Traditional Chinese medicine spray for disinfecting animal breeding environment

Abstract

The invention discloses a traditional Chinese medicine spray applied to disinfection of an animal breeding environment, and belongs to the technical field of traditional Chinese medicine sprays. According to the technology, ZnO quantum dots are tightly connected with baicalin molecules through coordinate bonds, traditional Chinese medicine active ingredients, nano materials and biotechnologies are creatively fused, a multi-mechanism synergistic effect system is constructed, efficient sterilization, resistance inhibition and environment friendliness are achieved, and by means of a pH response type microcapsule carrier, the pH response type baicalin microcapsule is prepared. The precise targeted release of the active ingredients in the breeding environment is realized; harmful microorganisms are continuously removed by utilizing the dynamic disinfection and killing characteristics of the genetically engineered bacteria and the bacteriophages; the unique photocatalytic and antibacterial properties of ZnO quantum dots further strengthen the sterilization effect, meanwhile, the spray is made of a full-biodegradable material, the ecological and environment-friendly advantages are fully manifested while the long-acting disinfection safety is ensured, and the technology can significantly reduce the disinfection frequency of the farm and reduce the use dependence of antibiotics, so that the method is suitable for large-scale popularization and application. The sustainable development of the green and healthy breeding industry is powerfully promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine sprays, and in particular relates to a traditional Chinese medicine spray for disinfecting an animal breeding environment.

[0002] As the animal husbandry industry rapidly advances towards intensification and scale, pathogen contamination in the farming environment is becoming increasingly serious. The proliferation and widespread spread of pathogens such as bacteria, viruses, and fungi has become a key factor in triggering large-scale animal disease outbreaks, significantly reducing production performance, and posing serious food safety risks. In intensive farming models, precise control of environmental parameters and effective, scientific hygiene management play a crucial role in establishing a biosafety system. Specifically, overly dense farming arrangements can severely restrict air circulation; failure to promptly remove livestock waste creates favorable conditions for the spread of pathogens; inaccurate temperature and humidity control can easily lead to the proliferation of pathogens; and aerosol transmission and contaminated drinking water systems further accelerate the spread of harmful microorganisms in the farming environment. These environmental factors pose a serious threat to animal health. Therefore, using specialized disinfectants to control the environmental microbial load is crucial for preventing pathogen transmission, ensuring animal welfare, and maintaining the economic benefits of the farming industry.

[0003] In the fields of animal husbandry and public health disinfection, chemical disinfectants such as chlorine-based disinfectants, aldehyde-based disinfectants, and phenol-based disinfectants have long dominated. However, these traditional disinfectants have exposed numerous drawbacks in practical application: First, they present a high risk of toxicity. Chlorine-based disinfectants, when used in high concentrations or in poorly ventilated environments, can cause severe irritation and damage to the respiratory mucosa and skin tissues of humans and animals. Aldehyde-based disinfectants not only have a pungent odor but also carry a potential risk of carcinogenesis. Second, they exhibit a short disinfection effect. Disinfectants lack a long-lasting effect in the environment, and their effective disinfection duration is limited, often requiring frequent reapplications, increasing both human and material costs. Third, they rapidly develop resistance. Pathogens, through repeated exposure to disinfectants, can rapidly develop adaptive mutations and develop resistance, leading to a continuous decline in disinfection effectiveness. This not only drives up disinfection costs but also further increases the risk of disease transmission. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention provides a traditional Chinese medicine spray for disinfecting animal breeding environments, which solves the problems of high toxicity, short efficacy and rapid resistance in the existing technology.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: a traditional Chinese medicine spray for disinfecting animal breeding environments, comprising the following components in percentage by mass: 5%-10% of a baicalin-ZnO quantum dot complex, wherein the surface of the ZnO quantum dots is connected to baicalin molecules via coordination bonds, and the particle size distribution is 3.0±0.5nm; 3%-8% pH-responsive microcapsules, comprising a core material and a wall material, wherein the core material is a mixture of eugenol and citral in a mass ratio of 2.5:1-4:1, and the wall material is formed by cross-linking carboxymethyl chitosan and ε-polylysine in a molar ratio of 6:1-8:1; 1.5-5×10 6 CFU / mL of a genetically engineered yeast agent, wherein the yeast chromosome is integrated with an andrographolide synthesis gene cluster regulated by a PHO5 promoter; 1.2-3×10 9 The PFU / mL phage mixture contains T4 phage, φKZ phage, and ST1 phage, with a titer ratio of (5±0.5): (2.5±0.3):1; 0.8%-2% houttuynia cordata-rosemary composite extract, with houttuynia cordata content ≥15mg / g; 0.01%-0.1% quorum sensing inhibitor, composed of an N-butyryl homoserine lactone analog and an Agr antagonist peptide in a ratio of 1:2-1:3; The balance is water for injection and at least one stabilizer selected from trehalose and hydroxypropyl cyclodextrin.

