A disinfectant for animal husbandry environments and its preparation method

Abstract

This invention belongs to the field of environmental disinfectant technology, specifically relating to a disinfectant for animal husbandry environments and its preparation method. This invention induces Bacillus subtilis fermentation through a compound extract of hawthorn, codonopsis, and astragalus. The three extracts synergistically upregulate the expression of genes related to antibacterial lipopeptide synthesis, significantly enhancing the antibacterial potency of the fermentation broth. The highly active antibacterial fermentation broth is then incorporated into a sodium alginate-xanthan gum composite gel network. Utilizing the slow oxidation properties of ascorbic acid under environmental humidity and oxygen, the gel network gradually undergoes proton dissociation, achieving a stable and long-lasting release of the antibacterial active ingredients. Simultaneously, xanthan gum swells upon contact with water to form a highly viscoelastic gel film, significantly enhancing the disinfectant's adhesion and retention capacity on humid surfaces. The resulting disinfectant possesses comprehensive properties including high antibacterial activity, long-lasting action, and strong environmental adhesion. The preparation process is simple and controllable, making it suitable for large-scale application in livestock farming environments.

Description

Technical Field

[0001] This invention belongs to the field of environmental disinfectant technology, specifically relating to an animal breeding environment disinfectant and its preparation method. Background Technology

[0002] Animal farming environments are complex ecosystems where pathogenic microorganisms continuously exist. When the number of pathogens in the environment accumulates and exceeds the defense capacity of the livestock and poultry immune system, it is easy to trigger an outbreak of disease. Environmental disinfection, by destroying the living environment of pathogenic microorganisms and cutting off the disease transmission chain, is a key link in the biosecurity control system of farms. Scientific and reasonable disinfection can effectively kill various pathogens on the ground, walls, drinking water system, feeding tools and body surface of the pen, and control environmental microorganisms at a safe level. It has irreplaceable and important value for ensuring the healthy development of the livestock industry.

[0003] Currently, chemical disinfectants are the primary method for disinfecting aquaculture environments. Chlorine-containing disinfectants, aldehyde disinfectants, peroxides, and quaternary ammonium salts are widely used due to their low cost and ease of purchase. However, these disinfectants have several drawbacks in practical applications: most chemical disinfectants are highly corrosive and irritating, severely corroding metal equipment and damaging floor materials. Frequent contact can irritate the respiratory tract of livestock and poultry, as well as the skin and mucous membranes of operators. In addition, long-term and excessive use of the same disinfectant can induce drug resistance in pathogenic microorganisms, leading to a significant decrease in sterilization effectiveness. Furthermore, chemical disinfectants have a short duration of action, and the environment can be re-contaminated within hours after disinfection.

[0004] To overcome the drawbacks of chemical disinfectants, biological disinfection has gradually gained attention. Beneficial environmental bacteria such as Bacillus subtilis can inhibit pathogenic microorganisms through competitive site occupation, the formation of protective biofilms, and the secretion of antibacterial substances, and possess advantages such as being non-toxic, non-irritating, and not inducing drug resistance. However, existing biological disinfectants still suffer from drawbacks such as generally lower potency than chemical disinfectants, easy loss of active ingredients in humid environments, and short effective duration. Therefore, developing a specialized disinfectant for aquaculture environments that combines high antibacterial activity, intelligent long-lasting release, and strong environmental adhesion is a pressing technical challenge in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a disinfectant for animal breeding environments and its preparation method, so as to solve the above-mentioned technical problems.

[0006] To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows: A method for preparing a disinfectant for animal husbandry environments includes the following steps: S1 Add ascorbic acid to deionized water and stir until dissolved at 5-15°C under light-protected conditions to obtain an ascorbic acid solution; S2 Add ascorbic acid solution to sodium alginate-xanthan gum composite solution and stir for 15-30 minutes to obtain drug loading solution; S3. Add the highly active antibacterial fermentation broth to the drug-carrying solution to obtain a mixture; add the mixture dropwise to the calcium chloride solution, and after the addition is complete, let it stand for 15-30 minutes, filter, wash the precipitate with deionized water, and then vacuum dry or freeze dry to obtain an animal breeding environment disinfectant.

[0007] As a further improvement, the preparation method of the highly active antibacterial fermentation broth is as follows: Bacillus subtilis is inoculated into LB medium and cultured at 37°C with shaking until OD... 600 The OD value is 0.6–0.8. After centrifugation, the supernatant is discarded, and the precipitate is resuspended in a culture medium containing the compound Chinese medicine extract until the OD value is reached. 600 The concentration was 2.0–5.0. The mixture was cultured at 22–30℃ with shaking for 12–24 h, centrifuged, and the supernatant was collected. The supernatant was then concentrated under reduced pressure to 1 / 5 of its volume to obtain a highly active antibacterial fermentation broth.

