Agrochemical-free multifunctional environment purification spray harmless to pets and preparation method of agricultural-chemical-free multifunctional environment purification spray

Abstract

The invention relates to the technical field of environment purification, in particular to agricultural-chemical-free multifunctional environment purification spray harmless to pets and a preparation method of the agricultural-chemical-free multifunctional environment purification spray. According to the invention, the ratio of a wormwood culture solution to a bacillus subtilis culture solution to an aspergillus oryzae culture solution is 2: 1: 1; the wormwood culture solution is obtained by distillation extraction at 100-105 DEG C and contains 1-3% of glucose and 0.5-1.5% of a yeast extract; the bacillus subtilis culture solution and the aspergillus oryzae culture solution are respectively prepared by culturing food-grade safe strains in respective special culture mediums until the activity peak value is reached; finally, the mixed liquid is sterilized for 20 min at the temperature of 121 DEG C and the pressure of 0.12 MPa, sterile filling is conducted, the purifying power lasts for 12 months, and the pet cleaning agent is specially designed for pet families.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of environmental purification, in particular to a multifunctional environmental purification spray harmless to pets without pesticide and a preparation method thereof. BACKGROUND

[0002] The multifunctional environmental purification spray harmless to pets without pesticide and the preparation method thereof are a technical solution based on natural raw materials and combined with microbial synergistic effect, aiming to purify the environment while ensuring the safety of pets.

[0003] Traditional environmental purification products have many shortcomings: on the one hand, many chemical purification sprays contain pesticide residues, formaldehyde releasing agents, irritating disinfectants and other components, although they can achieve bactericidal or deodorizing effect in a short period of time, these components are easy to remain in the environment, and pets may cause skin allergy, respiratory irritation and even chronic poisoning after contacting, especially for pets that often lick their hair or move close to the ground, the potential harm is greater; on the other hand, some purification products with natural ingredients only rely on single plant extracts, which have limited purification effect and short duration, and cannot effectively deal with complex pathogenic bacteria (such as E. coli and Staphylococcus aureus) and odors (such as ammonia and hydrogen sulfide) in the home environment, and the preparation process often uses simple soaking extraction, resulting in low utilization rate of effective components, which further weakens the purification efficiency. In addition, traditional products often have single strain selection, lack of synergistic effect, and cannot fully decompose various pollutants in the environment, which cannot meet the dual needs of safety and high efficiency purification in pet families.

[0004] Therefore, the present application provides a multifunctional environmental purification spray harmless to pets without pesticide and a preparation method thereof, by selecting wormwood without pesticide residues as a natural raw material, using distillation extraction process to fully obtain its effective components, and combining with the synergistic culture of bacillus subtilis and aspergillus oryzae, using various enzymes produced by the strains to enhance the decomposition ability of pathogenic bacteria and odors, and by optimizing the raw material ratio (wormwood culture solution: bacillus subtilis culture solution: aspergillus oryzae culture solution = 2:1:1) and process parameters (such as 121℃ high pressure sterilization and 200-300r / min sufficient stirring), the product has multiple effects of no pesticide residues, no irritation to pet skin, high efficiency of sterilization (E. coli sterilization rate of 99.2%, Staphylococcus aureus sterilization rate of 98.8%) and strong deodorization (ammonia removal rate of 95.5%), effectively solving the problem that traditional products cannot balance safety and purification efficiency. SUMMARY

[0005] The technical problem solved by the present application is that the existing environmental purification products cannot balance safety and efficiency in pet family applications.

[0006] In view of the deficiencies of the prior art, the present application provides a multifunctional environment purification spray harmless to pets without agricultural chemicals, and a preparation method thereof, thereby solving the technical problems mentioned in the background art.

[0007] To achieve the above object, the present application is implemented by the following technical solutions: A multifunctional environment purification spray harmless to pets without agricultural chemicals is prepared by mixing the following culture solutions in a volume ratio: Artemisia argyi culture solution: Bacillus subtilis culture solution: Aspergillus oryzae culture solution = 2:1:1; The Artemisia argyi culture solution is obtained by distillation extraction at 100-105 DEG C and contains 1-3% glucose and 0.5-1.5% yeast extract; The Bacillus subtilis culture solution and the Aspergillus oryzae culture solution are respectively prepared by culturing food-grade safe strains in their respective special culture media to the active peak value; The final mixed solution is sterilized at 121 DEG C and 0.12 MPa for 20 min, and then aseptically filled.

[0008] Further, after mixing the three culture solutions, stirring is carried out at 200-300 r / min for 10-15 min, and then precision filtration is carried out at 0.2-0.3 MPa, and finally aseptic filling is carried out.

[0009] Further, the sterilization rate of Escherichia coli is ≥99%, the sterilization rate of Staphylococcus aureus is ≥98%, the removal rate of ammonia gas is ≥95%, and the removal rate of hydrogen sulfide is ≥94%.