[0006] Preferably, the XRD spectrum of the ZnO quantum dots shows a characteristic peak of the (101) crystal plane at 2θ=36.2±0.2°.

[0007] Preferably, the cross-linking degree of the wall material of the microcapsule is controlled at 85%-90%, and the release rate thereof in a 30-min environment at a pH value ≤ 6.5 is ≥ 78%.

[0008] Preferably, the stabilizer is a complex of trehalose and hydroxypropyl-β-cyclodextrin in a mass ratio of 2:1, and the total addition amount is 0.5%-1.2%.

[0009] A method for preparing a traditional Chinese medicine spray for disinfecting an animal breeding environment comprises the following steps: S1. Quantum Dot Synthesis 0.5 mol / L zinc nitrate solution and 8% baicalin solution were mixed in a volume ratio of 1:3-1:5, and hydrothermally reacted at 175°C-185°C for 11h-13h under nitrogen protection. After centrifugation, a composite quantum dot suspension was obtained; S2. Microcapsule Preparation A coaxial microfluidic device was used to control the flow rate of the core material to 1.2±0.1mL / min and the flow rate of the wall material to 3.2±0.2mL / min, and cross-linking and curing were carried out in a 2.5mol / L CaCl2 solution for 25min-35min. S3. Yeast Culture Two-stage cultivation was carried out in a 30-L bioreactor. In the first stage, the dissolved oxygen was controlled to be ≥30% and the pH value was 5.5-6.0. In the second stage, induced expression was initiated when the residual sugar concentration dropped to 5±0.5 g / L. S4. Phage Enhancement The original phage was alternately inoculated into ESBL-producing Escherichia coli and methicillin-resistant Staphylococcus aureus for 5-7 generations of directed evolution and concentrated by tangential flow filtration to a titer of ≥1.5×10¹ 0 PFU / mL; S5. Preparation Integration The products of steps 1-4 above were mixed with the composite extract in proportion, and subjected to microfluidization treatment at 150 MPa-180 MPa for three times to obtain a homogeneous dispersion with a particle size of D90 ≤ 450 nm.

[0010] Preferably, the specific parameters of the two-stage culture in step S3 are: Stage 1: temperature 30±0.5°C, stirring speed 300-350 rpm, ventilation volume 1.2 vvm, lasting 12-14 hours; The second stage: temperature 28±0.5℃, stirring speed 400rpm-450rpm, ventilation volume 1.5vvm, lasting 24h-26h.

[0011] Preferably, the composite extract in step S5 is prepared by the following method: a) Houttuynia cordata and rosemary were mixed in a mass ratio of 4:1 and extracted with 65% ethanol in a dynamic countercurrent extraction process three times for 40 minutes each time. b) The extract was enriched on a molecularly imprinted adsorption column, and the target component was eluted with an acetonitrile-water (70:30, v / v) gradient.

[0012] Preferably, the tangential flow filtration in step S4 uses a 100 kDa ultrafiltration membrane, the transmembrane pressure difference is controlled at 0.8 bar-1.2 bar, and the temperature is maintained at 4° C.-8° C.

[0013] Preferably, the method further comprises a post-processing step: mixing the prepared waste liquid with an Aspergillus niger spore suspension in a volume ratio of 1:0.08, aerobically fermenting at 48±2° C. for 72 hours, and converting the mixture into a liquid organic fertilizer with a pH value of 6.8-7.2.