[0008] As a further improvement, the preparation method of the culture medium containing the compound traditional Chinese medicine extract is as follows: weigh hawthorn, codonopsis and astragalus, pulverize and sieve them, mix them evenly to obtain a mixed powder; add the mixed powder to deionized water at a mass ratio of 1:10-15, heat to 90-100℃, reflux and extract 2-3 times, each time for 1-1.5 hours; filter, concentrate the filtrate under reduced pressure to 1 / 20 of the filtrate volume, filter to remove bacteria, and obtain the compound traditional Chinese medicine extract; mix the compound traditional Chinese medicine extract and sterile LB culture medium at a volume ratio of 0.5-1:10 to obtain the culture medium containing the compound traditional Chinese medicine extract.

[0009] As a further improvement, the mass ratio of hawthorn, codonopsis and astragalus is 1:2-3:2-3.

[0010] As a further improvement, the preparation method of the sodium alginate-xanthan gum composite solution is as follows: dissolve sodium alginate powder in deionized water to prepare a sodium alginate solution with a mass concentration of 2% to 3%; dissolve xanthan gum powder in deionized water at 50 to 65°C to prepare a xanthan gum solution with a mass concentration of 0.5% to 1%; mix the sodium alginate solution and the xanthan gum solution and stir evenly to obtain the sodium alginate-xanthan gum composite solution.

[0011] As a further improvement, the volume ratio of the sodium alginate solution to the xanthan gum solution is 3 to 6:1.

[0012] As a further improvement, in step S1, the mass ratio of ascorbic acid to deionized water is 1:15 to 25.

[0013] As a further improvement, in step S2, the volume ratio of the ascorbic acid solution and the sodium alginate-xanthan gum composite solution is 1:8 to 12.

[0014] As a further improvement, in step S3, the volume ratio of the highly active antibacterial fermentation broth to the drug-loaded solution is 1:4 to 6; and the concentration of the calcium chloride solution is 2% to 4%.

[0015] The present invention also provides a disinfectant for animal breeding environments.

[0016] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows: 1. This invention reveals that the combination of hawthorn, codonopsis, and astragalus synergistically regulates the expression level of key genes for antimicrobial lipopeptide synthesis in Bacillus subtilis, significantly enhancing the production capacity of antimicrobial substances in Bacillus subtilis at the transcriptional level; this traditional Chinese medicine composition acts as a metabolic signaling molecule to activate the secondary metabolic pathways of Bacillus subtilis, increasing the content of antimicrobial substances in Bacillus subtilis fermentation products and enhancing the antimicrobial potency of Bacillus subtilis fermentation products.

[0017] 2. This invention utilizes the slow oxidation property of ascorbic acid under the action of environmental humidity and oxygen to construct a gel network combining ascorbic acid and sodium alginate; ascorbic acid is embedded between the sodium alginate gel network, and during the use stage, it continuously and gently produces acid, driving the gel to gradually dissociate from the outside to the inside, thereby achieving a stable and long-lasting release of antibacterial active ingredients.

[0018] 3. This invention uses xanthan gum and sodium alginate to form a network structure, which significantly improves the adhesion and retention capacity of disinfectant particles on humid surfaces; xanthan gum absorbs water and swells to form a highly viscous gel film layer, which strongly anchors to the substrate of the aquaculture environment, enabling the disinfectant particles to firmly adhere to the surface of aquaculture substrates such as cement and plastic, thus achieving a synergistic effect of slow release and retention. Attached Figure Description

[0019] Figure 1 The results of the long-lasting antibacterial performance experiments of Example 1, Comparative Example 1, and Comparative Example 4 are shown in the figure. Detailed Implementation

[0020] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or manufacturer's conditions shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0021] Example 1: A method for preparing a disinfectant for animal husbandry environments, comprising the following steps: 1. Take hawthorn, codonopsis, and astragalus at a mass ratio of 1:3:3, pulverize and sieve them separately, mix them evenly to obtain a mixed powder; add the mixed powder to 10 times its mass of deionized water, heat to 95℃, and perform reflux extraction 3 times, 1.5h each time; filter, concentrate the filtrate under reduced pressure to 1 / 20 of the original volume of the filtrate; filter and sterilize in a clean bench using a 0.22μm filter membrane to obtain a compound traditional Chinese medicine extract; mix the compound traditional Chinese medicine extract and sterile LB medium at a ratio of 1:10 to obtain a culture medium containing the compound traditional Chinese medicine extract; 2. Inoculate Bacillus subtilis onto LB medium, place in an incubator, and incubate with shaking at 37°C until OD reaches [value missing]. 600 The OD value was 0.6. Centrifuge at 4000 r / min for 20 min, discard the supernatant, and resuspend the precipitate in a culture medium containing the compound traditional Chinese medicine extract until the OD value was 0.6. 600 The value was 3.0. The mixture was put back into the incubator and cultured at 25°C with shaking for 24 hours. After centrifugation at 4000 r / min for 20 min, the supernatant was collected and concentrated under reduced pressure to 1 / 5 of the original volume of the supernatant to obtain a highly active antibacterial fermentation broth. 3. Dissolve sodium alginate powder in deionized water to prepare a sodium alginate solution with a mass concentration of 3%; dissolve xanthan gum powder in deionized water at 60℃ to prepare a xanthan gum solution with a mass concentration of 1%; mix the sodium alginate solution and the xanthan gum solution at a volume ratio of 5:1 and stir evenly to obtain a sodium alginate-xanthan gum composite solution. 4. Add ascorbic acid to deionized water at a mass ratio of 1:15, and stir at 10°C in the dark until the ascorbic acid dissolves to obtain an ascorbic acid solution; add the ascorbic acid solution to a sodium alginate-xanthan gum composite solution at a volume ratio of 1:10, and stir for 30 minutes to obtain a drug-loaded solution; add the highly active antibacterial fermentation broth to the drug-loaded solution at a volume ratio of 1:5 to obtain a mixed solution; add the mixed solution dropwise to a 3% calcium chloride solution, and after the addition is complete, let it stand for 30 minutes, filter and collect the precipitate, wash the precipitate with deionized water, and freeze-dry at -30°C for 12 hours to obtain an animal breeding environment disinfectant.

