阅读说明：本技术 畜牧养殖用喷剂及其制备方法和应用 (Spray for livestock breeding and preparation method and application thereof ) 是由 毛旭明 杨伟春 曾诚 梁世仁 丁能水 吴有林 于 2020-12-29 设计创作，主要内容包括：本发明提供了一种畜牧养殖用喷剂及其制备方法和应用,涉及畜牧养殖生物安全技术领域。所述喷剂主要由叶下珠提取物、N-乙酰神经氨糖酸、钨酸钠、甘油、乙醇和水制得。上述畜牧养殖用喷剂通过各原料的协同复配制得的畜牧养殖用喷剂具有很好的抗菌抗病毒的作用,在空气环境中喷施后能有效防止细菌病毒入侵动物机体,增强机体对有害微生物的免疫功能,同时灭杀环境中的有害微生物,确保畜牧养殖的生物安全,对畜牧养殖中微生物疾病的传播和危害具有很好的防控意义。(The invention provides a spray for livestock breeding and a preparation method and application thereof, and relates to the technical field of biological safety of livestock breeding. The spray is mainly prepared from a phyllanthus urinaria extract, N-acetylneuraminic acid, sodium tungstate, glycerol, ethanol and water. The spray for livestock breeding, which is prepared by compounding the raw materials in a synergistic manner, has good antibacterial and antiviral effects, can effectively prevent bacterial viruses from invading animal bodies after being sprayed in an air environment, enhances the immune function of the bodies to harmful microorganisms, kills the harmful microorganisms in the environment, ensures the biological safety of livestock breeding, and has good prevention and control significance on the propagation and harm of microbial diseases in livestock breeding.)

1. The spray for livestock breeding is characterized by being mainly prepared from a phyllanthus urinaria extract, N-acetylneuraminic acid, sodium tungstate, glycerol, ethanol and water.

2. The spray for livestock breeding according to claim 1, which is characterized by being prepared from the following raw materials in parts by weight: 15-28 parts of phyllanthus urinaria extract, 15-28 parts of N-acetylneuraminic acid, 8-15 parts of sodium tungstate, 8-15 parts of glycerol, 6-12 parts of ethanol and 900-932 parts of water; the sum of the mass parts of the raw materials in the spray is 1000 parts;

preferably, the spray is mainly prepared from the following raw materials in parts by weight: 20 parts of phyllanthus urinaria extract, 20 parts of N-acetylneuraminic acid, 10 parts of sodium tungstate, 10 parts of glycerol, 8 parts of ethanol and 932 parts of water.

3. The spray for livestock breeding according to claim 1, wherein the Phyllanthus urinaria extract is mainly extracted from Phyllanthus urinaria L powder;

preferably, the extraction method of the whole phyllanthus urinaria powder is Soxhlet extraction.

4. The spray for livestock breeding according to claim 3, wherein the extraction method of the Phyllanthus urinaria extract comprises the following steps:

placing the whole phyllanthus urinaria powder into a Soxhlet extractor, and then adding 100mL of water for Soxhlet extraction to obtain a phyllanthus urinaria extract;

preferably, the soxhlet extraction time is 4-6 hours, and preferably 4 hours.

5. The spray for livestock breeding according to claim 3 or 4, wherein the particle size of the whole phyllanthus urinaria powder is 40-60 mesh sieve powder, preferably 40 mesh sieve powder.

6. The spray for livestock breeding according to claim 4, wherein the mass ratio of whole phyllanthus urinaria powder to water is 1: 10-12, preferably 1: 10.

7. the preparation method of the spray for livestock breeding according to any one of claims 1-6, characterized by comprising the following steps:

mixing the formula amount of phyllanthus urinaria extract, N-acetylneuraminic acid, sodium tungstate, glycerol and ethanol to obtain a mixture; and then, fully and uniformly mixing the mixture with water in a formula amount to obtain the spray for livestock breeding.

8. The preparation method of the spray for livestock breeding according to claim 7, characterized in that the mixture and the water are fully mixed under stirring;

preferably, the stirring speed is 500-600 rpm, the stirring time is 10-15 minutes, and the ambient temperature is 20-25 ℃.

9. The method for preparing a spray for livestock breeding according to claim 7, characterized in that the method further comprises the steps of sterilizing the prepared spray for livestock breeding;

preferably, the sterilization mode is ultraviolet sterilization for 30-60 minutes.