[0014] The traditional Chinese medicine spray for disinfecting animal breeding environments provided by this invention innovatively integrates traditional Chinese medicine active ingredients, nanomaterials, and bioengineering technology to construct a synergistic disinfection system with environmental responsiveness and resistance management functions, creating a new technical path for breeding environment management. Its core advantages are as follows: First, a multi-mechanism synergistic disinfection system breaks through traditional limitations. A highly effective bactericidal network is constructed using a baicalin-ZnO quantum dot complex. Nanosized ZnO quantum dots (particle size 3.0nm ± 0.5nm) are bound to the traditional Chinese medicine active ingredient baicalin through coordination bonds, forming a triple bactericidal mechanism of "photocatalytic oxidation - cell membrane damage - metabolic pathway inhibition." Compared to the single mode of action of traditional disinfectants, this significantly improves broad-spectrum disinfection efficiency against bacteria, viruses, and fungi. The pH-responsive microcapsule carrier intelligently responds to the pH of the aquaculture environment, releasing active ingredients in a targeted manner within the humid microenvironment where pathogens are concentrated, increasing active ingredient utilization by over 40%. Second, dynamic resistance management technology inhibits the development of drug resistance. The biocontrol module, comprised of genetically engineered bacteria and directed-evolved phage, possesses dual resistance management capabilities: the engineered bacteria continuously secrete metabolites such as antimicrobial peptides and lysozyme, disrupting pathogen cell wall synthesis; while the phage, through directed evolution of tail fiber proteins, recognizes and binds to surface receptors of mutant pathogens in real time, achieving specific lysis of resistant strains. This dynamic disinfection system can extend the evolutionary cycle of microbial resistance by more than three times, effectively addressing the rapid development of resistance caused by traditional disinfectants. Thirdly, a full-chain eco-friendly design promotes green farming. Microcapsules are made of fully biodegradable chitosan-polylysine materials. The degradation products are carbon dioxide, water, and oligosaccharide molecules, which promote the proliferation of beneficial bacteria such as lactic acid bacteria and Bacillus, creating a benign microecological cycle of antibacterial and growth-promoting effects. The integration of continuous flow microreactor technology and microfluidic encapsulation technology achieves nanoscale uniform dispersion of baicalin-ZnO quantum dots and precise control of microcapsule particle size, ensuring batch-to-batch performance stability of less than 5%. Through its long-lasting, sustained-release mechanism, a single spray can achieve an effective disinfection cycle of 7-10 days (compared to only 2-3 days with traditional disinfectants), reducing disinfection frequency by over 60%. Bioactive ingredients replace chemical agents, eliminating the respiratory irritation and carcinogenic risks of chlorine- and aldehyde-based disinfectants at the source, ensuring dual safety protection for both livestock and poultry. This technology completely overturns the technical bottlenecks of traditional disinfectants, characterized by high toxicity, short duration, and rapid resistance development. It establishes a closed-loop disinfection system combining precise release, dynamic disinfection, and ecological restoration, offering a revolutionary solution to the challenge of microbial contamination in intensive aquaculture. It has significant industrial and environmental benefits in reducing reliance on antibiotics, ensuring the healthy growth of livestock and poultry, and improving the economic efficiency of aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a schematic diagram of the preparation process of the present invention. DETAILED DESCRIPTION

[0016] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0017] like Figure 1 As shown, an embodiment of the present invention provides a traditional Chinese medicine spray for disinfecting animal breeding environments, which is composed of the following components in percentage by mass: 5%-10% of a baicalin-ZnO quantum dot complex, wherein the surface of the ZnO quantum dots is connected to the baicalin molecules through coordination bonds, and the particle size distribution is 3.0±0.5nm; 3%-8% pH-responsive microcapsules, comprising a core material and a wall material, wherein the core material is a mixture of eugenol and citral in a mass ratio of 2.5:1-4:1, and the wall material is formed by cross-linking carboxymethyl chitosan and ε-polylysine in a molar ratio of 6:1-8:1; 1.5-5×10 6 CFU / mL of genetically engineered yeast agent, the yeast chromosome is integrated with the andrographolide synthesis gene cluster controlled by the PHO5 promoter; 1.2-3×10 9 The PFU / mL phage mixture contains T4 phage, φKZ phage, and ST1 phage, with a titer ratio of (5±0.5): (2.5±0.3):1; 0.8%-2% houttuynia cordata-rosemary composite extract, with houttuynia cordata content ≥15mg / g; 0.01%-0.1% quorum sensing inhibitor, composed of an N-butyryl homoserine lactone analog and an Agr antagonist peptide in a ratio of 1:2-1:3; The balance is water for injection and at least one stabilizer selected from trehalose and hydroxypropyl cyclodextrin.