[0022] Example 2: A method for preparing a disinfectant for animal husbandry environments, comprising the following steps: 1. Take hawthorn, codonopsis, and astragalus at a mass ratio of 1:2:2, pulverize and sieve them separately, mix them evenly to obtain a mixed powder; add the mixed powder to 15 times its mass of deionized water, heat to 100℃, and perform reflux extraction twice, 1 hour each time; filter, concentrate the filtrate under reduced pressure to 1 / 20 of the original volume of the filtrate; filter and sterilize using a 0.22μm filter membrane in a clean bench to obtain a compound traditional Chinese medicine extract; mix the compound traditional Chinese medicine extract and sterile LB medium at a ratio of 0.5:10 to obtain a culture medium containing the compound traditional Chinese medicine extract; 2. Inoculate Bacillus subtilis onto LB medium, place in an incubator, and incubate with shaking at 37°C until OD reaches [value missing]. 600 The OD value was 0.8. Centrifuge at 4000 r / min for 20 min, discard the supernatant, and resuspend the precipitate in a culture medium containing the compound Chinese medicine extract until the OD value was 0.8. 600 The value was 5.0. The mixture was returned to the incubator and cultured at 30℃ with shaking for 20 hours. After centrifugation at 4000 r / min for 20 minutes, the supernatant was collected and concentrated under reduced pressure to 1 / 5 of the original volume of the supernatant to obtain a highly active antibacterial fermentation broth. 3. Dissolve sodium alginate powder in deionized water to prepare a sodium alginate solution with a mass concentration of 2%; dissolve xanthan gum powder in deionized water at 65℃ to prepare a xanthan gum solution with a mass concentration of 0.5%; mix the sodium alginate solution and the xanthan gum solution at a volume ratio of 3:1 and stir evenly to obtain a sodium alginate-xanthan gum composite solution. 4. Add ascorbic acid to deionized water at a mass ratio of 1:25, and stir at 5°C in the dark until the ascorbic acid dissolves to obtain an ascorbic acid solution; add the ascorbic acid solution to a sodium alginate-xanthan gum composite solution at a volume ratio of 1:8, and stir for 20 minutes to obtain a drug-loaded solution; add the highly active antibacterial fermentation broth to the drug-loaded solution at a volume ratio of 1:6 to obtain a mixed solution; add the mixed solution dropwise to a 4% calcium chloride solution, and after the addition is complete, let it stand for 20 minutes, filter and collect the precipitate, wash the precipitate with deionized water, and freeze-dry at -20°C for 24 hours to obtain an animal breeding environment disinfectant.

