic thermophilic bacteria protease, the fermentation supernatant is incubated at 60℃ to 70℃ for 30 to 40 minutes. This denatures and precipitates heat-sensitive proteins, which are then removed by centrifugation, achieving preliminary purification and enrichment of the target enzyme. The temperature of 60℃ to 70℃ is higher than the denaturation temperature of the proteins but lower than the optimal temperature for the volcanic thermophilic bacteria protease.

[0047] S33. Selective Adsorption and Elution: Selective adsorption is performed using a thermally stable adsorption medium at 45-60℃. This selective adsorption operation utilizes the adsorption differences between the target enzyme and impurity proteins to achieve separation. Preferably, the thermally stable adsorption medium is ceramic hydroxyapatite or adsorption resin.

[0048] S34. Ion Exchange Chromatography: Select a suitable ion exchange resin based on the isoelectric point characteristics of the target enzyme. Under optimized pH and ionic strength conditions, a gradient elution method can further improve the purity of the target enzyme.

[0049] S35. Gel filtration chromatography: Using Sephadex or Superdex series gel filtration columns, the target enzyme is finally separated based on the difference in molecular weight to obtain a high-purity enzyme solution with a purity of over 95%.

[0050] S36. Spray drying or freeze drying: The purified enzyme solution is concentrated by ultrafiltration, and then spray-dried or freeze-dried to prepare a stable enzyme powder. Preferably, in this embodiment, an automated program and software are used to precisely control the inlet and outlet air temperatures. The automated program is an automatic control program for the freeze-drying or spray-drying process of the enzyme preparation.

[0051] SEQ ID NO:2 is: MKLLVVSTLLLGLFASARASGPTDTAPKTIQDAKTAYLADSGKDTVVLAGGGFFQTLSTADNGTIFIQKDSTDLAVEGVGGTTGSITKGDTTVLTPSNNGTAFISSVADKDLTVGAPVLGGTQYITGTSGTPIVT ATDGSKVVYDAVGDSGTVLVGSSGSTVVAGADGSTTVVVNAAGDSVVVAHNGTSASVTVSSVGKDVTVQASGDGSTTVVGSTGDATLVVSADGSVTVTASNGTDGTVVVAANGTSVTVTSADGSKVTVQANGTDG S4, Mixing of composite active components: Weigh out the deep-sea pressure-resistant bacterial lysin, volcanic thermophilic protease, triterpenoid glycyrrhetinic acid, and phenolic gallic acid in the following mass ratios: (1.8-2.2):(0.9-1.1):(1.3-1.7):(0.9-1.1). Mix the components using a three-dimensional mixer at 20 rpm for 60 minutes under light-protected conditions at 4-6°C and relative humidity below 15%, until all components are homogeneous. In this embodiment, the specific mass ratio of the deep-sea pressure-resistant bacterial lysin, volcanic thermophilic protease, triterpenoid glycyrrhetinic acid, and phenolic gallic acid ensures the synergistic bactericidal effect of the four components, avoiding problems such as decreased disinfection efficiency and enzyme activity loss due to excessive or insufficient amounts of any single component.

[0052] S5, Total Mixture of Formulation

[0053] The uniformly mixed composite active components are dispersed in a composite biological polysaccharide carrier colloidal solution, and an appropriate amount of pH adjuster is added to maintain the pH of the system within a suitable range. After stirring evenly, the solution is filtered through a microporous membrane for sterilization to obtain the finished disinfectant preparation. Here, "appropriate amount" refers to an amount sufficient to adjust the pH to the desired range.

[0054] As shown in the nucleotide sequence listing, this invention provides two novel antimicrobial protein sequences, each with an amino acid length of less than 200 residues, exhibiting short and efficient characteristics. The sequences are rich in alanine (Ala), valine (Val), and leucine (Leu), ensuring structural invariance under high hydrostatic pressure of 50–55 MPa and stability at low temperatures of 4–6 °C, making them suitable for low-temperature purification processes. Both sequences completely retain the GDT catalytic motif, enabling specific lysis of the cell wall of pathogenic bacteria, demonstrating extreme enzyme properties. Simultaneously, these proteins do not attack normal human flora, exhibiting extremely high biocompatibility. Both sequences are original designs by the applicant, and are registered under SEQ ID NO:1 and SEQ ID NO:2, respectively. In this invention, the core amino acid sequence of the deep-sea pressure-resistant bacterial lysin is shown in SEQ ID NO:1, and the core amino acid sequence of the volcano thermophilic protease is shown in SEQ ID NO:2. Any derived polypeptides with at least 90% homology to the above sequences and possessing the same cleavage activity are within the scope of protection of this invention. The above limitations ensure both the flexibility of technology implementation and the rigor of patent protection.

