Natural plant-extracted essential oil composition for antibiotic-free breeding and application of natural plant-extracted essential oil composition

Abstract

The invention provides a natural plant extraction essential oil composition for antibiotic-free breeding and application. The natural plant extraction essential oil composition is prepared from pelargonium roseum oil, cedar leaf oil, citrus lemon peel oil, bergamot oil and tea tree oil. Experiments prove that the natural plant extract essential oil composition disclosed by the invention can effectively kill or inhibit various common pathogenic microorganisms in poultry breeding, including bacteria, viruses and fungi, so that the natural plant extract essential oil composition can be applied to preparation of common pathogenic microorganism disinfectants in poultry breeding. The raw materials are simple, easy to obtain, non-toxic, harmless, green and environment-friendly, and the preparation method is simple to operate. The natural plant extract essential oil disinfectant is convenient, safe and efficient to use in livestock and poultry breeding, can replace antibiotics to prevent and treat livestock and poultry diseases, improves the survival rate and meat percentage of livestock and poultry, realizes green antibiotic-free breeding, and guarantees the safety and health of livestock and poultry meat.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibiotic-free breeding, and specifically relates to a natural plant essential oil composition for antibiotic-free breeding and an application thereof, namely a natural plant essential oil composition having the function of inhibiting pathogenic bacteria, fungi and viruses in breeding and an application thereof in antibiotic-free breeding. Background Art

[0002] With the development of society and the improvement of economic level, the traditional free-range farming model is difficult to meet the meat demand of my country's 1.4 billion people. my country's farming model has quickly shifted to an intensive and large-scale high-density farming model. However, the dense farming environment is prone to the spread of pathogenic bacteria, fungi and viruses, causing large-scale illness or death of farmed economic animals, resulting in huge economic losses. In order to reduce the incidence of diseases in economic animal farming, various broad-spectrum antibiotics with inhibitory effects on bacteria, fungi and viruses are widely used in high-density farming. In addition, antibiotics are also widely added to livestock feed as growth-promoting ingredients, which improves the economic benefits of the farming industry. These factors have led to a serious dependence on the use of antibiotics in the farming industry. In recent years, countries around the world have realized that the abuse of antibiotics in the farming industry will cause the risk of drug-resistant pathogens. At the same time, long-term sub-dose use of antibiotics will reduce the immunity of farmed economic animals and easily cause cross-infection of pathogenic bacteria, which is a huge long-term risk in the farming industry. More seriously, the antibiotics remaining in the farmed products not only affect their quality and taste, but may also lead to the formation of drug-resistant bacteria in the human body, directly threatening human health. In order to promote the healthy development of the aquaculture industry and protect human health, it is urgent to develop safe, non-toxic and harmless aquaculture methods and reduce or eliminate the use of antibiotics.

[0003] Under the current large-scale breeding environment, density, and epidemic prevention conditions in my country, directly stopping the use of antibiotics will significantly increase the morbidity and mortality of economic animals, reduce breeding efficiency, and bring greater economic losses. At present, some products that replace antibiotics have emerged on the market, mainly feed additives, including acidifiers, microecological preparations, bioactive peptides, Chinese herbal medicine preparations, etc. Its mode of action is similar to that of antibiotics. It is added to the feed and ingested by farmed animals, and has a certain antibacterial effect or improves its immunity. However, these products have high production costs and are difficult to promote on a large scale. At the same time, they cannot fundamentally solve the problem of high morbidity in high-density breeding. In addition, the method of use of these products is still to add them to the feed, and there is also a risk of residues in the body.

[0004] Pathogenic microorganisms, including pathogenic bacteria, fungi and viruses, are widely present and easily spread in the breeding environment. When farmed animals are infected by these microorganisms, they will quickly become ill and cause large-scale infection and death. At present, the main pathogenic bacteria in large-scale breeding include Riemerella anatipestifer, Salmonella pullorum, Escherichia coli, Staphylococcus aureus, Pasteurella multocida, Campylobacter jejuni, Clostridium perfringens, Haemophilus paragallinarum, Mycoplasma gallisepticum, Enterococcus, Streptococcus, etc., pathogenic fungi include Candida albicans, Aspergillus fumigatus, Aspergillus niger, etc., pathogenic viruses include duck plague virus, Newcastle disease virus, avian influenza virus, fowl pox virus, avian adenovirus, duck hepatitis A virus, infectious bronchitis virus, bursal disease virus, porcine coronavirus, parvovirus, Tembusu virus, classical swine fever virus, etc. The core issue of antibiotic-free farming is to reduce the number of pathogenic microorganisms such as bacteria, fungi and viruses under the "antibiotic-free approach", block their spread and infection of farmed economic animals, reduce the morbidity of farmed economic animals, and improve the economic benefits of farming. Natural plant essential oils rich in active substances such as ketones, terpenes, aldehydes, alcohols, etc. have the activities of enhancing immunity, inhibiting bacteria, and antiviral, and are widely used in products such as medicines, antibacterial agents, and antiviral agents. In addition, natural plant essential oils are non-toxic and harmless and have an aromatic smell. They are environmentally friendly and do not pose safety risks. They are widely used in the field of air disinfection. Therefore, the development of natural plant essential oil compositions to inhibit or kill pathogenic bacteria, fungi and viruses in the breeding environment can reduce the morbidity and mortality of economic animals, reduce breeding risks, and achieve antibiotic-free farming, which has huge economic value and social benefits. Summary of the invention