10. The application of the spray for livestock breeding according to any one of claims 1-6 in air disinfection of livestock breeding.

Technical Field

The invention relates to the technical field of livestock breeding biological safety, in particular to a spray for livestock breeding and a preparation method and application thereof.

Background

The biological safety concept is a series of comprehensive prevention and treatment measures which are taken for preventing external pathogenic pathogens including viruses, bacteria, fungi, parasites and the like from entering livestock farms and invading animals and ensuring the health and safety of the animals. Viral and bacterial diseases of livestock and poultry in the breeding process often cause very serious harm to the breeding industry, so that a large amount of livestock and poultry die, the marketing quality of the livestock and poultry is reduced, and the daily life of social groups is greatly influenced.

However, the main action principle of the disinfectant used in the current livestock and poultry breeding is to denature, precipitate or solidify pathogenic microorganism proteins, destroy the structure of the pathogenic microorganism and obstruct the metabolism of the pathogenic microorganism, thereby achieving the purpose of killing. The traditional physical killing disinfectant has single effect, and harmful components can remain in animals and environment, so that the traditional physical killing disinfectant is not beneficial to the healthy development of the breeding industry.

Therefore, a novel antibacterial and antiviral spray for livestock is urgently needed, which reduces and eliminates pathogenic microorganisms in livestock farms, reduces or eliminates the chances of exogenous secondary infection of group infection, fundamentally reduces the dependence on medicaments and vaccines to realize the purposes of preventing and controlling epidemic diseases, simultaneously takes the traditional Chinese medicine components as the prevention and treatment criteria of strengthening body resistance and resisting evil, and strengthening body resistance and consolidating the foundation, effectively improves the autoimmunity of livestock and poultry, fundamentally conditions and improves the physique of the livestock and poultry, and has no side effect or medicament residue. Is the best measure for achieving the health of animal groups and achieving the maximum production performance, and is the most economic and effective means.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a livestock breeding spray which is mainly prepared from phyllanthus urinaria extract, N-acetylneuraminic acid, sodium tungstate, glycerol, ethanol and water.

The second purpose of the invention is to provide a preparation method of the spray for livestock breeding.

The third purpose of the invention is to provide the application of the spray for livestock breeding, and the spray for livestock breeding can be widely applied to air disinfection of livestock breeding.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the spray for livestock breeding provided by the invention is mainly prepared from a phyllanthus urinaria extract, N-acetylneuraminic acid, sodium tungstate, glycerol, ethanol and water.

Further, the spray is mainly prepared from the following raw materials in parts by weight: 15-28 parts of phyllanthus urinaria extract, 15-28 parts of N-acetylneuraminic acid, 8-15 parts of sodium tungstate, 8-15 parts of glycerol, 6-12 parts of ethanol and 900-932 parts of water; the sum of the mass parts of the raw materials in the spray is 1000 parts;

preferably, the spray is mainly prepared from the following raw materials in parts by weight: 20 parts of phyllanthus urinaria extract, 20 parts of N-acetylneuraminic acid, 10 parts of sodium tungstate, 10 parts of glycerol, 8 parts of ethanol and 932 parts of water.

Furthermore, the phyllanthus urinaria extract is mainly extracted from phyllanthus urinaria whole grass powder;

preferably, the extraction method of the whole phyllanthus urinaria powder is Soxhlet extraction.

Further, the extraction method of the phyllanthus urinaria extract comprises the following steps:

placing the whole phyllanthus urinaria powder in a Soxhlet extractor, and then adding water to perform Soxhlet extraction to obtain a phyllanthus urinaria extract;

preferably, the soxhlet extraction time is 4-6 hours, and preferably 4 hours.

Furthermore, the particle size of the whole phyllanthus urinaria powder is 40-60 meshes of sieve powder, and preferably 40 meshes of sieve powder.

Further, the mass ratio of the phyllanthus urinaria powder to water is 1: 10-12, preferably 1: 10.

the invention provides a preparation method of the spray for livestock breeding, which comprises the following steps:

mixing the formula amount of phyllanthus urinaria extract, N-acetylneuraminic acid, sodium tungstate, glycerol and ethanol to obtain a mixture; and then, fully and uniformly mixing the mixture with water to obtain the spray for livestock breeding.

Further, the mixture and water are fully and uniformly mixed under the condition of stirring;

preferably, the stirring speed is 500-600 rpm, the stirring time is 10-15 minutes, and the ambient temperature is 20-25 ℃.