[0023] Example 3: A method for preparing a disinfectant for animal husbandry environments, comprising the following steps: 1. Take hawthorn, codonopsis, and astragalus at a mass ratio of 1:2.5:3, pulverize and sieve them separately, mix them evenly to obtain a mixed powder; add the mixed powder to 12 times its mass of deionized water, heat to 90℃, and perform reflux extraction 3 times, 1.5h each time; filter, concentrate the filtrate under reduced pressure to 1 / 20 of the original volume of the filtrate; filter and sterilize in an ultra-clean bench using a 0.22μm filter membrane to obtain a compound traditional Chinese medicine extract; mix the compound traditional Chinese medicine extract and sterile LB medium at a ratio of 0.8:10 to obtain a culture medium containing the compound traditional Chinese medicine extract; 2. Inoculate Bacillus subtilis onto LB medium, place in an incubator, and incubate with shaking at 37°C until OD reaches [value missing]. 600 The OD value was 0.7. Centrifuge at 4000 r / min for 20 min, discard the supernatant, and resuspend the precipitate in a culture medium containing the compound traditional Chinese medicine extract until the OD value was 0.7. 600 The value was 2.0. The mixture was put back into the incubator and cultured at 22℃ with shaking for 12 hours. After centrifugation at 4000r / min for 20 minutes, the supernatant was collected and concentrated under reduced pressure to 1 / 5 of the original volume of the supernatant to obtain a highly active antibacterial fermentation broth. 3. Dissolve sodium alginate powder in deionized water to prepare a sodium alginate solution with a mass concentration of 2.4%; dissolve xanthan gum powder in deionized water at 50℃ to prepare a xanthan gum solution with a mass concentration of 0.8%; mix the sodium alginate solution and the xanthan gum solution at a volume ratio of 6:1 and stir evenly to obtain a sodium alginate-xanthan gum composite solution. 4. Add ascorbic acid to deionized water at a mass ratio of 1:20, and stir at 15°C in the dark until the ascorbic acid dissolves to obtain an ascorbic acid solution; add the ascorbic acid solution to a sodium alginate-xanthan gum composite solution at a volume ratio of 1:12, and stir for 15 minutes to obtain a drug-loaded solution; add the highly active antibacterial fermentation broth to the drug-loaded solution at a volume ratio of 1:4 to obtain a mixed solution; add the mixed solution dropwise to a 2% calcium chloride solution, and after the addition is complete, let it stand for 15 minutes, filter and collect the precipitate, wash the precipitate with deionized water, and vacuum dry at 50°C for 24 hours to obtain an animal breeding environment disinfectant.

[0024] Comparative Example 1: A method for preparing a disinfectant for animal husbandry environments, differing from Example 1 in that sodium metabisulfite is used instead of ascorbic acid, comprising the following steps: 1. Take hawthorn, codonopsis, and astragalus at a mass ratio of 1:3:3, pulverize and sieve them separately, mix them evenly to obtain a mixed powder; add the mixed powder to 10 times its mass of deionized water, heat to 95℃, and perform reflux extraction 3 times, 1.5h each time; filter, concentrate the filtrate under reduced pressure to 1 / 20 of the original volume of the filtrate; filter and sterilize in a clean bench using a 0.22μm filter membrane to obtain a compound traditional Chinese medicine extract; mix the compound traditional Chinese medicine extract and sterile LB medium at a ratio of 1:10 to obtain a culture medium containing the compound traditional Chinese medicine extract; 2. Inoculate Bacillus subtilis onto LB medium, place in an incubator, and incubate with shaking at 37°C until OD reaches [value missing]. 600 The OD value was 0.6. Centrifuge at 4000 r / min for 20 min, discard the supernatant, and resuspend the precipitate in a culture medium containing the compound traditional Chinese medicine extract until the OD value was 0.6. 600 The value was 3.0. The mixture was put back into the incubator and cultured at 25°C with shaking for 24 hours. After centrifugation at 4000 r / min for 20 min, the supernatant was collected and concentrated under reduced pressure to 1 / 5 of the original volume of the supernatant to obtain a highly active antibacterial fermentation broth. 3. Dissolve sodium alginate powder in deionized water to prepare a sodium alginate solution with a mass concentration of 3%; dissolve xanthan gum powder in deionized water at 60℃ to prepare a xanthan gum solution with a mass concentration of 1%; mix the sodium alginate solution and the xanthan gum solution at a volume ratio of 5:1 and stir evenly to obtain a sodium alginate-xanthan gum composite solution. 4. Following the principle of equimolar amounts as in Example 1, weigh sodium metabisulfite, add it to deionized water, and stir at 25°C until dissolved to obtain a sodium metabisulfite solution; add the sodium metabisulfite solution to the sodium alginate-xanthan gum composite solution at a volume ratio of 1:10, and stir rapidly to obtain a drug-loaded solution; add the highly active antibacterial fermentation broth to the drug-loaded solution at a volume ratio of 1:5 to obtain a mixed solution; immediately add the mixed solution dropwise to a 3% calcium chloride solution, and after the addition is complete, let it stand for 30 minutes, filter and collect the precipitate, wash the precipitate with deionized water, and freeze-dry at -30°C for 12 hours to obtain an animal breeding environment disinfectant.