[0055] This disinfectant preparation was obtained according to the above method. The following precautions should be taken: This disinfectant preparation should be stored strictly in a cool, dry place and protected from direct sunlight. Given that this preparation uses a microencapsulated composite biological polysaccharide carrier system, physical micro-sedimentation may occur after standing. Therefore, it must be thoroughly shaken before use to ensure the homogeneity of the disinfectant suspension and the atomization effect. Regarding usage, this preparation must not be mixed with chlorine-containing disinfectants, peroxides, or other strong oxidizing agents, as this may cause irreversible denaturation of the target enzyme protein molecular structure, leading to complete inactivation. Furthermore, regarding application procedures, although this product has high biocompatibility, operators should still wear basic protective equipment such as masks and gloves when adding and preparing the concentrate. Additionally, individuals with a known history of allergy to extreme environmental proteases such as deep-sea pressure-resistant bacteria or volcanic thermophilic bacteria should strictly avoid direct contact with this preparation.

[0056] The second embodiment of the present invention is an optimal formulation and standard preparation method. This embodiment aims to obtain a biological disinfectant with the most stable performance against pathogenic microorganisms in enclosed and semi-enclosed spaces, comprising: CO2 concentration-responsive composite biological polysaccharide carrier, the carrier constitutes a microcapsule system with a porous network structure, and a composite active component encapsulated by the carrier; wherein, the composite active component includes microbial lysin, protease, triterpenoids and phenolic compounds, which are used to produce a synergistic bactericidal effect against pathogenic microorganisms.

[0057] In this embodiment, the composite biological polysaccharide carrier is composed of sodium alginate, natural polysaccharides, and hyaluronic acid.

[0058] In this embodiment, the natural polysaccharide is xanthan gum; the composite biological polysaccharide carrier is composed of sodium alginate, xanthan gum, and hyaluronic acid in a mass ratio of 3:1:2.

[0059] In this embodiment, the microbial lysin is a deep-sea pressure-resistant bacterial lysin; the protease is a volcanic thermophilic bacterial protease; the triterpenoid compound is glycyrrhetinic acid; and the phenolic compound is gallic acid.

[0060] In this embodiment, the volcanic thermophilic bacteria protease is a derived polypeptide with 91% homology to SEQ ID NO: 2 and cleavage activity, and the activity of this homologous mutant can reach 91% of the original enzyme. The deep-sea pressure-resistant bacteria lyase is a derived polypeptide with 92% homology to SEQ ID NO: 1 and cleavage activity, and the activity of this homologous mutant can reach 94% of the original enzyme. The mass ratio of the deep-sea pressure-resistant bacteria lyase, the volcanic thermophilic bacteria protease, the triterpenoid glycyrrhetinic acid, and the phenolic gallic acid is 2:1:1.5:1. The specific activity of all the above enzymes (including their homologous mutants) must meet the activity threshold ≥20000 U / mg.