[0018] The XRD spectrum of ZnO quantum dots shows a characteristic peak of (101) crystal plane at 2θ=36.2±0.2°, and the grafting rate of baicalin is ≥85%. The cross-linking degree of the wall material of the microcapsule is controlled at 85%-90%, and its 30min release rate is ≥78% under a pH value ≤6.5. The stabilizer is a complex composed of trehalose and hydroxypropyl-β-cyclodextrin in a mass ratio of 2:1, with a total addition amount of 0.5%-1.2%. A method for preparing a traditional Chinese medicine spray for disinfecting an animal breeding environment comprises the following steps: S1. Quantum Dot Synthesis 0.5 mol / L zinc nitrate solution and 8% baicalin solution were mixed in a volume ratio of 1:3-1:5, and hydrothermally reacted at 175°C-185°C for 11h-13h under nitrogen protection. After centrifugation, a composite quantum dot suspension was obtained; S2. Microcapsule Preparation Using a coaxial microfluidic device, the core material flow rate was controlled at 1.2±0.1mL / min and the wall material flow rate was controlled at 3.2±0.2mL / min. Cross-linking and curing were carried out in a 2.5mol / L CaCl2 solution for 25-35min. S3. Yeast Culture Two-stage cultivation was carried out in a 30-L bioreactor. In the first stage, the dissolved oxygen was controlled to be ≥30% and the pH value was 5.5-6.0. In the second stage, induced expression was initiated when the residual sugar concentration dropped to 5±0.5 g / L. S4. Phage Enhancement The original phage was alternately inoculated into ESBL-producing Escherichia coli and methicillin-resistant Staphylococcus aureus for 5-7 generations of directed evolution and concentrated by tangential flow filtration to a titer of ≥1.5×10¹ 0 PFU / mL; S5. Preparation Integration The products of steps 1-4 above were mixed with the composite extract in proportion, and subjected to microfluidization treatment at 150 MPa-180 MPa for three times to obtain a homogeneous dispersion with a particle size of D90 ≤ 450 nm.

[0019] The specific parameters of the two-stage culture in step S3 are: Stage 1: temperature 30±0.5°C, stirring speed 300-350 rpm, ventilation volume 1.2 vvm, lasting 12-14 hours; The second stage: temperature 28±0.5℃, stirring speed 400rpm-450rpm, ventilation volume 1.5vvm, lasting 24h-26h.

[0020] The composite extract in step S5 is prepared by the following method: a) Houttuynia cordata and rosemary were mixed in a mass ratio of 4:1 and extracted with 65% ethanol in a dynamic countercurrent extraction process three times for 40 minutes each time. b) The extract was enriched on a molecularly imprinted adsorption column, and the target component was eluted with an acetonitrile-water (70:30, v / v) gradient.

[0021] In step S4, the tangential flow filtration uses a 100 kDa ultrafiltration membrane, the transmembrane pressure difference is controlled at 0.8 bar-1.2 bar, and the temperature is maintained at 4° C.-8° C.

[0022] The method also includes a post-processing step: mixing the prepared waste liquid with the Aspergillus niger spore suspension in a volume ratio of 1:0.08, aerobically fermenting at 48±2°C for 72 hours, and converting the mixture into liquid organic fertilizer with a pH value of 6.8-7.2.

[0023] 3.5nm ZnO quantum dots generate high-energy electron-hole pairs under visible light through surface plasmon resonance, directly disrupting the double helix structure of pathogen DNA (experiments show a 92% DNA damage rate in E. coli). Baicalin and eugenol form a π-π conjugated system, synergistically blocking the LuxS quorum sensing system (QS inhibition rate ≥ 85%). This improves the efficiency of evolved T4 bacteriophage in breaking through biofilm barriers by three times (biofilm clearance rate increases from the traditional 35% to 82%).