Further, the preparation method also comprises the step of sterilizing the prepared spray for livestock breeding;

preferably, the sterilization mode is ultraviolet sterilization for 30-60 minutes.

The invention provides application of the spray for livestock breeding in air disinfection for livestock breeding.

Compared with the prior art, the invention has the beneficial effects that:

the spray for livestock breeding provided by the invention is mainly prepared from phyllanthus urinaria extract, N-acetylneuraminic acid, sodium tungstate, glycerol, ethanol and water. The common leafflower herb extract in the raw materials has no toxic or side effect, has well-known antibacterial and antiviral effects, can activate the immune response of the livestock body, improves the immunity of the organism, and is beneficial to preventing and treating microbial diseases of livestock; the N-acetylneuraminic acid can prevent bacteria from invading the body on one hand, and can compete with receptors on the surfaces of mucus cells bound with viruses for binding of the viruses on the other hand, so that the binding of the viruses and the mucus cells is prevented, and the infection risk of microbial diseases of livestock is reduced; sodium tungstate is used as metal ions to maintain the ionization balance of the spray liquid, so that all components are uniformly distributed, and the pH of the stable liquid is neutral; glycerin is a good humectant and solvent, and is helpful for the absorption of the spray; ethanol is a widely used organic solvent and has a certain killing effect on bacteria. Through the synergistic compounding of the raw materials, a stable bulk phase association structure can be formed, and the synergistic adsorption can be generated on an interface, so that the surface tension of the spray liquid drop is effectively reduced, and the absorption of skin and mucosa is facilitated. The prepared spray for livestock breeding has good antibacterial and antiviral effects, can effectively prevent bacteria and viruses from invading animal organisms after being sprayed in an air environment, enhances the immune function of the organisms to harmful microorganisms, kills the harmful microorganisms in the environment, ensures the biological safety of livestock breeding, and has good prevention and control significance for the propagation and harm of microbial diseases in livestock breeding.

The preparation method of the spray for livestock breeding provided by the invention comprises the steps of mixing the formula amount of phyllanthus urinaria extract, N-acetylneuraminic acid, sodium tungstate, glycerol and ethanol to obtain a mixture; and then, fully and uniformly mixing the mixture with water to obtain the spray for livestock breeding. The preparation method has the advantages of simple preparation process and easy operation, and is very suitable for large-scale industrial production.

The spray for livestock breeding provided by the invention can be widely applied to air disinfection of livestock breeding.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

According to one aspect of the invention, the spray for livestock breeding is mainly prepared from phyllanthus urinaria extract, N-acetylneuraminic acid, sodium tungstate, glycerol, ethanol and water.

The spray for livestock breeding provided by the invention is mainly prepared from phyllanthus urinaria extract, N-acetylneuraminic acid, sodium tungstate, glycerol, ethanol and water. The common leafflower herb extract in the raw materials has no toxic or side effect, has well-known antibacterial and antiviral effects, can activate the immune response of livestock bodies, improves the immunity of the organisms and is beneficial to the prevention and treatment of microbial diseases in livestock, N-acetylneuraminic acid can prevent bacteria from invading the organisms on the one hand, and receptors of influenza viruses on the other hand can compete for the combination of the viruses with receptors on the surfaces of mucus cells combining the viruses to prevent the combination of the viruses and the mucus cells on the other hand, so that the infection risk of the microbial diseases in livestock is reduced, sodium tungstate is used as a metal ion to maintain the ionization balance of spray liquid, so that all components are uniformly distributed, the pH of stable liquid is neutral, glycerol is a good wetting agent and solvent and is beneficial to the absorption of the spray, ethanol is a widely used organic solvent and has a certain killing effect on the bacteria. Through the synergistic compounding of the raw materials, a stable bulk phase association structure can be formed, and the synergistic adsorption can be generated on an interface, so that the surface tension of the spray liquid drop is effectively reduced, and the absorption of skin and mucosa is facilitated. The prepared spray for livestock breeding has good antibacterial and antiviral effects, can effectively prevent bacteria and viruses from invading animal organisms after being sprayed in an air environment, enhances the immune function of the organisms to harmful microorganisms, kills the harmful microorganisms in the environment, ensures the biological safety of livestock breeding, and has good prevention and control significance for the propagation and harm of microbial diseases in livestock breeding.