[0061] A standard preparation method for a biological disinfectant agent targeting pathogenic microorganisms in enclosed and semi-enclosed spaces includes, S1. Preparation of the composite biological polysaccharide carrier: Sodium alginate, xanthan gum, and hyaluronic acid were weighed as raw materials at a mass ratio of 3:1:2. The mixture was stirred at 30 rpm / min for 30 min at 80℃ to ensure complete dissolution in medical deionized water. Then, 0.5 times the standard dosage of the cross-linking agent calcium chloride (CaCl2) was added to the mixed solution, and intermolecular cross-linking was carried out at room temperature and pressure to form a colloid. Calcium chloride was chosen because it is a standard, commonly used, and safe cross-linking agent for sodium alginate systems, has good compatibility with xanthan gum and hyaluronic acid, and does not affect enzyme activity. Using half-dose cross-linking avoids the problems of overly dense networks, weak CO2 response, and insufficient release caused by full-dose cross-linking, thus forming a moderately cross-linked flexible porous network structure. This structure balances carrier stability and resistance to collapse, triggering swelling and secondary release at CO2 concentrations greater than 1000 ppm, and ensuring effective release of active components without excessive encapsulation. The colloid was then subjected to high-speed homogenization: homogenization was performed at 12000–15000 rpm for 3–5 min at room temperature (25±2℃) to refine the colloidal particles from 50–100 μm to 2–5 μm, forming a stable, fine, and uniform three-dimensional network colloid. The resulting colloid had a particle size of 2–5 μm, an atomized particle size ≤4.8 μm (meeting the ≤5 μm requirement), a suspension time ≥8 h, and a colloidal stability of over 18 months without stratification or precipitation, with an activity retention rate ≥95%.

[0062] S2. Isolation and purification of deep-sea pressure-resistant bacterial lysin: The strain was subjected to high-pressure fermentation at 50 MPa and 4°C, and the supernatant was collected by centrifugation. If some enzyme remained intracellularly, it was disrupted by low-temperature high-pressure homogenization at 4°C or by sonication. Subsequently, affinity chromatography was performed using a designed His-tag (histidine tag) or Strep-tag (streptavidin tag) bound to a specific resin for capture. Gradient salting and ultrafiltration concentration and desalting were carried out at 4-6°C, followed by fine purification using ion exchange and gel filtration chromatography (ensuring enzyme purity ≥95% and enzyme activity ≥20000 U / mg). Finally, trehalose, mannitol, and an amino acid complex protectant were added, and the mixture was freeze-dried under vacuum to form enzyme powder.

[0063] S3. Isolation and purification of thermophilic protease from volcanoes: High-temperature fermentation is carried out at 60-80°C, and the fermentation supernatant is collected. The supernatant is then incubated at 60-70°C for 30 minutes to denature and precipitate heat-sensitive proteins, followed by centrifugation for removal. This step increases the purity of the target enzyme from 62% to over 95%, achieving an activity ≥20000 U / mg. Next, selective adsorption is performed at 45-60°C using ceramic hydroxyapatite or adsorption resin. Ion exchange chromatography is then performed using DEAE-cellulose or CM-cellulose, eluting for 10-20 column volumes at pH 5.5-6.5 with a linear ionic strength gradient increasing from 0 to 0.5 M. Finally, gel filtration chromatography using Sephadex or Superdex series chromatography is performed to ensure a purity ≥95% and an activity ≥20000 U / mg. After ultrafiltration concentration, the inlet and outlet air temperatures are precisely controlled using automated programs and software. The automated programs are for the lyophilization or spray drying processes of enzyme preparations, and the accompanying software includes: Spray Drying Unit Simulation Software V1.0 for the spray drying process, and a dedicated lyophilization PLC control system for the freeze-drying process. This program can adjust temperature parameters in real time and record process curves, thereby ensuring stable enzyme activity.

[0064] S4, Mixing of composite active components:

[0065] Weigh out deep-sea pressure-resistant bacterial lyase, volcanic thermophilic bacterial protease, triterpenoid glycyrrhetinic acid, and phenolic gallic acid in a mass ratio of 2:1:1.5:1. Mix them in a three-dimensional mixer at 20 rpm for 60 minutes under conditions of darkness, 4-6°C, and relative humidity below 15%, until completely homogeneous.

[0066] S5, Total Mixture of Formulation: The uniformly mixed composite active components are dispersed in the composite biological polysaccharide carrier colloidal solution prepared in the first step. An appropriate amount of phosphate buffer is added as a pH adjuster to maintain the pH of the system between 6.5 and 7.5. After stirring evenly, the mixture is filtered through a microporous membrane for sterilization to obtain the finished disinfectant preparation.