[0010] Further, the steps include the following steps in sequence: S1: raw material preparation and pretreatment S1.1: preparation of Artemisia argyi culture solution Fresh, pest-free, and agricultural chemical residue-free Artemisia argyi is crushed to 1-2 cm small pieces, distilled water is added at a solid-liquid ratio of 1:5-1:10, and distillation is carried out at 100-105 DEG C for 2-4 h; after collecting the distillate, glucose and yeast extract are added, and stirring is carried out at 150 r / min for 8-12 min until complete dissolution; S1.2: preparation of Bacillus subtilis culture solution Activated Bacillus subtilis is inoculated into a culture medium containing 8-12 g / L proteose peptone, 3-7 g / L beef extract, 3-7 g / L sodium chloride, and 15-25 g / L agar at an inoculation amount of 0.5 mL / 100 mL, and the pH is 7.0-7.2; the culture is incubated at 37 DEG C for 24-48 h until a milky white bacterial film is formed on the surface; S1.3: preparation of Aspergillus oryzae culture solution Inoculate Aspergillus oryzae spores into solid-state culture medium containing bran 60-100 g, sucrose 10-30 g, sodium nitrate 2-4 g, potassium dihydrogen phosphate 0.5-1.5 g, magnesium sulfate 0.3-0.7 g, potassium chloride 0.3-0.7 g, and water content 60-65%, and cultivate at 30°C for 48-72 h until the yellow-green spores grow fully, then add sterile water to stir and filter to obtain a spore-containing liquid; S2: Mix the three culture solutions in a volume ratio of 2:1:1, and stir at 200-300 r / min for 10-15 min; S3: After the mixed solution is filtered through a 0.2-0.3 MPa plate and frame, collect the filtrate into a sterile storage tank; S4: Sterilize at 121°C for 20 min, aseptically fill, seal, label, and box to obtain the finished product Further, all culture media and filling equipment are sterilized at 121°C and 0.12 MPa for 20 min to ensure no contamination by miscellaneous bacteria.

[0011] Further, the filtrate has a transmittance of ≥90% in the filtration process, and the filter cloth is replaced every 50 L to ensure the clarity and purity of the product.

[0012] Compared with the prior art, the beneficial effects are: In the present scheme, by scientifically selecting natural raw materials and optimizing the process design, the synergistic unity of the non-agricultural characteristics of environmental purification spray, the safety of pets, and the high-efficiency purification function is achieved. From the selection of raw materials, the method takes fresh and disease-free wormwood as the core natural ingredient, which has no agricultural residues and is harmless to pets. The effective components (such as volatile oil) extracted by distillation have natural bactericidal and odor inhibiting effects. At the same time, the selected Bacillus subtilis and Aspergillus oryzae are both safe strains for pets, and they can produce various enzymes to decompose chemical residues and foul odors in the environment, avoiding the potential harm of agricultural ingredients or harmful chemicals in traditional chemical purifiers to pets. In the process design, wormwood is extracted by distillation (all of Examples 1-3 use this method), which can more fully obtain effective components compared to direct water immersion (Comparative Example 1), ensuring the purification foundation; the two strains of Bacillus subtilis and Aspergillus oryzae are synergistically cultured, which form a complementary effect, and their culture solutions are mixed in a ratio of 2:1:1 (Examples 1-3 have a fixed ratio), after sufficient stirring (200-300 r / min, 10-15 min), the strain activity and enzyme decomposition ability are maximized, which can efficiently decompose pathogenic bacteria such as Escherichia coli and Staphylococcus aureus (Example 1 has a sterilization rate of 99.2% and 98.8%) and odor gases such as ammonia and hydrogen sulfide (ammonia removal rate 95.5%). In addition, the whole preparation process does not use any pesticide, chemical preservative or stimulating additive, and is ensured to be free of bacterial contamination by 121℃ high pressure sterilization for 20 minutes (all of Examples 1-3 adopt), and the final product is verified to have 0 irritation score on rabbit skin, and fully meets the requirement of being harmless to pets. This design not only retains the purification advantage of natural ingredients, but also enhances the functional effect through microbial synergistic effect, solves the problems of pesticide residue, toxicity to pets or insufficient purification effect of traditional chemical purification products, and realizes the dual protection of safety and efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0013] The above description is only a summary of the technical solutions of the present application. In order to more clearly understand the technical means of the present application, and to implement the content of the description, the following will be described in detail with reference to the preferred embodiments of the present application and in conjunction with the drawings.

[0014] Figure 1 The overall method step flow chart of the present application. DETAILED DESCRIPTION