[0025] Comparative Example 2: A method for preparing an animal husbandry environment disinfectant, which differs from Example 1 in that xanthan gum is not added, and includes the following steps: 1. Take hawthorn, codonopsis, and astragalus at a mass ratio of 1:3:3, pulverize and sieve them separately, mix them evenly to obtain a mixed powder; add the mixed powder to 10 times its mass of deionized water, heat to 95℃, and perform reflux extraction 3 times, 1.5h each time; filter, concentrate the filtrate under reduced pressure to 1 / 20 of the original volume of the filtrate; filter and sterilize in a clean bench using a 0.22μm filter membrane to obtain a compound traditional Chinese medicine extract; mix the compound traditional Chinese medicine extract and sterile LB medium at a ratio of 1:10 to obtain a culture medium containing the compound traditional Chinese medicine extract; 2. Inoculate Bacillus subtilis onto LB medium, place in an incubator, and incubate with shaking at 37°C until OD reaches [value missing]. 600 The OD value was 0.6. Centrifuge at 4000 r / min for 20 min, discard the supernatant, and resuspend the precipitate in a culture medium containing the compound traditional Chinese medicine extract until the OD value was 0.6. 600 The value was 3.0. The mixture was put back into the incubator and cultured at 25°C with shaking for 24 hours. After centrifugation at 4000 r / min for 20 min, the supernatant was collected and concentrated under reduced pressure to 1 / 5 of the original volume of the supernatant to obtain a highly active antibacterial fermentation broth. 3. Dissolve sodium alginate powder in deionized water to prepare a 3% sodium alginate solution. Add ascorbic acid to deionized water at a mass ratio of 1:15 and stir at 10°C in the dark until the ascorbic acid dissolves to obtain an ascorbic acid solution. Add the ascorbic acid solution to the sodium alginate solution at a volume ratio of 1:10 and stir for 30 minutes to obtain a drug-loaded solution. Add the highly active antibacterial fermentation broth to the drug-loaded solution at a volume ratio of 1:5 to obtain a mixed solution. Add the mixed solution dropwise to a 3% calcium chloride solution. After the addition is complete, let it stand for 30 minutes, filter and collect the precipitate. Wash the precipitate with deionized water and freeze-dry at -30°C for 12 hours to obtain an animal breeding environment disinfectant.

[0026] Comparative Example 3: A method for preparing an animal husbandry environment disinfectant, differing from Example 1 in that Bacillus subtilis was not treated with mixed traditional Chinese medicine powder, and includes the following steps: 1. Inoculate Bacillus subtilis onto LB medium, place in an incubator, and incubate with shaking at 37°C until OD reaches [value missing]. 600 The value was 0.6. Centrifuge at 4000 r / min for 20 min, discard the supernatant, and resuspend the precipitate in sterile LB medium until the OD value was 0.6. 600 The value was 3.0. The mixture was returned to the incubator and cultured at 25°C with shaking for 24 hours. After centrifugation at 4000 r / min for 20 min, the supernatant was collected and concentrated under reduced pressure to 1 / 5 of the original volume of the supernatant to obtain the antibacterial fermentation broth. 2. Dissolve sodium alginate powder in deionized water to prepare a sodium alginate solution with a mass concentration of 3%; dissolve xanthan gum powder in deionized water at 60℃ to prepare a xanthan gum solution with a mass concentration of 1%; mix the sodium alginate solution and the xanthan gum solution at a volume ratio of 5:1 and stir evenly to obtain a sodium alginate-xanthan gum composite solution. 3. Add ascorbic acid to deionized water at a mass ratio of 1:15, and stir at 10°C in the dark until the ascorbic acid dissolves to obtain an ascorbic acid solution; add the ascorbic acid solution to a sodium alginate-xanthan gum composite solution at a volume ratio of 1:10, and stir for 30 minutes to obtain a drug-loaded solution; add the antibacterial fermentation broth to the drug-loaded solution at a volume ratio of 1:5 to obtain a mixed solution; add the mixed solution dropwise to a 3% calcium chloride solution, and after the addition is complete, let it stand for 30 minutes, filter and collect the precipitate, wash the precipitate with deionized water, and freeze-dry at -30°C for 12 hours to obtain an animal breeding environment disinfectant.

[0027] Comparative Example 4: A method for preparing a disinfectant for animal husbandry environments, differing from Example 1 in that the components are simply physically mixed, including the following steps: 1. Take hawthorn, codonopsis, and astragalus at a mass ratio of 1:3:3, pulverize and sieve them separately, mix them evenly to obtain a mixed powder; add the mixed powder to 10 times its mass of deionized water, heat to 95℃, and perform reflux extraction 3 times, 1.5h each time; filter, concentrate the filtrate under reduced pressure to 1 / 20 of the original volume of the filtrate; filter and sterilize in a clean bench using a 0.22μm filter membrane to obtain a compound traditional Chinese medicine extract; mix the compound traditional Chinese medicine extract and sterile LB medium at a ratio of 1:10 to obtain a culture medium containing the compound traditional Chinese medicine extract; 2. Inoculate Bacillus subtilis onto LB medium, place in an incubator, and incubate with shaking at 37°C until OD reaches [value missing]. 600 The OD value was 0.6. Centrifuge at 4000 r / min for 20 min, discard the supernatant, and resuspend the precipitate in a culture medium containing the compound traditional Chinese medicine extract until the OD value was 0.6. 600 The value was 3.0. The mixture was put back into the incubator and cultured at 25°C with shaking for 24 hours. After centrifugation at 4000 r / min for 20 min, the supernatant was collected and concentrated under reduced pressure to 1 / 5 of the original volume of the supernatant to obtain a highly active antibacterial fermentation broth. 3. Dissolve sodium alginate powder in deionized water to prepare a sodium alginate solution with a mass concentration of 3%; dissolve xanthan gum powder in deionized water at 60℃ to prepare a xanthan gum solution with a mass concentration of 1%; mix the sodium alginate solution and the xanthan gum solution at a volume ratio of 5:1 and stir evenly to obtain a sodium alginate-xanthan gum composite solution. 4. Add ascorbic acid to deionized water at a mass ratio of 1:15, and stir at 10°C in the dark until the ascorbic acid dissolves to obtain an ascorbic acid solution; add the ascorbic acid solution to a sodium alginate-xanthan gum composite solution at a volume ratio of 1:10, and stir for 30 minutes to obtain a drug-loaded solution; add the highly active antibacterial fermentation broth to the drug-loaded solution at a volume ratio of 1:5 to obtain a mixed solution; concentrate the mixed solution and freeze-dry it to obtain a mixed dry powder, which is an animal breeding environment disinfectant.