[0067] The third embodiment of the present invention presents the experimental verification results of the formulation. To further verify the broad-spectrum antibacterial properties of the disinfectant described in this invention, Escherichia coli (representing Gram-negative bacteria) and Staphylococcus aureus (representing Gram-positive bacteria) were selected as test strains in this embodiment for quantitative antibacterial effect testing and qualitative inhibition zone testing.

[0068] 1. Experimental Materials

[0069] (1) Test sample: Disinfectant prepared according to the second embodiment of the present invention; (2) Test strains: Escherichia coli, Staphylococcus aureus; 2. Experimental Methods (1) Quantitative antibacterial effect test Two test bacterial solutions were prepared using the test strain, with a concentration of 1×10⁻⁶. 8 CFU / mL. The test bacterial suspension was mixed thoroughly with the disinfectant prepared according to the second embodiment of this invention. The experimental conditions were: incubation at room temperature (37℃) for 30 minutes. After the incubation period, samples were serially diluted and spread onto solid agar plates. The plates were then incubated at 37℃ for 24 hours, followed by colony counting to calculate the inhibition rate. Sterile water without disinfectant was used as a blank control group.

[0070] (2) Antibacterial zone test

[0071] Two test bacterial solutions were prepared using the test strain (the concentration of the test bacterial solutions was the same as above, 1×10⁻⁶). 8 Prepare agar plates (CFU / mL). Place a sterile filter paper soaked in the pure biological disinfectant stock solution of this invention in the center of the plate. Incubate the plates at 37°C for 24 hours. Remove the plates and observe them, measuring the diameter of the inhibition zone using calipers.

[0072] 3. Experimental Results

[0073] Experimental results are as follows Figure 2 As shown, Figure 2 This is a graph demonstrating the antibacterial effect of a pure biological disinfectant against Escherichia coli and Staphylococcus aureus.

[0074] (1) such as Figure 2 A and Figure 2 As shown in Figure C, the quantitative test results indicate that, compared with the control group, the pure biological disinfectant of this invention is effective against Escherichia coli (E. coli). Figure 2 A) and Staphylococcus aureus ( Figure 2 C) All showed significant killing / inhibiting effects.

[0075] (2) such as Figure 2 B and Figure 2 As shown in Figure D, the inhibition zone test results clearly demonstrate that the pure biological disinfectant of this invention forms a clear, transparent, and well-defined inhibition zone on the agar plate. Among them, as... Figure 2 As shown in Figure B, the inhibition zone diameter against *E. coli* reached 22.3 ± 1.2 mm, with an inhibition rate of 99.7%; Figure 2 As shown in Figure D, the inhibition zone diameter against Staphylococcus aureus reached 20.8 ± 0.9 mm, with an inhibition rate of 99.6%. This indicates that the effective antibacterial component of this disinfectant has good diffusion properties and strong antibacterial activity.

[0076] 4. Experimental Conclusions

[0077] In summary, the pure biological disinfectant provided by this invention has excellent and rapid inhibitory and killing effects on both Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus), proving that the product has excellent broad-spectrum antibacterial properties and has extremely high practical application and promotion value.

[0078] The second type of embodiment is the application scenario, method, and effect embodiment of the present invention.

[0079] In the fourth embodiment of the present invention, this embodiment provides a standard application method of the biological disinfectant preparation of the present invention, which is particularly suitable for enclosed or semi-enclosed spaces where the CO2 concentration is easily raised to >1000ppm due to dense crowds or poor air circulation.

[0080] The specific application methods are as follows: Step 1: Determine the concentration of the disinfectant agent based on the size of the space to be treated and the estimated degree of contamination, and add it to the storage tank of the piezoelectric microporous atomizer. Strictly apply the disinfectant agent suspension at a rate of 0.5 mL to 1.2 mL per cubic meter.

[0081] Step 2: Activate the piezoelectric microporous atomizer to continuously generate ultrafine droplets with a particle size ≤5μm for spatial atomization. Thanks to the excellent physicochemical properties of the composite biopolysaccharide carrier in this formulation, ultrafine droplets of this particle size can not only maintain stable suspension in the air for a long time, but also diffuse to every dead corner and corner of a confined space.

[0082] Step 3: After the initial atomization process has been completed for a period of time, stop atomization and keep the space sealed for 30 to 60 minutes.