[0015] The preferred embodiments of the present application will be described in detail with reference to the accompanying drawings, but the present application can be realized in various different forms, and therefore the present application is not limited to the embodiments described below; The technical solutions in the embodiments of the present application are to solve the problems in the above background art, and the general idea is as follows: Please refer to Figure 1 As shown in the figure, a preparation method of a multifunctional environmental purification spray harmless to pets without pesticide is introduced, including the following steps: I. Selection of raw materials: Artemisia argyi: fresh, free of pests and diseases, natural and free of pesticide residues, harmless to pets, which can avoid the risk of pet allergy caused by chemical agents on the one hand, and contains volatile oil, which can kill and inhibit bacteria, and adsorb odors; and artemisia argyi is widely distributed and easy to obtain, and grows without pesticides, which is suitable for large-scale production and provides a safe and efficient basis for the purification spray; Strains: Bacillus subtilis and Aspergillus oryzae; Bacillus subtilis can secrete protease, lipase and other enzymes, which can efficiently decompose protein pollutants in the environment (such as nitrogen-containing organic matter in pet excrement), thereby reducing the generation of odors from the source; at the same time, its metabolic products have broad-spectrum antibacterial properties, which can enhance the inhibition ability to pathogenic bacteria; Aspergillus oryzae is good at secreting amylase, cellulase and other enzymes, which can decompose carbohydrate impurities and part of organic odor molecules in the environment, and form a functional complement with Bacillus subtilis, which significantly improves the decomposition efficiency of complex pollutants, and solves the problem of limited purification capacity of single strain; Both are food-grade safe strains, widely used in fermented food field, their metabolites are non-toxic, even if the pet contact or small intake will not cause health problems, in sharp contrast to the irritating components of traditional chemical disinfectants; Culture medium ingredients: Bacillus subtilis culture medium: peptone (8-12 g), beef extract (3-7 g), sodium chloride (3-7 g), agar (15-25 g); peptone and beef extract provide nitrogen source, carbon source and growth factors for the strain, meet the nutritional needs of Bacillus subtilis, promote its mass propagation and secrete active enzymes; sodium chloride maintains the osmotic pressure balance of the culture medium, ensures the activity of the strain; agar as a coagulant provides a stable growth carrier for the strain, ensures uniform propagation of the strain during culture; Aspergillus oryzae culture medium: bran (60-100 g), sucrose (10-30 g), sodium nitrate (2-4 g), potassium dihydrogen phosphate (0.5-1.5 g), magnesium sulfate (0.3-0.7 g), potassium chloride (0.3-0.7 g); bran is rich in cellulose and vitamins, sucrose provides carbon source, sodium nitrate provides nitrogen source, together meet the nutritional needs of Aspergillus oryzae, promote its enzyme production capacity; inorganic salts such as potassium dihydrogen phosphate, magnesium sulfate and potassium chloride adjust the pH of the culture medium, maintain the metabolic balance of the strain and enhance the activity of the enzyme; Artemisia argyi culture solution additives: glucose (1-3%), yeast extract (0.5-1.5%); glucose provides a stable environment for beneficial components in artemisia distillation solution (such as volatile oil dissolved substances), and supplements carbon source for microbial activity when mixed with subsequent strain culture solution; yeast extract is rich in amino acids and vitamins, which can enhance the nutritional compatibility of artemisia culture solution, promote the synergistic effect of double strains after mixing, and avoid antagonistic reaction between components; II. Treatment of raw materials: 1. Artemisia argyi treatment Pretreatment: After picking fresh artemisia argyi, remove rotten leaves, dry stems and attached soil, weeds and other impurities by manual screening; then rinse with flowing water for 3-4 times until no visible stains on the surface, and finally dry to remove excess surface water to avoid mold caused by residual raw water; Grinding and distillation: grind the pretreated artemisia argyi to 1-2 cm pieces with a high-speed grinder (speed 3000 r / min), add distilled water at a solid-liquid ratio of 1:5-10, mix evenly, then transfer to a SZZ-550 type distillation device; set the distillation temperature to 100-105℃, the distillation time to 2-4 hours, collect the distillation liquid every 30 minutes, combine and avoid light for 2 hours, then remove the upper floating scum; 2. Culture medium treatment Bacillus subtilis culture medium: protein peptone (8-12 g), beef extract (3-7 g), sodium chloride (3-7 g), agar (15-25 g) were weighed according to the proportion, and distilled water was added according to the solid-liquid ratio of 1:30 and stirred until completely dissolved, and the pH was adjusted to 7.0-7.2 with 1 mol / L sodium hydroxide solution; after adding agar, heat and boil to melt the agar, and divide into 250 mL triangular bottles (100 mL per bottle), and seal the bottle opening with a breathable cotton plug; Aspergillus oryzae culture medium: bran (60-100 g), sucrose (10-30 g), sodium nitrate (2-4 g), potassium dihydrogen phosphate (0.5-1.5 g), magnesium sulfate (0.3-0.7 g), potassium chloride (0.3-0.7 g) were weighed according to the proportion, and the bran was passed through an 80-mesh screen to remove coarse residue, and mixed with sucrose, sodium nitrate, potassium dihydrogen phosphate, magnesium sulfate and potassium chloride, and distilled water was added in a mass ratio of 1:2 and stirred until the water content was 60-65% (hand into a group without dripping water), and divided into 500 mL wide-mouth bottles (150 g per bottle), and the bottle opening was covered with double-layer gauze; 3. Sterilization treatment All the divided culture media were placed in a YXQ-LS-50SII type high-pressure steam sterilization pot, set to a pressure of 0.12 MPa and a temperature of 121℃, and sterilized for 20 minutes; after sterilization, the Bacillus subtilis culture medium was cooled to about 45℃ in a sterile operation table, and the Aspergillus oryzae culture medium was cooled to 30℃ to avoid excessive temperature affecting the subsequent strain inoculation activity; 4. Additive treatment Glucose and yeast extract were accurately weighed with an electronic balance (error ≤0.1 g) respectively, glucose was dried at 80℃ for 2 hours to remove crystallization water, and yeast extract was passed through a 100-mesh screen to remove impurities; both were added to the wormwood distillate according to the proportion, and then stirred with a JJ-1 type stirrer at 150 r / min for 8-12 minutes to ensure complete dissolution and form a uniform and stable wormwood culture solution; III. Selection of equipment: Distillation device: SZZ-550 type distillation device, used for wormwood distillation to extract distillate; Stirrer: JJ-1 type stirrer, used for dissolution of glucose and yeast extract in wormwood culture solution; JBJ-50 type liquid stirrer, used for mixing of three kinds of culture solution; Sterilization equipment: YXQ-LS-50SII type high-pressure steam sterilization pot, used for sterilization of culture medium; Culture equipment: SPX-250B-Z type constant temperature incubator, used for culture of Bacillus subtilis and Aspergillus oryzae; Filtering equipment: BMY3 / 450-30U type plate and frame filter, used for filtering of mixed solution to remove impurities; Filling equipment: YG-100 type liquid filling machine, used for filling of filtered purified spray; Four, preparation steps: S1: raw material preparation and pretreatment S1.1: wormwood culture solution preparation The pretreated and crushed wormwood (1-2 cm pieces) was added to distilled water at a solid-liquid ratio of 1:5-10, poured into a SZZ-550 type distillation device, sealed and heated, and maintained at 100-105°C for 2-4 hours. Distilled liquid was collected every 30 minutes, combined and filtered with double-layer filter paper to remove fine residues. To the filtered wormwood distillation liquid, dried and sieved glucose (1-3%) and yeast extract (0.5-1.5%) were added in proportion, and placed on a JJ-1 type stirrer to stir at 150 r / min for 8-12 minutes until completely dissolved to obtain a uniform wormwood culture solution, which was stored in the dark for later use. S1.2: strain culture Bacillus subtilis culture: In a sterile operation table, 0.5 mL of activated Bacillus subtilis liquid was taken with a pipette and inoculated into Bacillus subtilis culture medium cooled to 45°C (1 inoculum per 100 mL of culture medium), shaken gently, and then placed in a SPX-250B-Z type constant temperature incubator for 24-48 hours of static culture at 37°C. A white bacterial film was formed on the surface of the culture medium, and the Bacillus subtilis culture solution was obtained. Aspergillus oryzae culture: In a sterile environment, a small amount of Aspergillus oryzae spores was taken with an inoculation loop and evenly spread on the surface of Aspergillus oryzae culture medium cooled to 30°C. It was placed in a constant temperature incubator at 30°C for 48-72 hours of culture until the surface of the culture medium was covered with yellow-green spores. 100 mL of sterile water was added and stirred, and the spore-containing bacterial liquid was collected by filtration, which was the Aspergillus oryzae culture solution. S2: mixing and homogenization The wormwood culture solution, Bacillus subtilis culture solution and Aspergillus oryzae culture solution were poured into the sterile stirring barrel of a JBJ-50 type liquid stirrer in the order of 2:1:1 volume ratio. After the barrel cover was tightly closed, the stirring parameters were set as follows: speed 200-300 r / min, stirring time 10-15 minutes, to ensure that the three liquids were fully mixed to form a uniform mixture. During the stirring process, samples were taken every 5 minutes for observation. If stratification or precipitation occurred, the stirring time was extended by 2-3 minutes until the mixture was clear and yellow with no obvious particles. S3: precision filtration A BMY3 / 450-30U type plate and frame filter was opened, and the filter cloth and filter channel were sterilized with 75% alcohol for 30 minutes, then rinsed with sterile water until no alcohol residue was left. The mixture was slowly injected into the filter through the feed pump, the filtration pressure was controlled at 0.2-0.3 MPa, the flow rate was 10-15 L / h, and the filtrate was collected in a sterile storage tank. Check the filter cloth every 50L of mixed solution, if the filtrate is turbid (transmittance <90%), stop immediately and replace the filter cloth to avoid impurities affecting the purity of the product; S4: aseptic filling and packaging Before filling, sterilize the pipeline and needle of YG-100 type liquid filling machine with 121℃ high pressure steam for 20 minutes, and then connect the aseptic storage tank after cooling; Set the filling parameters: capacity 500mL / bottle, filling speed 30 bottles / minute, open the automatic filling mode, and randomly take 1 bottle for weighing every 100 bottles, the error needs to be controlled within ±5mL; After filling, immediately seal the bottle opening with a sterile capping machine, and then put it into a sterilized carton after labeling, and store it in a cool and dry place (temperature 15-25℃, relative humidity <60%) for 12 months.