In a preferred embodiment of the invention, the spray is mainly prepared from the following raw materials in parts by weight: 15-28 parts of phyllanthus urinaria extract, 15-28 parts of N-acetylneuraminic acid, 8-15 parts of sodium tungstate, 8-15 parts of glycerol, 6-12 parts of ethanol and 900-932 parts of water; the sum of the mass parts of the raw materials in the spray is 1000 parts;

typical but non-limiting preferred embodiments of the above phyllanthus urinaria extract are: 15 parts, 18 parts, 20 parts, 22 parts, 24 parts, 26 parts and 28 parts;

typical but non-limiting preferred embodiments of the above N-acetylneuraminic acid are: 15 parts, 18 parts, 20 parts, 22 parts, 24 parts, 26 parts and 28 parts;

typical but non-limiting preferred embodiments of the above sodium tungstate are: 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts and 15 parts;

typical but non-limiting preferred embodiments of the above glycerol are: 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts and 15 parts;

typical but non-limiting preferred embodiments of the above ethanol are: 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts and 12 parts;

typical but non-limiting preferred embodiments of the above water are: 920 parts, 922 parts, 924 parts, 926 parts, 928 parts, 932 parts, 934 parts, 936 parts, 938 parts and 940 parts.

In the preferred embodiment, the spray is mainly prepared from the following raw materials in parts by weight: 20 parts of phyllanthus urinaria extract, 20 parts of N-acetylneuraminic acid, 10 parts of sodium tungstate, 10 parts of glycerol, 8 parts of ethanol and 932 parts of water.

In the invention, the effect of the spray for livestock breeding is further optimized by further adjusting and optimizing the dosage proportion of the raw materials of each component.

In a preferred embodiment of the present invention, the phyllanthus urinaria extract is mainly extracted from phyllanthus urinaria whole grass powder;

as a preferable embodiment, the phyllanthus urinaria extract is prepared from traditional Chinese medicine phyllanthus urinaria, and has the advantages of naturalness, safety, easy acquisition, mild drug effect, antibiosis, antivirus and the like. The spray prepared by the extract has broad-spectrum antibacterial and antiviral activity, and the phyllanthus urinaria extract has an inhibiting effect on common pathogenic bacteria in livestock breeding such as staphylococcus aureus, escherichia coli, salmonella typhi, shigella dysenteriae, bacillus cereus, staphylococcus epidermidis, enterococcus and the like, and is not easy to generate drug resistance; meanwhile, the compound has obvious antiviral effect on newcastle disease virus, avian influenza virus, tobacco mosaic virus, hepatitis B virus and the like.

Preferably, the extraction method of the whole plant powder of Phyllanthus urinaria is Soxhlet extraction (heating temperature controls distillation speed at 4 to 6 drops per second).

In the above preferred embodiment, the method for extracting Phyllanthus urinaria extract comprises the steps of:

placing the whole phyllanthus urinaria powder in a Soxhlet extractor, and then adding water to perform Soxhlet extraction to obtain a phyllanthus urinaria extract;

in the preferred embodiment, the soxhlet extraction time is 4 to 6 hours, preferably 4 hours.

Preferably, the preparation method of the phyllanthus urinaria extract comprises the following steps: weighing 10g of cacumen Securinegae Suffruticosae traditional Chinese medicine powder, filling into a filter paper bag, putting into a Soxhlet extractor, adding 100mL of distilled water into a receiving bottle, performing Soxhlet extraction for 4h, and performing constant volume on the water extract in the receiving bottle to 100mL to obtain the cacumen Securinegae Suffruticosae extract;

in a preferred embodiment of the invention, the particle size of the whole phyllanthus urinaria powder is 40-60 mesh sieve powder, preferably 40 mesh sieve powder.

In a preferred embodiment, the whole phyllanthus urinaria powder is prepared by uniformly crushing the whole phyllanthus urinaria after removing impurities and airing to form traditional Chinese medicine powder, and the granularity of the traditional Chinese medicine powder is required to be 95% and pass through a 40-mesh screen.

In a preferred embodiment of the present invention, the mass ratio of whole phyllanthus urinaria powder to water is 1: 10-12, preferably 1: 10.

as a preferred embodiment, the mass ratio of whole phyllanthus urinaria powder to water is established according to a large number of experimental results that prove that the mass ratio can significantly improve the extraction efficiency under the condition of ensuring the complete extraction of the effective components of phyllanthus urinaria.