[0083] During this period, this method fully utilizes a scenario-based avoidance mechanism for intelligent regulation: when the pollution risk is high in a confined space, dense crowds or poor air circulation lead to CO2 accumulation. Only when the CO2 concentration exceeds 1000 ppm will a secondary release mechanism be triggered. The released active ingredients then synergistically degrade pathogenic microorganisms in the air and corners. This application method significantly reduces ineffective consumption in non-target scenarios, minimizing potential ecological impacts.

[0084] In the fifth embodiment of the present invention, the application method and effect of the disinfectant preparation of the present invention in medium-sized enclosed spaces and medium levels of pollution are demonstrated.

[0085] For a submersible cabin environment with a volume of 150 cubic meters, an initial CO2 concentration of 800 ppm, and moderate contamination from a mixture of Escherichia coli and Staphylococcus aureus, the biological disinfectant of this invention was added to a piezoelectric microporous atomizer at a concentration of 0.5 mL / m³. Atomization was performed for 30 minutes with a particle size of 3 μm, followed by 45 minutes of space sealing, and finally 20 minutes of ventilation. Furthermore, when the particle size is ≤5 μm, the suspension time can reach >8 hours. During this period, the droplets slowly disperse with the airflow, effectively covering dead corners. If the particle size is >5 μm, the settling speed is faster, the disinfection range is limited, and the disinfection effect is significantly reduced.

[0086] The efficacy verification showed that the total number of bacteria in the cabin space decreased by more than 99.2% after disinfection; in particular, when the CO2 concentration increased to 1200ppm, the carrier successfully triggered the response mechanism to release the compound active components a second time, and the release amount of the compound active components increased by 2.3 times compared with the initial state, achieving the sterilization purpose of on-demand enhancement. Furthermore, no irritating odor or any allergic reaction was detected after personnel entered the cabin.

[0087] In the sixth embodiment of the present invention, the application method and effect of the disinfectant preparation of the present invention in small semi-enclosed spaces and in the context of viral contamination are demonstrated.

[0088] For elevator cars with a volume of 2.5 cubic meters, where CO2 concentrations can fluctuate drastically between 600 ppm and 1500 ppm during peak commuting hours, and where the target microorganisms are influenza virus H1N1 and Staphylococcus aureus, this disinfectant is added at a concentration of 1.2 mL / m³ (the effective dosage range of this invention is 0.5–1.2 mL / m³). The system is set to an atomization particle size of 4 μm, with automatic atomization for 10 minutes each morning and evening, followed by 20 minutes of sealed operation and then 5 minutes of automatic ventilation.

[0089] The efficacy verification showed that, after 7 consecutive days of efficacy verification, during peak hours when the CO2 concentration was approximately 1000–1500 ppm, the virus titer reduction value in the elevator reached ≥4.5 log, and the bacterial kill rate remained stable at over 98.5%. In addition, the verification results also showed that the carrier material had no corrosive effect on the elevator's metal inner wall, and the CO2 concentration in the car could be restored to normal levels within 30 minutes after atomization.

[0090] In the seventh embodiment of the present invention, the application method and effect of the disinfectant preparation of the present invention in a long-term enclosed space are demonstrated.

[0091] For long-term enclosed or semi-enclosed spaces where CO2 concentrations can easily rise to more than 1000 ppm, such as underground fortifications, specific laboratories, long-distance transport vehicles, aircraft cabins, elevator cars, and small conference rooms.

[0092] This formulation fully utilizes the sustained-release properties of the composite biological polysaccharide carrier. After a single initial ultrafine atomization, it can continuously exert a disinfection effect in the enclosed space for up to 8 to 12 hours. Its core mechanism lies in the fact that the microgel structure formed by the carrier material after atomization can slowly release active ingredients. When the concentration of microorganisms in the space rebounds, or when the environmental CO2 concentration exceeds the threshold of 1000 ppm again, the carrier can automatically recognize the environmental changes and trigger a new round of active ingredient release, thereby maintaining the dynamic disinfection balance of the space.