[0016] Example 1: This example introduces a preparation method of a multifunctional environmental purification spray harmless to pets without agricultural chemicals, which comprises the following steps: S1: treatment of raw materials S1.1: treatment of wormwood: Pretreatment: manually remove rotten leaves, dry stems and impurities, rinse with flowing water for 3-4 times, and dry to remove excess surface water; Pulverization and distillation: pulverize to 1-2cm small pieces with a high-speed pulverizer (3000r / min), add distilled water according to a solid-liquid ratio of 1:8, transfer into a SZZ-550 type distillation device, and distill at 100-105℃ for 3 hours, collect the distillation liquid every 30 minutes, combine, and then avoid light and stand for 2 hours to remove the upper floating scum; S1.2: treatment of culture medium: Bacillus subtilis culture medium: weigh 10g of proteose peptone, 5g of beef extract, 5g of sodium chloride, and 20g of agar, add distilled water according to a solid-liquid ratio of 1:30, stir until completely dissolved, adjust pH to 7.0-7.2 with 1mol / L sodium hydroxide, heat and boil to melt the agar, divide into 250mL triangular flasks (100mL per flask), and seal with a breathable cotton plug; Aspergillus oryzae culture medium: mix 80g of bran (passed through an 80 mesh sieve), 20g of sucrose, 3g of sodium nitrate, 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, and 0.5g of potassium chloride, add distilled water according to a mass ratio of 1:2, stir until the water content is 60-65%, divide into 500mL wide-mouth bottles (150g per bottle), and cover with double-layer gauze; S1.3: sterilization treatment: Put all the culture media into a YXQ-LS-50SII type high pressure steam sterilization pot, sterilize at 0.12MPa and 121℃ for 20 minutes; cool the Bacillus subtilis culture medium to 45℃, and cool the Aspergillus oryzae culture medium to 30℃; S1.4: Additive treatment: Glucose was dried at 80℃ for 2 hours, and yeast extract was sieved through a 100-mesh sieve; the artemisia distillate was added in a ratio of 2% glucose and 1% yeast extract, and a JJ-1 type stirrer was used to stir at 150 r / min for 10 minutes until complete dissolution, forming an artemisia culture solution, which was stored in the dark; S2: Strain culture S2.1: Bacillus subtilis culture: In a sterile operation table, 0.5 mL of activated bacterial solution was taken with a pipette gun and inoculated into 100 mL of Bacillus subtilis culture medium, which was gently shaken and then placed in an SPX-250B-Z type constant temperature incubator for 37℃ static culture for 36 hours until a milky white bacterial film was formed on the surface of the culture medium, and the Bacillus subtilis culture solution was obtained; S2.2: Aspergillus oryzae culture: Under sterile conditions, a small amount of Aspergillus oryzae spores was taken with an inoculation loop and evenly spread on the surface of the Aspergillus oryzae culture medium, which was placed in a constant temperature incubator for 30℃ culture for 60 hours until the surface was covered with yellow-green spores; 100 mL of sterile water was added and stirred, and the spore-containing bacterial solution was collected by filtration, which was the Aspergillus oryzae culture solution; S3: Mixing and homogenization The artemisia culture solution, Bacillus subtilis culture solution, and Aspergillus oryzae culture solution were poured into the sterile stirring barrel of a JBJ-50 type liquid stirrer in a volume ratio of 2:1:1, the barrel cover was tightly closed, and the stirring speed was set to 250 r / min for 12 minutes; during this period, samples were taken every 5 minutes for observation until the mixed solution was clear and light yellow with no obvious particles, and the stirring was stopped; S4: Filtration and filling S4.1: Precision filtration: A BMY3 / 450-30U type plate and frame filter was opened, and the filter cloth and channel were first sterilized with 75% alcohol for 30 minutes, and then washed with sterile water until no alcohol residue was left; The mixed solution was injected into the filter through the feed pump, and the pressure was controlled at 0.2-0.3 MPa and the flow rate was 10-15 L / h, and the filtrate was collected in a sterile storage tank; every 50 L of filtration was checked for filter cloth, and if the transmittance of the filtrate was <90%, the filter cloth was immediately replaced; S4.2: Aseptic filling and packaging: Before filling, the pipeline and needle of a YG-100 type liquid filling machine were sterilized with 121℃ high-pressure steam for 20 minutes, and then connected to the sterile storage tank after cooling; The parameters were set as follows: 500 mL / bottle, filling speed 30 bottles / minute, automatic filling was started; every 100 bottles were randomly inspected, and the error was controlled within ±5 mL; After filling, the bottles were immediately sealed with a sterile capping machine, labeled, and then placed in a sterilized carton, stored in an environment with a temperature of 15-25℃ and a relative humidity of <60%, with a shelf life of 12 months.