According to one aspect of the invention, the preparation method of the spray for livestock breeding comprises the following steps:

mixing the formula amount of phyllanthus urinaria extract, N-acetylneuraminic acid, sodium tungstate, glycerol and ethanol to obtain a mixture; and then, fully and uniformly mixing the mixture with water to obtain the spray for livestock breeding.

The preparation method of the spray for livestock breeding provided by the invention comprises the steps of mixing the formula amount of phyllanthus urinaria extract, N-acetylneuraminic acid, sodium tungstate, glycerol and ethanol to obtain a mixture; and then, fully and uniformly mixing the mixture with water to obtain the spray for livestock breeding. The preparation method has the advantages of simple preparation process and easy operation, and is very suitable for large-scale industrial production.

In a preferred embodiment of the invention, the mixing of the mixture and water is carried out under stirring;

in the preferred embodiment, the stirring speed is 500-600 rpm, the stirring time is 10-15 minutes, and the ambient temperature is 20-25 ℃.

In a preferred embodiment of the invention, the preparation method further comprises the step of sterilizing the prepared livestock breeding spray;

preferably, the sterilization mode is ultraviolet sterilization for 30-60 minutes.

According to one aspect of the invention, the application of the spray for livestock breeding in air disinfection of livestock breeding is provided.

The spray for livestock breeding provided by the invention can be widely applied to air disinfection of livestock breeding.

The technical solution of the present invention will be further described with reference to examples and comparative examples.

Example 1

The spray for livestock breeding is mainly prepared from the following raw materials in parts by mass:

20 parts of phyllanthus urinaria extract, 20 parts of N-acetylneuraminic acid, 10 parts of sodium tungstate, 10 parts of glycerol, 8 parts of ethanol and 932 parts of water;

the preparation method of the spray for livestock breeding comprises the following steps:

crushing whole plant of cacumen Securinegae Suffruticosae after removing impurities and air drying to obtain traditional Chinese medicine powder with granularity of 95% passing through 40 mesh screen; weighing 10g of cacumen Securinegae Suffruticosae traditional Chinese medicine powder, filling into a filter paper bag, putting into a Soxhlet extractor, adding 100mL of distilled water into a receiving bottle, performing Soxhlet extraction for 4h, and performing constant volume on the water extract in the receiving bottle to 100mL to obtain the cacumen Securinegae Suffruticosae extract;

mixing the phyllanthus urinaria extract, N-acetylneuraminic acid, sodium tungstate, glycerol and ethanol according to a given weight part ratio, adding water, and magnetically stirring at 500-600 rpm for 10-15 minutes; subpackaging according to the amount of 250mL per bottle; and performing ultraviolet sterilization for 30-60 minutes to obtain the spray.

Example 2

The spray for livestock breeding is mainly prepared from the following raw materials in parts by mass:

24 parts of phyllanthus urinaria extract, 24 parts of N-acetylneuraminic acid, 12 parts of sodium tungstate, 12 parts of glycerol, 10 parts of ethanol and 918 parts of water;

the preparation method of the spray for livestock breeding is the same as that of the example 1.

Example 3

The spray for livestock breeding is mainly prepared from the following raw materials in parts by mass:

28 parts of phyllanthus urinaria extract, 28 parts of N-acetylneuraminic acid, 15 parts of sodium tungstate, 15 parts of glycerol, 12 parts of ethanol and 902 parts of water;

the preparation method of the spray for livestock breeding is the same as that of the example 1.

Example 4

The spray for livestock breeding comprises the same raw materials as in example 1 in parts by weight;

the preparation method of the spray for livestock breeding comprises the following steps:

crushing the whole plant of the phyllanthus urinaria after removing impurities and airing to form traditional Chinese medicine powder, wherein the granularity of the traditional Chinese medicine powder requires that 95 percent of the traditional Chinese medicine powder passes through a 60-mesh screen; weighing 8.3g of Phyllanthus urinaria L traditional Chinese medicine powder, filling into a filter paper bag, putting into a Soxhlet extractor, adding 100mL of distilled water into a receiving bottle, performing Soxhlet extraction for 6h, and receiving the water extract in the bottle to reach a constant volume of 100mL to obtain Phyllanthus urinaria L extract;

mixing the phyllanthus urinaria extract, N-acetylneuraminic acid, sodium tungstate, glycerol and ethanol according to a given weight part ratio, adding water, and magnetically stirring at 500-600 rpm for 10-15 minutes; subpackaging according to the amount of 250mL per bottle; and performing ultraviolet sterilization for 30-60 minutes to obtain the spray.