[0093] In such high-risk, long-term enclosed environments, daily supplemental atomization only needs to be performed 1 to 2 times a day to significantly reduce the labor costs of repeated disinfection operations and achieve a continuous and highly effective antibacterial effect for more than 72 hours.

[0094] The third type of embodiment is the comparative experiment of the present invention.

[0095] In the eighth embodiment of the present invention, this comparative example aims to verify the differences of the present invention in terms of safety and residue by comparing it with traditional chemical agents.

[0096] Traditional methods mentioned in the background section are used, such as the use of chemical agents including chlorine-containing disinfectants, alcohol, or hydrogen peroxide, and traditional disinfection methods such as fumigation and wiping are used to disinfect enclosed spaces.

[0097] Experimental results show that although the aforementioned traditional chemical agents have certain bactericidal effects, they generally suffer from serious drug residues, strong corrosiveness, and significant irritation to human skin and mucous membranes in practical applications. In contrast, this invention uses a composite biological polysaccharide carrier as the basic carrier. Its core function is to endow the disinfectant with excellent atomization performance and suspension stability in the air, effectively prolonging the suspension time of atomized particles in space, thereby ensuring that the drug-loaded particles can achieve more complete and uniform contact with airborne microorganisms. This composite biological polysaccharide carrier is composed entirely of biologically derived components, has good environmental compatibility, will not cause chemical residues or environmental pollution, and possesses high biosafety, making it particularly suitable for safe use in indoor environments where people are present.

[0098] In the ninth embodiment of the present invention, this comparative example aims to verify the effect of enzymes with special components used in the present invention on the microecological balance by comparing them with enzyme preparations from conventional sources.

[0099] Disinfectant preparations were formulated using enzyme proteins derived from common microorganisms (such as lysozyme and protease) and tested under the same conditions.

[0100] Experimental results show that while conventional enzyme preparations can degrade microbial structures, they also have a strong inhibitory effect on the normal flora beneficial to the human body, easily disrupting the microecological balance. Furthermore, due to their common origin, they pose a high risk of allergens. In contrast, this invention utilizes a deep-sea pressure-resistant bacterial lyase (SEQ ID NO:1) and a thermophilic volcano protease (SEQ ID NO:2) with unique amino acid sequences. The specific origin of these components reduces the broad-spectrum inhibitory effect on the normal human flora and lowers the risk of common allergens. The production process also has low dependence on conventional environmental resources.

[0101] In the tenth embodiment of the present invention, this comparative example aims to verify the role of CO2 concentration response mechanism in release by comparing it with a common polysaccharide carrier that does not have CO2 sensitivity.

[0102] A common polysaccharide carrier without CO2 sensitivity was prepared and coated with the same composite active component.

[0103] Experiments revealed that the control group formulation lacked an intelligent response mechanism in practical applications, failing to sense changes in CO2 concentration and provide feedback. This hindered on-demand, scenario-based release and failed to meet the continuous, adaptive, and dynamic disinfection requirements of enclosed spaces. In contrast, this invention utilizes the sensitivity of a composite biopolysaccharide carrier to CO2 concentration. When the CO2 concentration exceeds 1000 ppm, the carrier structure undergoes a pH response or swelling change, triggering a secondary release of the encapsulated components. This significantly improves the effective concentration and efficiency of the active components in the target environment. Furthermore, the composite biopolysaccharide carrier is used to immobilize and sustain the release of the composite active components, enhancing their stability in high-temperature and high-humidity environments.

[0104] In the eleventh embodiment of the present invention, this comparative example aims to verify the synergistic bactericidal effect among the composite active components.

[0105] A bactericidal experiment was conducted by preparing a control group lacking either the triterpenoid glycyrrhetinic acid or the phenolic gallic acid.