[0017] Example 2: This embodiment introduces a preparation method of a multifunctional environmental purification spray harmless to pets without agricultural chemicalization, comprising the following steps: S1: treatment of raw materials S1.1: wormwood treatment: pretreatment: same as example 1; crushing and distillation: crushed to 1-2 cm small pieces by high-speed crusher (3000 r / min), added with distilled water according to solid-liquid ratio 1:5, transferred into SZZ-550 type distillation device, distilled at 100-105℃ for 2 hours, collected every 30 minutes, combined and placed in dark for 2 hours to remove the upper floating scum; S1.2: treatment of culture medium: Bacillus subtilis culture medium: weigh 8 g of proteose peptone, 3 g of beef extract, 3 g of sodium chloride and 15 g of agar, add distilled water according to solid-liquid ratio 1:30, stir until completely dissolved, adjust pH to 7.0-7.2 with 1 mol / L sodium hydroxide, heat and boil to melt agar, divide into 250 mL triangular bottles (100 mL per bottle), seal with air-permeable cotton plug; Aspergillus oryzae culture medium: bran 60 g passed through 80 mesh sieve, mixed with sucrose 10 g, sodium nitrate 2 g, potassium dihydrogen phosphate 0.5 g, magnesium sulfate 0.3 g, potassium chloride 0.3 g, added with distilled water according to mass ratio 1:2, stirred until water content is 60-65%, divided into 500 mL wide-mouth bottles (150 g per bottle), covered with double-layer gauze; S1.3: sterilization treatment: same as example 1; S1.4: additive treatment: glucose was dried at 80℃ for 2 hours, and yeast extract was passed through 100 mesh sieve; wormwood distillate was added according to the proportion of 1% glucose and 0.5% yeast extract, stirred by JJ-1 type stirrer at 150 r / min for 8 minutes until completely dissolved, to form wormwood culture solution, which was stored in dark; S2: strain culture S2.1: Bacillus subtilis culture: In a sterile operation table, 0.5 mL of activated bacterial solution was taken by a pipette gun and inoculated into 100 mL of Bacillus subtilis culture medium, which was gently shaken and then placed in a SPX-250B-Z type constant temperature incubator for static culture at 37℃ for 24 hours, until a milky white bacterial film was formed on the surface of the culture medium, to obtain Bacillus subtilis culture solution; S2.2: Aspergillus oryzae culture: Under sterile environment, a small amount of Aspergillus oryzae spores was taken by an inoculation loop and evenly coated on the surface of Aspergillus oryzae culture medium, which was placed in a constant temperature incubator for culture at 30℃ for 48 hours, until the surface was covered with yellow-green spores; 100 mL of sterile water was added and stirred, and the bacterial solution containing spores was collected by filtration, to obtain Aspergillus oryzae culture solution; S3: Mixing and homogenization The wormwood culture solution, bacillus subtilis culture solution, and aspergillus oryzae culture solution were poured into a sterile stirring barrel of a JBJ-50 liquid stirrer in a volume ratio of 2:1:1, the barrel cover was tightly covered, the stirring speed was set to 200 r / min, and stirring was performed for 10 minutes; during the stirring, samples were taken every 5 minutes for observation until the mixed solution was clear and light yellow without obvious particles, and the stirring was stopped; S4: Filtration and filling S4.1: Precision filtration: same as in Example 1; S4.2: Sterile filling and packaging: same as in Example 1.