[0028] Performance testing 1. Effects of traditional Chinese medicine compound on the expression of genes related to the antibacterial properties of Bacillus subtilis By using quantitative reverse transcription polymerase chain reaction (qRT-PCR), we detected changes in the mRNA expression levels of key antibacterial lipopeptide synthase genes and regulatory genes in Bacillus subtilis after induction with compound extracts of different combinations of traditional Chinese medicines, elucidating the molecular mechanism of the enhanced efficacy of traditional Chinese medicines at the transcriptional level.

[0029] According to the Chinese herbal medicine ratios shown in Table 1, and in accordance with the principle of consistent total mass, the Chinese herbal medicines were compounded.

[0030] Table 1. Quality Proportioning Table of Traditional Chinese Medicines

[0031] Take the appropriate mass of traditional Chinese medicine, pulverize and sieve to obtain a mixed powder; add the mixed powder to 10 times its mass of deionized water, heat to 95℃, and perform reflux extraction 3 times, 1.5h each time; filter, concentrate the filtrate under reduced pressure to 1 / 20 of the original volume of the filtrate; filter and sterilize in a clean bench using a 0.22μm filter membrane to obtain a compound traditional Chinese medicine extract; mix the compound traditional Chinese medicine extract and sterile LB medium at a ratio of 1:10 to obtain a medium containing the compound traditional Chinese medicine extract (the blank control group is pure sterile LB medium without any added traditional Chinese medicine components). Bacillus subtilis was inoculated onto LB medium and placed in an incubator, where it was cultured at 37°C with shaking until OD reached. 600 The OD value was 0.6. Centrifuge at 4000 r / min for 20 min, discard the supernatant, and resuspend the precipitate in a culture medium containing the compound traditional Chinese medicine extract until the OD value was 0.6. 600 The value was 3.0. The cells were returned to the incubator and cultured at 25°C with shaking for 8 hours. 2 mL of bacterial culture was taken from each group, flash-frozen in liquid nitrogen, and then transferred to -80°C for storage until use.

[0032] Total RNA was extracted from each sample using a bacterial total RNA extraction kit, and RNA concentration was measured. Reverse transcription was performed, and qPCR was conducted using primers. 16S rRNA was used as an internal reference gene. Three replicates were set up for each sample group. Statistical analysis was performed, and the upregulation fold of mRNA expression levels was calculated compared with the blank control group. The experimental results are shown in Table 2. Table 2. Statistical analysis of gene expression upregulation in each experimental group

[0033] As shown in Table 2, the three herbal compound groups (groups 7-11) had a greater upregulation effect on the expression of genes related to the antibacterial properties of Bacillus subtilis than the single-drug groups or the two-drug compound groups. This indicates that Astragalus membranaceus, Codonopsis pilosula, and Crataegus pinnatifida have a clear synergistic effect in promoting the antibacterial metabolism of Bacillus subtilis: by regulating and promoting the key genes srfA, fenB, and ppsA of Bacillus subtilis, they promote the synthesis of various antibacterial lipopeptides, including surfactants, iturin, and fenosin, thereby significantly improving the antibacterial properties of Bacillus subtilis.

[0034] The three-component traditional Chinese medicine compound described in this invention, under a specific ratio, produced a significant synergistic upregulation effect on the expression of key antibacterial lipopeptide synthesis genes such as srfA, fenB, and ppsA. The upregulation factor far exceeded the simple sum of the effects of each component, thereby achieving an essential enhancement of the antibacterial ability of Bacillus subtilis at the transcriptional level.

[0035] As can be seen from the data in Table 2, Group 8 (Astragalus membranaceus, Codonopsis pilosula, and Crataegus pinnatifida = 2:2:1) and Group 10 (Astragalus membranaceus, Codonopsis pilosula, and Crataegus pinnatifida = 3:3:1) showed the best and most stable overall upregulation effect, and the upregulation effect of the single Crataegus pinnatifida group was poor. This indicates that Astragalus membranaceus and Codonopsis pilosula play a leading role in this compound system, while Crataegus pinnatifida plays a synergistic role in a specific ratio. Therefore, the optimal ratio of Astragalus membranaceus, Codonopsis pilosula, and Crataegus pinnatifida is 2:2:1 or 3:3:1.