[0106] The results showed that the overall bactericidal effect and the persistence of enzyme activity decreased when the plant extract components were missing. Specifically, the deep-sea pressure-resistant bacterial lyase (SEQ ID NO: 1) specifically lyses the cell wall or membrane structure of certain pathogenic microorganisms (such as Gram-negative or Gram-positive bacteria), leading to cell rupture and death. Thermophilic protease (SEQ ID NO: 2) efficiently degrades the structural and functional proteins of microorganisms, disrupting their metabolism and reproductive capacity. The triterpenoid glycyrrhetinic acid possesses anti-inflammatory, antibacterial, and antiviral activities, enhancing the bactericidal effect and potentially alleviating inflammatory responses caused by microbial metabolites. The phenolic gallic acid has broad-spectrum antibacterial and antioxidant effects, inhibiting bacterial growth, scavenging free radicals, and protecting enzyme activity. This invention achieves a synergistic bactericidal effect by utilizing the specific lysis of cell walls or membrane structures by deep-sea pressure-resistant bacteria lyase, the degradation of functional proteins by volcanic thermophilic bacteria protease, the enhancement of bactericidal effect by triterpenoid glycyrrhetinic acid, and the inhibition of bacterial growth and scavenging of free radicals by phenolic gallic acid to protect enzyme activity. This significantly improves the disinfection efficiency in complex environments.

[0107] The present invention has the following beneficial effects: First, this invention utilizes microbial enzymes isolated from extreme environments (such as high-temperature, high-salt, or high-pressure habitats) combined with triterpenoids and phenolic compounds as core bactericidal components. These components have unique origins and high specificity and stability, enabling them to degrade the structural and functional proteins of pathogens. This ensures that the disinfectant maintains its activity in complex environments. While effectively avoiding the problems of drug residues, strong corrosion, and mucosal irritation caused by traditional chemical disinfectants, it significantly reduces the broad-spectrum inhibition of beneficial normal flora in the human body and overcomes the defect of conventional enzyme preparations that easily disrupt the microecological balance.

[0108] Secondly, this invention constructs a CO2 concentration-responsive composite biopolysaccharide carrier composed of sodium alginate, xanthan gum, and hyaluronic acid, enabling disinfectant preparations to intelligently sense and respond to environmental changes. When the risk of pollution increases due to poor air circulation in a confined space, the carrier structure undergoes microscopic swelling or pH response, thereby triggering the secondary release of the encapsulated components. This solves the problem of existing technologies lacking a scenario binding mechanism and making it difficult to achieve on-demand release.

[0109] Furthermore, the porous microcapsule encapsulation structure of the biopolysaccharide carrier, combined with the antioxidant and free radical scavenging effects of phenolic gallic acid in the composite active components, greatly enhances the physicochemical stability of the extreme enzyme protein in complex temperature and humidity environments, achieving scientific sustained release of active ingredients and multi-target synergistic sterilization, and can maintain dynamic disinfection balance for a relatively long time in a closed space.

[0110] Finally, this invention optimizes the distribution and control of disinfectants by working in conjunction with ventilation systems and environmental monitoring equipment. Specifically, relying on the excellent film-forming and air-suspending properties of the composite carrier, this invention enables disinfectant particles to penetrate deep into every corner of enclosed spaces, effectively compensating for the shortcomings of traditional physical methods such as ultraviolet irradiation, which have blind spots. While reducing ineffective consumption in non-target areas and the overall ecological burden, it safely and efficiently achieves three-dimensional dynamic disinfection in a human-machine coexistence state.

[0111] The embodiments described above are merely further illustrations of the present invention and are not intended to limit the present invention in any other way. The present invention may have many other embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding modifications and changes based on the present invention, but all such modifications and changes should fall within the protection scope of the present invention.

Claims

1. A biological disinfectant agent targeting pathogenic microorganisms in enclosed and semi-enclosed spaces, characterized in that, include: CO2 concentration-responsive composite biopolysaccharide carrier, wherein the carrier constitutes a microcapsule system with a porous network structure, and a composite active component is encapsulated by the carrier; The composite active components include microbial lysins, proteases, triterpenoids, and phenolic compounds.

2. The biological disinfectant agent for pathogenic microorganisms in enclosed and semi-enclosed spaces according to claim 1, characterized in that, The composite biological polysaccharide carrier is composed of sodium alginate, natural polysaccharides, and hyaluronic acid.

3. The biological disinfectant agent for pathogenic microorganisms in enclosed and semi-enclosed spaces according to claim 2, characterized in that, The natural polysaccharide is xanthan gum; The composite biopolysaccharide carrier is composed of sodium alginate, xanthan gum, and hyaluronic acid in a mass ratio of (2.5-3.5):(0.8-1.2):(1.8-2.2).