[0018] Example 3: This example introduces a preparation method of a multifunctional environmental purification spray harmless to pets without agricultural chemical treatment, which comprises the following steps: S1: Treatment of raw materials S1.1: Wormwood treatment: Pre-treatment: same as in Example 1; Crushing and distillation: crushed to 1-2 cm small pieces by a high-speed crusher (3000 r / min), distilled water was added at a solid-liquid ratio of 1:10, transferred into a SZZ-550 type distillation device, and distilled at 100-105°C for 4 hours; the distilled liquid was collected every 30 minutes, combined, and placed in the dark for 2 hours to remove the upper floating scum; S1.2: Treatment of culture medium: Bacillus subtilis culture medium: 12 g of proteose peptone, 7 g of beef extract, 7 g of sodium chloride, and 25 g of agar were weighed, distilled water was added at a solid-liquid ratio of 1:30, and stirred until completely dissolved; 1 mol / L sodium hydroxide was used to adjust the pH to 7.0-7.2, and the agar was melted by heating and boiling; 250 mL triangular bottles (100 mL per bottle) were used for sub-packaging, and the bottles were sealed with air-permeable cotton plugs; Aspergillus oryzae culture medium: bran 100 g was sieved through an 80 mesh sieve, mixed with sucrose 30 g, sodium nitrate 4 g, potassium dihydrogen phosphate 1.5 g, magnesium sulfate 0.7 g, and potassium chloride 0.7 g, distilled water was added at a mass ratio of 1:2, and stirred until the water content was 60-65%; 500 mL wide-mouth bottles (150 g per bottle) were used for sub-packaging, and the bottles were covered with double-layer gauze; S1.3: Sterilization treatment: same as in Example 1; S1.4: Additive treatment: Glucose was dried at 80°C for 2 hours, and yeast extract was sieved through a 100 mesh sieve; the wormwood distillate was added with 3% glucose and 1.5% yeast extract, a JJ-1 stirrer was used for stirring at 150 r / min for 12 minutes until complete dissolution, and the wormwood culture solution was formed and stored in the dark; S2: Strain culture S2.1: Bacillus subtilis culture: In the sterile operation platform, 0.5 mL of activated bacteria solution was taken by a pipette gun and inoculated into 100 mL of Bacillus subtilis culture medium. After being shaken gently, it was placed in a SPX-250B-Z type constant temperature incubator and cultured at 37°C for 48 hours until a milky white bacterial film was formed on the surface of the culture medium. Thus, the Bacillus subtilis culture solution was obtained. S2.2: Aspergillus oryzae culture: Under sterile conditions, a small amount of Aspergillus oryzae spores was taken with an inoculation loop and evenly coated on the surface of the Aspergillus oryzae culture medium. It was placed in a constant temperature incubator and cultured at 30°C for 72 hours until the surface was covered with yellow-green spores. Then 100 mL of sterile water was added and stirred. The spore-containing bacterial solution was collected by filtration, which was the Aspergillus oryzae culture solution. S3: Mixing and homogenization The wormwood culture solution, Bacillus subtilis culture solution and Aspergillus oryzae culture solution were poured into the sterile stirring barrel of a JBJ-50 type liquid stirrer in a volume ratio of 2:1:1. After the barrel cover was tightly closed, the stirring speed was set to 300 r / min and stirred for 15 minutes. During this period, samples were taken every 5 minutes for observation until the mixed solution was clear and light yellow with no obvious particles, and the stirring was stopped. S4: Filtration and filling S4.1: Precision filtration: same as Example 1; S4.2: Sterile filling and packaging: same as Example 1.

[0019] Comparative Example 1: (the extraction method of wormwood is replaced by water immersion) I. Variable design The distillation extraction process of wormwood was replaced by 60°C warm water immersion extraction to explore the influence of different extraction methods on the utilization rate of effective components of wormwood and the purification efficiency of the product. II. Preparation steps S1, treatment of raw materials S1.1: Wormwood treatment: Pretreatment: same as Example 1; Crushing and leaching: crushed to 1-2 cm small pieces by a high-speed crusher (3000 r / min), 60°C warm water was added at a solid-liquid ratio of 1:8, soaked for 2 hours, then filtered with double-layer filter paper, collected the leaching solution, avoid light and stand for 2 hours to remove the upper floating scum; S1.2: Culture medium treatment: same as Example 1; S1.3: Sterilization treatment: same as Example 1; S1.4: Additive treatment: same as Example 1 (wormwood leaching solution was added with 2% glucose and 1% yeast extract, stirred and dissolved); S2, strain culture: same as Example 1; S3, mixing and homogenization: same as Example 1; S4, filtration and filling: same as Example 1; III. Comparative significance By comparing with the distillation process of Example 1, the advantage of distillation method in extracting effective components such as artemisia vulgaris volatile oil is verified; the experimental results show that the water immersion method leads to a significant decrease in sterilization rate (E. coli 82.5%) and odor removal rate (ammonia 72.1%), proving that the distillation process is the key to ensuring the purification performance of the product.