[0036] 2. Overall antibacterial efficacy assessment The total antibacterial efficacy of the animal husbandry environment disinfectants prepared in Example 1, Comparative Example 3, and Comparative Example 4 was determined.

[0037] Take 200 mg of the animal breeding environment disinfectant prepared in Example 1, Comparative Example 3 and Comparative Example 4, add 10 mL of sterile phosphate buffer, sonicate at 300 W for 5 min in an ice bath, pulse for 2 s with a 5 s interval, centrifuge at 1000 r / min for 10 min, and take the supernatant as the test stock solution.

[0038] Staphylococcus aureus, Escherichia coli, and Salmonella were used as experimental strains. The bacterial suspensions were evenly spread on agar plates, Oxford cups were placed on them, and 100 μL of the test stock solution was added to each group. Three parallel experiments were set up for each group. The plates were incubated at 37℃ for 24 h, and the diameter of the inhibition zone (including the outer diameter of the cup) was accurately measured using calipers. The average value of the parallel experimental data was taken. The experimental results are shown in Table 3. Table 3 Results of Overall Antibacterial Efficacy Test

[0039] As shown in Table 3, the inhibition zone diameters of the three tested pathogens in Example 1 were significantly larger than those in Comparative Example 3, indicating that the compound extract of hawthorn, codonopsis and astragalus can induce and enhance the antibacterial ability of Bacillus subtilis, thereby significantly enhancing the antibacterial activity of the final animal breeding environment disinfectant product. There was no significant difference in the antibacterial performance between the samples of Example 1 and Comparative Example 4, indicating that the releasable antibacterial components contained in the two groups of samples are similar, suggesting that the cross-linking process of sodium alginate and calcium chloride ions used in Example 1 does not affect the activity of the antibacterial components.

[0040] 3. Long-lasting antibacterial properties Long-lasting antibacterial performance tests were conducted on Example 1, Comparative Example 1, and Comparative Example 4. 200 mg of disinfectant samples from each of Example 1, Comparative Example 1, and Comparative Example 4 were taken and evenly coated onto sterilized filter paper discs of each group. Another group of sterilized filter paper discs without disinfectant coating was taken as a blank control. The drug-loaded filter paper discs were placed in a constant temperature and humidity chamber (35℃, RH75%) to simulate the breeding environment.

[0041] At designated time points (4h, 8h, 12h, 24h, 72h, 144h, 168h), drug-loaded filter paper discs were removed and directly placed on agar plates inoculated with indicator bacteria (Staphylococcus aureus). The agar plates were then placed in a bacterial incubator and incubated for 16h. The number of colonies grown was counted and compared with a blank control to calculate the inhibition rate at each time point. The test results are as follows: Figure 1 As shown.

[0042] from Figure 1 It can be seen that Example 1 can maintain a high level of antibacterial effect of more than 80% within the set time period; the antibacterial effect of Comparative Example 1 is strong in the initial stage, but gradually decreases after 12 hours; while the antibacterial ability of Comparative Example 4 reaches its peak at 4 hours, and the antibacterial ability subsequently shows a linear decreasing trend over time, with the lowest antibacterial ability among the three at 168 hours.

[0043] Comparative Example 1 used sodium metabisulfite instead of ascorbic acid. Sodium metabisulfite oxidizes much faster than ascorbic acid, causing the gel network to rapidly disintegrate, resulting in the leakage of antibacterial active ingredients and a loss of sustained antibacterial effect. In contrast, Example 1 used ascorbic acid, which, under the influence of ambient humidity and oxygen, continuously releases H+ through slow oxidation. + This process achieves gentle and controllable protonation degradation of the gel network from the surface inwards, causing the sodium alginate gel network to gradually dissociate, thereby achieving a stable and long-lasting release of antibacterial components.

[0044] 4. Environmental Adhesion Adhesion experiments were conducted using the disinfectants prepared in Example 1 and Comparative Example 2 to verify the enhancing effect of the xanthan gum component added in this invention on the adhesion and retention capacity in the aquaculture environment.

[0045] Cement sheets and PVC sheets were selected as test boards. The surface of the cement board was sanded smooth, rinsed with water, and then dried for later use. The PVC board was disinfected by wiping with alcohol, rinsed with water, and then dried for later use.