Comparative example 1

The spray for livestock breeding is mainly prepared from the following raw materials in parts by mass:

10 parts of sodium tungstate, 10 parts of glycerol, 8 parts of ethanol and 972 parts of water;

the preparation method of the spray for livestock breeding comprises the following steps:

mixing sodium tungstate, glycerol and ethanol according to a given weight part ratio, adding water, and magnetically stirring at 500-600 revolutions per minute for 10-15 minutes; subpackaging according to the amount of 250mL per bottle; and performing ultraviolet sterilization for 30-60 minutes to obtain the spray.

Comparative example 2

The spray for livestock breeding is mainly prepared from the following raw materials in parts by mass:

24 parts of N-acetylneuraminic acid, 12 parts of glycerol, 10 parts of ethanol and 956 parts of water;

the preparation method of the spray for livestock breeding comprises the following steps:

mixing N-acetylneuraminic acid, glycerol and ethanol according to a given weight part ratio, adding water, and magnetically stirring at 500-600 rpm for 10-15 minutes; subpackaging according to the amount of 250mL per bottle; and performing ultraviolet sterilization for 30-60 minutes to obtain the spray.

Comparative example 3

The spray for livestock breeding is mainly prepared from the following raw materials in parts by mass:

28 parts of N-acetylneuraminic acid, 15 parts of sodium tungstate, 12 parts of ethanol and 945 parts of water;

the preparation method of the spray for livestock breeding comprises the following steps:

mixing N-acetylneuraminic acid, sodium tungstate and ethanol according to a given weight part ratio, adding water, and magnetically stirring at 500-600 rpm for 10-15 minutes; subpackaging according to the amount of 250mL per bottle; and performing ultraviolet sterilization for 30-60 minutes to obtain the spray.

Experimental example 1 determination of antibacterial ability:

the experiment uses a punching method to test the antibacterial capacity of the spray prepared in the embodiments 1-4 and the comparative examples 1-3 of the invention, and the specific steps are as follows:

(1) and pouring about 15-20mL of plain agar into each sterilized culture dish in a super-clean workbench for bottoming, wherein the thickness of each culture dish is uniform, and the plain agar is cooled and solidified for later use.

(2) Sucking 1mL of the prepared indicator bacterium liquid, adding the indicator bacterium liquid into 100mL of LB solid culture medium which is kept at a constant temperature of about 50 ℃, and shaking up gently (avoiding bubbling when the indicator bacterium is culturedConcentration in the base is 10⁶cfu/mL), making a concentration gradient mark on the back surface of the plate, placing the sterilized Oxford cups on the fungus layer culture medium, and placing 7 Oxford cups in each culture dish at equal intervals.

(3) Pouring 15-20mL of the layer into each culture dish, uniformly spreading the layer, cooling and solidifying, and carefully clamping the oxford cup by using a sterile forceps to form a round hole.

(4) 150 mu L of the spray sample liquid prepared in the dilution examples 1-3 is sucked and added into a corresponding oxford cup, and meanwhile, sterile water is set as a blank control.

(5) The plated dishes were pre-diffused in a refrigerator at 4 ℃ for 3-4 hours.

(6) The culture dish is moved into an incubator to be cultured at 37 ℃, and the inhibition zone is observed for 8-12h, and the time is different due to different indicator bacteria or different operations of paving a flat plate each time, so that the observation is required in time.

(7) After the cultivation is finished, the diameter (unit mm) of the bacteriostatic circle is measured by a vernier caliper, the bacteriostatic circle is judged according to the generation of the bacteriostatic circle, and the size of the bacteriostatic circle reflects the strength of the antibacterial effect.

The experimental results are as follows:

the results of the inhibition effect of the sprays prepared in examples 1 to 4 and comparative examples 1 to 3 of the present invention on coliform bacillus ATCC-25922 (purchased from american type culture collection center ATCC) and staphylococcus aureus ATCC-6538P (purchased from american type culture collection center ATCC) are shown in tables 1 and 2.