[0020] Comparative Example 2: (single strain instead of double strain synergy) I. Variable design Only Bacillus subtilis single strain is used, and Aspergillus oryzae culture and addition are cancelled to explore the influence of double strain synergy on pollutant decomposition ability; II. Preparation steps S1, treatment of raw materials: same as Example 1 (artemisia vulgaris distillation, culture medium preparation and sterilization are unchanged); S2, strain culture S2.1: Bacillus subtilis culture: same as Example 1; S2.2: cancel the Aspergillus oryzae culture step; S3, mixing and homogenization Artemisia vulgaris culture solution and Bacillus subtilis culture solution are poured into a JBJ-50 type liquid mixer at a volume ratio of 2:1, stirred at 250 r / min for 12 minutes, and the mixed solution is clear and light yellow; S4, filtration and filling: same as Example 1; III. Comparison significance By comparing with the double strain system of Example 1, the functional complementarity of Aspergillus oryzae and Bacillus subtilis is verified; the experimental results show that the sterilization rate (E. coli 78.6%) and odor removal rate (ammonia 68.3%) of single strain group decrease by about 25%, proving that double strain synergy is the core to improve performance and safety.

[0021] Comparative Example 3: (mixed ratio adjustment to reduce artemisia vulgaris culture solution proportion) I. Variable design The mixed ratio of the three culture solutions is adjusted to artemisia vulgaris culture solution: Bacillus subtilis culture solution: Aspergillus oryzae culture solution = 1:1:2, and the influence of artemisia vulgaris culture solution proportion on the basic purification ability of the product is explored; II. Preparation steps S1, treatment of raw materials: same as Example 1; S2, strain culture: same as Example 1; S3, mixing and homogenization Artemisia vulgaris culture solution, Bacillus subtilis culture solution and Aspergillus oryzae culture solution are poured into a JBJ-50 type liquid mixer at a volume ratio of 1:1:2, stirred at 250 r / min for 12 minutes, and the mixed solution is uniform; S4, filtration and filling: same as Example 1; III. Comparative Significance By comparison with the 2:1:1 ratio of Example 1, the necessity of wormwood culture solution as the basic purification component is verified; the experiment shows that the decrease of wormwood proportion leads to about 15% decrease of sterilization rate (E. coli 85.3%) and odor removal rate (ammonia 78.5%), proving that the ratio of 2:1:1 can balance the synergistic effect of natural ingredients and microorganisms.

[0022] Comparative Example 4: (sterilization condition changed to low temperature and short time) I. Variable Design Adjust the sterilization condition of the culture medium to 110°C for 10 minutes to explore the influence of sterilization thoroughness on product safety and strain activity; II. Preparation Steps S1, Treatment of Raw Materials S1.1: Wormwood treatment: same as Example 1; S1.2: Culture medium treatment: same as Example 1; S1.3: Sterilization treatment All culture media were placed in a YXQ-LS-50SII type high-pressure steam sterilization pot and sterilized at 110°C for 10 minutes; the Bacillus subtilis culture medium was cooled to 45°C, and the Aspergillus oryzae culture medium was cooled to 30°C; S1.4: Additive treatment: same as Example 1; S2, Strain Cultivation: same as Example 1; S3, Mixing and Homogenization: same as Example 1; S4, Filtration and Filling: same as Example 1; III. Comparative Significance By comparison with the 121°C, 20-minute sterilization of Example 1, the importance of sterilization process for controlling miscellaneous bacteria is verified; the experiment shows that low-temperature and short-time sterilization leads to miscellaneous bacterial contamination, with about 40% decrease of sterilization rate (E. coli 65.2%) and odor removal rate (ammonia 55.6%), proving that strict sterilization is a prerequisite for product safety and effectiveness.

[0023] Comparative Example 5: (mixing and stirring condition changed to low speed and short time) I. Variable Design Adjust the mixing and stirring parameters to 100 r / min for 5 minutes to explore the influence of mixing thoroughness on the synergistic effect of ingredients; II. Preparation Steps S1, Treatment of Raw Materials: same as Example 1; S2, Strain Cultivation: same as Example 1; S3, Mixing and Homogenization Artemisia vulgaris culture solution, Bacillus subtilis culture solution, Aspergillus oryzae culture solution were poured into a JBJ-50 liquid stirrer at a volume ratio of 2:1:1, and stirred at 100 r / min for 5 minutes; S4, filtration and filling: same as example 1; III. Comparative significance By comparing with the 250 r / min, 12 minutes stirring of example 1, the influence of mixing process on the dispersion of ingredients was verified; the experiment showed that insufficient stirring led to about 10% decrease in sterilization rate (Escherichia coli 88.7%) and odor removal rate (ammonia 82.3%), proving that sufficient stirring is the key to ensure uniform action of natural ingredients and strain enzymes.

[0024] Experimental example: I. Experimental purpose To verify the differences in sterilization effect, odor removal ability and pet safety of the environmental purification sprays prepared in examples 1-3 and comparative examples 1-5, and to clarify the rationality of the optimal process parameters; II. Experimental materials Test samples: purification sprays prepared in examples 1, 2, 3 and comparative examples 1-5, 3 samples for each group; Experimental strains: Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC6538) (simulating environmental pathogenic bacteria); Odor source: 3% ammonia solution (simulating pet excrement odor), pet urine (dog urine, collected and stored at 4°C); Experimental animals: healthy adult beagle dogs (6, body weight 10-12 kg), healthy adult domestic cats (6, body weight 3-4 kg); III. Experimental method Sterilization effect test Plate counting method: 1 mL of sample was mixed with 9 mL of bacteria-containing liquid (10 6 CFU / mL), 37°C for 30 minutes, then 0.1 mL of the mixed solution was spread on nutrient agar plates, 37°C for 24 hours, and the number of colonies was counted to calculate the sterilization rate: Sterilization rate (%) = (control group colony count - experimental group colony count) / control group colony count x 100% Odor removal ability test Ammonia removal rate: 10 mL of 3% ammonia solution was added to a 10 L closed container, 10 mL of spray sample was sprayed, and after 2 hours, the residual concentration was measured with an ammonia gas detector to calculate the removal rate; Pet urine odor: 5 mL of dog urine was placed in a petri dish, 5 mL of spray sample was sprayed, and after 2 hours, 5 professional evaluators scored according to "0-5 points" (0=no odor, 5=strong odor), and the average value was taken; Pet safety test Skin irritation experiment: dilute the sample 10 times and apply it to the depilated area (2 cm x 2 cm) on the back of the dog / cat, once a day for 7 consecutive days, and observe whether there is erythema and edema on the skin; Acute oral toxicity: gavage the sample at 10 mL / kg body weight, and observe the behavior and health status of the animals within 7 days; IV. Experimental results (average ± standard deviation)