[0024] The disintegration rate of carboxymethyl chitosan wall material reaches 78% in 30 minutes in the acidic environment of pathogen metabolism (pH ≤ 6.5), realizing "release on demand". The hydroxyl groups on the surface of the microcapsule hydrate and swell at RH ≥ 75%, and the release rate is increased to 3.2 times that of dry conditions.

[0025] The synergistic effect of a shikimate pathway inhibitor (IC50 = 0.8 μM) and a phage lytic enzyme (specific activity ≥ 500 U / mg) makes it difficult for pathogens to escape through single gene mutations. The inhibition zone diameter for NDM-1-producing superbugs reaches 25 mm (compared to 8 mm for traditional quaternary ammonium salts).

[0026] The CRISPR-dCas9 system achieved a 94% efficiency in silencing the mecA gene by methylation, restoring MRSA's susceptibility to β-lactams. After 20 generations of continuous use, the pathogen's MIC value increased only 1.8-fold (compared to a 128-fold increase in the traditional disinfectant group).

[0027] Chitosan-based materials undergo enzymatic degradation in soil within 72 hours, reaching 95% and producing beneficial substances such as glucosamine. Their environmental half-life (DT50) is only 36 hours, far shorter than the 14 days of quaternary ammonium salts.

[0028] Seven days after spraying, the actinomycete / pathogen ratio in the farm soil increased from 0.6 to 2.3, forming a dominant community of probiotics. Its acute toxicity to aquatic organisms (zebrafish LC50 > 1000 mg / L) reached the highest OECD safety level.

[0029] Technology Comparison Table 1 This invention achieves the trinity of "precise release, dynamic disinfection, and ecological restoration," transforming disinfection from a hygienic procedure into an ecological management tool. It also promotes the transition of the aquaculture industry toward antibiotic-free farming by reducing disinfection frequency by 62.5% and antibiotic usage by 78%.

[0030] The embodiments of the present invention have been fully disclosed and presented through experimental verification and practical application. In accordance with the relevant provisions of the Patent Law, those skilled in the art may, based on the technical solutions, core technical principles, and innovative concepts disclosed in the present invention, make reasonable changes, modifications, substitutions, and improvements to the technical features in the embodiments, without violating the substantive protection of the present invention, including but not limited to derivative technical solutions such as optimization of process parameters, adjustment of functional modules, and improvement of structural design.

[0031] It should be clearly pointed out that the equivalent transformations and improvements based on the core technical concept of the present invention should be included in the scope of patent protection of the present invention. The scope of patent protection of the present invention is based on the attached claims and their legally equivalent technical solutions. Any technical solution that is substantially the same as the technical features in the claims in terms of function, structure, and principle, and has an equivalent effect, shall fall within the scope of protection of the present invention.

Claims

1. A Chinese medicine spray for disinfecting animal breeding environments, characterized in that: The invention is composed of the following components in the following mass percentages: 5%-10% of a baicalin-ZnO quantum dot complex, wherein the surface of the ZnO quantum dots is connected to the baicalin molecules via coordination bonds, and the particle size distribution is 3.0±0.5nm; 3%-8% pH-responsive microcapsules, comprising a core material and a wall material, wherein the core material is a mixture of eugenol and citral in a mass ratio of 2.5:1-4:1, and the wall material is formed by cross-linking carboxymethyl chitosan and ε-polylysine in a molar ratio of 6:1-8:1; 1.5-5×10 6 CFU / mL of a genetically engineered yeast agent, wherein the yeast chromosome is integrated with an andrographolide synthesis gene cluster regulated by a PHO5 promoter; 1.2-3×10 9 The PFU / mL of the phage mixture contained T4 phage, φKZ phage, and ST1 phage, with a titer ratio of (5±0.5):(2.5±0.3):1; 0.8%-2% houttuynia cordata-rosemary compound extract, with houttuynia cordata content ≥15mg / g; 0.01%-0.1% quorum sensing inhibitor, composed of an N-butyryl homoserine lactone analog and an Agr antagonist peptide in a ratio of 1:2-1:3; The balance is water for injection and at least one stabilizer selected from trehalose and hydroxypropyl cyclodextrin.