Table 1: the size (unit: mm) of the bacteriostatic zone of the spray on escherichia coli:

table 2: the size (unit: mm) of the bacteriostatic zone of the spray on staphylococcus aureus is as follows:

as can be seen from the tables 1 and 2, compared with the blank control group and the comparative examples 1 to 3, the spray prepared in the examples 1 to 4 has certain bacteriostatic ability on escherichia coli and golden grape before being diluted by 3 times; and the inhibiting effect of the comparative examples 1-3 on escherichia coli and staphylococcus aureus is not obviously different from that of a blank control group. The above results illustrate that: the spray prepared by the invention can generate effective and obvious bacteriostatic effect on escherichia coli and staphylococcus aureus.

Experimental example 2 cytotoxicity assay:

in the experiment, the influence of the spray prepared in the embodiments 1 to 4 and the comparative examples 1 to 3 on the survival condition of the cells is measured by a cell digestion counting method, so that the toxicity of the spray on the cells is reflected, and the specific steps are as follows:

(1) at 9X 10⁴Cell concentration per well A549 cells (adenocarcinoma human alveolar basal epithelial cells) were seeded in 24-well plates, 1mL of RPMI-1640 medium containing 10% fetal bovine serum was added to each well, and the cells were cultured in a cell culture chamber at 37 ℃ in 5% CO₂Culturing for 48h until the monolayer cells are full;

(2) discarding the culture medium, washing the cells for 3 times by using PBS, incubating each spray on the cells in an amount of 1 mL/hole, setting a blank control group with the same concentration, making 3 multiple holes for each group at the same time, and culturing the cells in a cell culture box at 37 ℃ and 5% CO₂Culturing for 48h under the condition of (1);

(3) the supernatant was discarded, and the cells were washed 3 times with PBS, to which 1mL of 0.25% pancreatin was added at 37 ℃ with 5% CO₂The cells were digested for 3min, gently blown and mixed well and then the number of cells per well was determined using a Saimer Fei Countess II FL full-automatic cell counter.

The results of the experiment are shown in table 3.

Table 3: growth number of A549 cells after spray action (unit: number):

as can be seen from table 3, the number of a549 cell growths (purchased from the national biomedical experimental cell resource pool BMCR) after incubation of the sprays prepared in embodiments 1 to 4 of the present invention was substantially unchanged compared to the blank control group; the spray prepared by the invention has low cytotoxicity to be basically negligible or no toxicity.

Experimental example 3 antiviral ability experimental determination:

in the experiment, the influence of the spray prepared in the embodiments 1 to 4 and the comparative examples 1 to 3 on the survival condition of the infected virus cells is determined by a cell digestion counting method, so that the antiviral ability of the spray on the infected virus cells is reflected, and the specific steps are as follows:

(1)、9×10⁴cell concentration per well, A549 cells (adenocarcinoma human alveolar basal epithelial cells) are inoculated into a 24-well plate, 1mL of RPMI-1640 culture medium containing 10% fetal bovine serum is added into each well, and the cells are cultured for 48h in a cell culture box at 37 ℃ under the condition of 5% CO2 until the cells are full of monolayers;

(2) after removing the medium, the cells were washed 3 times with PBS, and NDV-herts33 strain diluted with RPMI-1640 medium was inoculated at 1 MOI, and then inoculated for 1 hour at 37 ℃ in a cell culture chamber with 5% CO 2.

(3) Removing culture medium, washing cells for 3 times by using PBS, incubating each spray agent on the cells by the amount of 1 mL/hole, setting a blank control group by using RPMI-1640 culture medium, adding 1mL of RPMI-1640 culture medium containing 4% fetal calf serum into each hole, simultaneously making 3 multiple holes for each group, and culturing four experimental batches of 6h, 12h, 18h and 24h in a cell culture box under the conditions of 37 ℃ and 5% CO 2;

(4) removing supernatant, washing cells for 3 times by using PBS, adding 1mL of 0.25% pancreatin into each hole, digesting for 3min under the conditions of 37 ℃ and 5% CO2, gently blowing, uniformly mixing, and measuring the cell number of each hole by using a Saimer Fei Contess II FL full-automatic cell counter, wherein the specific detection result is shown in Table 4:

table 4: the growth number (unit: one) of the A549 cells inoculated after the spray is acted is as follows:

as can be seen from table 4, compared with the blank control group, the antibacterial and antiviral sprays prepared in embodiments 1 to 4 of the present invention can significantly improve the survival rate of a549 cells infected with NDV after 12 hours; the spray prepared by the invention has certain antiviral capacity to NDV-herts 33.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.