[0046] Cement sheets and PVC sheets were placed in a constant temperature and humidity chamber (25℃, RH 75%) for 2 hours to form a uniform wet film on the surface of the test plates. 5g of each sample powder was weighed and placed in a 0.5mm standard sieve. The test plate was placed at a fixed angle of 45° below the sieve to evenly distribute the powder on the test plate. The total mass of the test plate and the loaded sample was accurately weighed and recorded as M0 (g). The test plate with the loaded sample was placed in a constant temperature and humidity chamber (25℃, RH 75%) and allowed to stand for 30 minutes. Remove the test plate and fix it at a 45° angle. Using a constant pressure spray bottle, spray deionized water evenly at a vertical angle of 20cm from the test plate for 30s. Then, place the test plate flat in a drying oven and dry for 12 hours. Accurately weigh the total mass of the test plate and the residual sample, and record it as M1 (g). Calculate the adhesion residue rate according to the following formula: Adhesion residue rate (%) = [1-(M0-M1) / 5]*100%. Set up 3 parallel samples for each group, and repeat the experiment twice. Calculate the average as the final result. The experimental results are shown in Table 4. Table 4 Results of environmental adhesion test

[0047] As can be seen from Table 4, the adhesion of the sample in Example 1 to cement sheets and PVC sheets was stronger than that in Comparative Example 2, indicating that xanthan gum is the key component of sodium alginate-xanthan gum composite gel, which can enhance the adhesion of disinfectant in the humid environment of the farm. After contacting the humid surface, xanthan gum can quickly hydrate and swell to form a highly viscous adhesive layer, which enhances the interfacial bonding strength between the disinfectant and the substrate. The adhesive effect of xanthan gum has wide applicability to the complex and diverse environmental surfaces of the farm.

[0048] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a disinfectant for animal husbandry environments, characterized in that, Includes the following steps: S1 Add ascorbic acid to deionized water and stir until dissolved at 5-15°C under light-protected conditions to obtain an ascorbic acid solution; S2 Add ascorbic acid solution to sodium alginate-xanthan gum composite solution and stir for 15-30 minutes to obtain drug loading solution; S3. Add the highly active antibacterial fermentation broth to the drug-carrying solution to obtain a mixture; add the mixture dropwise to the calcium chloride solution, and after the addition is complete, let it stand for 15-30 minutes, filter, wash the precipitate with deionized water, and then vacuum dry or freeze dry to obtain an animal breeding environment disinfectant.

2. The method for preparing an animal husbandry environment disinfectant according to claim 1, characterized in that, The preparation method of the highly active antibacterial fermentation broth is as follows: Bacillus subtilis is inoculated into LB medium and cultured at 37°C with shaking until OD. 600 The OD value is 0.6–0.

8. After centrifugation, the supernatant is discarded, and the precipitate is resuspended in a culture medium containing the compound Chinese medicine extract until the OD value is reached. 600 The concentration was 2.0–5.

0. The mixture was cultured at 22–30℃ with shaking for 12–24 h, centrifuged, and the supernatant was collected. The supernatant was then concentrated under reduced pressure to 1 / 5 of its volume to obtain a highly active antibacterial fermentation broth.

3. The method for preparing an animal husbandry environment disinfectant according to claim 2, characterized in that, The preparation method of the culture medium containing the compound traditional Chinese medicine extract is as follows: weigh hawthorn, codonopsis and astragalus, pulverize and sieve them, mix them evenly to obtain a mixed powder; add the mixed powder to deionized water at a mass ratio of 1:10-15, heat to 90-100℃, reflux and extract 2-3 times, each time for 1-1.5 hours; filter, concentrate the filtrate under reduced pressure to 1 / 20 of the filtrate volume, filter to remove bacteria, and obtain the compound traditional Chinese medicine extract; mix the compound traditional Chinese medicine extract and sterile LB medium at a volume ratio of 0.5-1:10 to obtain the culture medium containing the compound traditional Chinese medicine extract.

4. The method for preparing an animal husbandry environment disinfectant according to claim 3, characterized in that, The mass ratio of hawthorn, codonopsis and astragalus is 1:2-3:2-3.

5. The method for preparing an animal husbandry environment disinfectant according to claim 1, characterized in that, The preparation method of the sodium alginate-xanthan gum composite solution is as follows: Sodium alginate powder is dissolved in deionized water to prepare a sodium alginate solution with a mass concentration of 2% to 3%; xanthan gum powder is dissolved in deionized water at 50 to 65°C to prepare a xanthan gum solution with a mass concentration of 0.5% to 1%; the sodium alginate solution and the xanthan gum solution are mixed and stirred evenly to obtain the sodium alginate-xanthan gum composite solution.

6. The method for preparing an animal husbandry environment disinfectant according to claim 5, characterized in that, The volume ratio of the sodium alginate solution to the xanthan gum solution is 3 to 6:

1.

7. The method for preparing an animal husbandry environment disinfectant according to claim 1, characterized in that, In step S1, the mass ratio of ascorbic acid to deionized water is 1:15 to 25.

8. The method for preparing an animal husbandry environment disinfectant according to claim 1, characterized in that, In step S2, the volume ratio of the ascorbic acid solution and the sodium alginate-xanthan gum composite solution is 1:8 to 12.

9. The method for preparing an animal husbandry environment disinfectant according to claim 1, characterized in that, In step S3, the volume ratio of the highly active antibacterial fermentation broth to the drug-loaded solution is 1:4 to 6; the concentration of the calcium chloride solution is 2% to 4%.

10. The animal breeding environment disinfectant prepared by the method of any one of claims 1 to 9.

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