2. A Chinese medicine spray for disinfecting an animal breeding environment according to claim 1, characterized in that: The XRD spectrum of the ZnO quantum dots shows a characteristic peak of the (101) crystal plane at 2θ=36.2±0.2°.

3. A Chinese medicine spray for disinfecting an animal breeding environment according to claim 1, characterized in that: The cross-linking degree of the wall material of the microcapsule is controlled at 85%-90%, and the release rate thereof in a 30-minute environment at a pH value of ≤6.5 is ≥78%.

4. A Chinese medicine spray for disinfecting an animal breeding environment according to claim 1, characterized in that: The stabilizer is a complex of trehalose and hydroxypropyl-β-cyclodextrin in a mass ratio of 2:1, and the total addition amount is 0.5%-1.2%.

5. A method for preparing a Chinese medicine spray for disinfecting an animal breeding environment according to any one of claims 1 to 4, characterized in that: The specific steps include: S1. Quantum Dot Synthesis 0.5 mol / L zinc nitrate solution and 8% baicalin solution were mixed in a volume ratio of 1:3-1:5, and hydrothermally reacted at 175°C-185°C for 11h-13h under nitrogen protection. After centrifugation, a composite quantum dot suspension was obtained; S2. Microcapsule Preparation A coaxial microfluidic device was used to control the flow rate of the core material to 1.2±0.1mL / min and the flow rate of the wall material to 3.2±0.2mL / min, and cross-linking and curing were carried out in a 2.5mol / L CaCl2 solution for 25min-35min. S3. Yeast Culture Two-stage cultivation was carried out in a 30-L bioreactor. In the first stage, the dissolved oxygen was controlled to be ≥30% and the pH value was 5.5-6.

0. In the second stage, induced expression was initiated when the residual sugar concentration dropped to 5±0.5 g / L. S4. Phage Enhancement The original phage was alternately inoculated into ESBL-producing Escherichia coli and methicillin-resistant Staphylococcus aureus for 5-7 generations of directed evolution and concentrated by tangential flow filtration to a titer of ≥1.5×10¹ 0 PFU / mL; S5. Preparation Integration The products of steps 1-4 above were mixed with the composite extract in proportion, and subjected to microfluidization treatment at 150 MPa-180 MPa for three times to obtain a homogeneous dispersion with a particle size of D90 ≤ 450 nm.

6. The method for preparing a Chinese medicine spray for disinfecting an animal breeding environment according to claim 5, wherein: The specific parameters of the two-stage culture in step S3 are: Stage 1: temperature 30±0.5°C, stirring speed 300-350 rpm, ventilation volume 1.2 vvm, lasting 12-14 hours; The second stage: temperature 28±0.5℃, stirring speed 400rpm-450rpm, ventilation volume 1.5vvm, lasting 24h-26h.

7. The method for preparing a Chinese medicine spray for disinfecting an animal breeding environment according to claim 5, wherein: The composite extract in step S5 is prepared by the following method: a) Houttuynia cordata and rosemary were mixed in a mass ratio of 4:1 and extracted with 65% ethanol in a dynamic countercurrent extraction process three times for 40 minutes each time. b) The extract was enriched on a molecularly imprinted adsorption column, and the target component was eluted with an acetonitrile-water (70:30, v / v) gradient.

8. The method for preparing a Chinese medicine spray for disinfecting an animal breeding environment according to claim 5, wherein: In step S4, the tangential flow filtration uses a 100 kDa ultrafiltration membrane, the transmembrane pressure difference is controlled at 0.8-1.2 bar, and the temperature is maintained at 4° C.-8° C.

9. The method for preparing a Chinese medicine spray for disinfecting an animal breeding environment according to claim 5, wherein: The method further comprises a post-processing step of mixing the prepared waste liquid with an Aspergillus niger spore suspension in a volume ratio of 1:0.08, aerobically fermenting the mixture at 48±2° C. for 72 hours, and converting the mixture into a liquid organic fertilizer with a pH value of 6.8-7.2.

Images