4. The biological disinfectant agent for pathogenic microorganisms in enclosed and semi-enclosed spaces according to claim 1, characterized in that, The microbial lysin is a deep-sea pressure-resistant bacterial lysin; the protease is a volcanic thermophilic bacterial protease; the triterpenoid compound is glycyrrhetinic acid; and the phenolic compound is gallic acid.

5. The biological disinfectant agent for pathogenic microorganisms in enclosed and semi-enclosed spaces according to claim 4, characterized in that, The amino acid sequence of the volcanic thermophilic protease is shown in SEQ ID NO: 2, or it is a derived polypeptide with more than 90% homology to SEQ ID NO: 2 and the same cleavage activity. The amino acid sequence of the deep-sea pressure-resistant bacterial lysin is shown in SEQ ID NO: 1, or it is a derived polypeptide with more than 90% homology to SEQ ID NO: 1 and the same lysing activity. The mass ratio of the deep-sea pressure-resistant bacterial lyase, the volcanic thermophilic bacterial protease, the triterpenoid glycyrrhetinic acid, and the phenolic gallic acid is (1.8-2.2):(0.9-1.1):(1.3-1.7):(0.9-1.1).

6. A method for preparing a biological disinfectant agent for pathogenic microorganisms in enclosed and semi-enclosed spaces according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Preparation of composite biological polysaccharide carrier; S2. Microbial lysin separation and purification; S3. Protease isolation and purification; S4. Mixing of composite active components: Weigh the microbial lysin, protease, triterpenoid compound and phenolic compound according to the proportion, and mix them evenly; S5. Formulation Mixing: The composite active components are uniformly dispersed in a composite biological polysaccharide carrier colloidal solution, and a pH adjuster is added to adjust the pH of the system to 6.5-7.

5. The mixture is then stirred to obtain the final product.

7. The preparation method according to claim 6, characterized in that, In step S1, sodium alginate, natural polysaccharide and hyaluronic acid are weighed in a mass ratio of (2.5-3.5):(0.8-1.2):(1.8-2.2) and dissolved in a solvent. A crosslinking agent is added to the mixed solution to form a colloid through intermolecular crosslinking. The solution is then homogenized to obtain a colloidal solution with a stable three-dimensional network structure. In step S2, the microbial lysin includes a deep-sea pressure-resistant bacterial lysin. In step S3, the protease includes thermophilic volcano protease; In step S4, the deep-sea pressure-resistant bacterial lysin, volcanic thermophilic bacterial protease, triterpenoid glycyrrhetinic acid and phenolic gallic acid are weighed in a mass ratio of (1.8-2.2):(0.9-1.1):(1.3-1.7):(0.9-1.1) and mixed evenly.

8. The preparation method according to claim 6, characterized in that, In step S2, the enzyme-producing strain is subjected to high-pressure fermentation culture at 50 MPa to 55 MPa and 4°C to 6°C; the fermentation supernatant is homogenized or ultrasonically disrupted at low temperature and high pressure at 4°C to 10°C, and then subjected to affinity chromatography capture. In step S3, the purification process includes pre-treatment of the fermentation supernatant by incubating it at 60°C to 70°C for 30 to 40 minutes for heat denaturation, followed by centrifugation and filtration to remove heat-sensitive contaminants.

9. A method for applying a biological disinfectant agent for pathogenic microorganisms in enclosed and semi-enclosed spaces as described in any one of claims 1-5, characterized in that, Includes the following steps: Step 1: Add the biological disinfectant to the piezoelectric microporous atomizer; Step 2: The application rate is 0.5 mL to 1.2 mL per cubic meter, which is atomized by generating droplets with a particle size ≤ 5 μm through the piezoelectric microporous atomizer; Step 3: After atomization, keep the space sealed for 30 to 60 minutes.

10. The application method according to claim 9, characterized in that, The application is suitable for enclosed or semi-enclosed spaces where CO2 concentration can easily rise to >1000ppm. When the CO2 concentration in the space is >1000ppm, the carrier structure changes, triggering a secondary release of the encapsulated composite active components.

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