[0025] V. Result analysis Bactericidal and purification efficiency: The bactericidal rate and odor removal rate of examples 1-3 are significantly higher than those of the comparative examples (P<0.05), and the performance of example 1 is the best, which shows that the intermediate process parameters can balance the utilization rate of raw materials and the performance; Comparative example 1 (water extraction) and comparative example 2 (single strain) have obvious performance decline due to insufficient active ingredients or lack of synergy; Pet safety: Examples 1-3 and comparative examples 1, 3 and 5 have no skin irritation, and comparative example 2 (single strain) and comparative example 4 (insufficient sterilization) have different degrees of irritation due to abnormal metabolites or contamination of miscellaneous bacteria, which verifies the necessity of double-strain system and strict sterilization; There is no death and no abnormal performance in acute oral toxicity, which is actually non-toxic.

[0026] Process rationality: The comprehensive performance of example 1 is better than that of example 2 (insufficient performance due to low parameters) and example 3 (parameters are too high but do not significantly improve the performance), which proves that the intermediate value parameters are the best choice, and the comparative examples further verify the key roles of distillation extraction, double-strain synergy, reasonable proportioning, strict sterilization and sufficient mixing; VI. Conclusion The environmental purification spray prepared by the preparation process of example 1 of the present application has the best performance in bactericidal effect, odor removal ability and pet safety, and the process parameters have scientificity and practicality.

[0027] Finally, it should be pointed out that: obviously, the above examples are only examples for clearly illustrating the present application, and are not limitations on the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, it is not necessary and impossible to exhaust all embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A multifunctional environmental purifying spray that is pesticide-free and harmless to pets, characterized in that, It is a mixture of culture media in the following volume ratios: Artemisia argyi culture medium; Bacillus subtilis culture medium; Aspergillus oryzae culture medium = 2:1:1; The Artemisia argyi culture medium was obtained by distillation at 100–105℃ and contained 1–3% glucose and 0.5–1.5% yeast extract. The Bacillus subtilis culture medium and Aspergillus oryzae culture medium were prepared by culturing food-grade safe strains in their respective dedicated culture media until the activity peak was reached. The final mixture is sterilized at 121℃ and 0.12 MPa for 20 minutes and then aseptically filled.

2. The purifying spray according to claim 1, characterized in that, After mixing the three culture media, stir at 200–300 r / min for 10–15 min, then filter through a precision filter at 0.2–0.3 MPa, and finally aseptically fill the containers.

3. The purifying spray according to any one of claims 1-2, characterized in that, It has a sterilization rate of ≥99% against Escherichia coli, a sterilization rate of ≥98% against Staphylococcus aureus, a removal rate of ≥95% for ammonia, and a removal rate of ≥94% for hydrogen sulfide.

4. A method for preparing a multifunctional environmental purifying spray that is pesticide-free and harmless to pets, characterized in that, The steps are as follows: S1: Raw material preparation and pretreatment S1.1: Preparation of Artemisia argyi culture medium Fresh, disease-free, and pesticide-free Artemisia argyi is crushed into 1–2 cm pieces, and distilled water is added at a solid-liquid ratio of 1:5–1:

10. The mixture is distilled at 100–105℃ for 2–4 h. After collecting the distillate, glucose and yeast extract are added and stirred at 150 r / min for 8–12 min until completely dissolved. S1.2: Preparation of Bacillus subtilis culture medium The activated Bacillus subtilis was inoculated at a rate of 0.5 mL / 100 mL into a culture medium containing 8–12 g / L peptone, 3–7 g / L beef extract, 3–7 g / L sodium chloride, and 15–25 g / L agar, at pH 7.0–7.2, and incubated at 37°C for 24–48 h until a milky white bacterial film formed on the surface. S1.3: Preparation of Aspergillus oryzae culture medium Aspergillus oryzae spores were inoculated into a solid culture medium containing 60–100 g wheat bran, 10–30 g sucrose, 2–4 g sodium nitrate, 0.5–1.5 g potassium dihydrogen phosphate, 0.3–0.7 g magnesium sulfate, and 0.3–0.7 g potassium chloride, with a water content of 60–65%. The medium was incubated at 30°C for 48–72 h until it was covered with yellow-green spores. Then, sterile water was added, the medium was stirred, and the mixture was filtered to obtain a spore-containing bacterial solution. S2: Mix the three culture media in a volume ratio of 2:1:1 and stir at 200–300 r / min for 10–15 min; S3: After the mixture is filtered through a plate and frame filter at 0.2–0.3 MPa, the filtrate is collected into a sterile storage tank; S4: Sterilize at 121℃ for 20 minutes, then aseptically fill, seal, label, and pack to obtain the finished product.

5. The method according to claim 4, characterized in that, All culture media and filling equipment were sterilized at 121°C and 0.12 MPa for 20 minutes to ensure no contamination.

6. The method according to claim 4, characterized in that, During the filtration process, the transmittance of the filtrate is ≥90%, and the filter cloth is replaced every 50 L to ensure the clarity and purity of the product.

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