[0005] The purpose of the present invention is to provide a natural plant essential oil composition for antibiotic-free breeding. The composition is prepared by using natural plant essential oils in different proportions as raw materials. The raw materials used are simple, easy to obtain, non-toxic, harmless, and green and environmentally friendly. The preparation method is simple to operate. The obtained natural plant essential oil composition is easy to use, has significant effects, and has no environmental pollution and safety hazards.

[0006] The natural plant essential oil composition used in the present invention is a mixture of geranium oil, cedar leaf oil, citrus lemon peel oil, bergamot oil and tea tree oil in a specific ratio, and the weight ratio of each component is 2-6:5-11:1-5:2-4:1-3.

[0007] In a preferred embodiment of the present invention, the weight ratio of the components is 4:8:3:3:2 for geranium oil:cedar leaf oil:citrus lemon peel oil:bergamot oil:tea tree oil.

[0008] In another preferred embodiment of the present invention, the weight ratio of the components of geranium oil: cedar leaf oil: citrus lemon peel oil: bergamot oil: tea tree oil is 6:6:1:4:3.

[0009] In another preferred embodiment of the present invention, the weight ratio of the components of geranium oil: cedar leaf oil: citrus lemon peel oil: bergamot oil: tea tree oil is 2:10:5:2:1.

[0010] The present invention also provides a method for preparing natural plant essential oils for antibiotic-free breeding: according to the mass ratio mentioned in the above formula, weigh the corresponding natural plant essential oils respectively, add them into a closed mixing kettle in turn, control the temperature at 15-45°C, stir and mix at a speed of 100-1000rpm for 1-24 hours under magnetic stirring, so that each essential oil component is fully mixed, and the plant essential oil mixture prepared in the previous step is ultrasonically dispersed for 0.1-1 hour and then allowed to stand to obtain a natural plant essential oil composition stock solution. The stock solution or its dilution can inhibit or kill pathogenic microorganisms in the breeding environment, and can also be directly used in breeding places to replace antibiotics to achieve antibiotic-free breeding. The natural plant essential oil composition stock solution obtained by this method can be directly used for packaging, use, and sales.

[0011] In the present invention, the temperature during the mixing process is controlled at 15-45°C, preferably 25-35°C; the stirring speed is 100-1000rpm, preferably 300-800rpm, and more preferably 500-600rpm; the stirring time is 1-24 hours, preferably 4-18 hours, and more preferably 8-12 hours; the ultrasonic dispersion time is 0.1-1 hour, preferably 0.2-0.8 hours, and more preferably 0.4-0.6 hours.

[0012] The components of the natural plant essential oil composition mentioned in the present invention are all obtained by general methods, such as geranium oil, also known as geranium oil, which is obtained by steam distillation of fresh leaves of geranium, cedar leaf oil is extracted from the leaves of cedar, red pine or red cedar by steam distillation or solvent extraction, citrus lemon peel oil is obtained by steam distillation of fresh lemon peel of Rutaceae Citrus, bergamot oil is obtained by steam distillation of bergamot fruit of Rutaceae Citrus, and tea tree oil is obtained by steam distillation of Melaleuca alternifolia leaves of Myrtaceae.

[0013] Another object of the present invention is to provide an application of the natural plant essential oil composition in the preparation of a disinfectant for common pathogenic microorganisms in poultry farming. After being placed in a livestock and poultry breeding site, the disinfectant is rapidly distributed in the air by direct volatilization, exerting an inhibitory effect on bacteria, viruses, and fungi, and can replace the role of antibiotics.

[0014] The natural plant extract essential oil composition provided by the present invention has significant killing or inhibiting activity against common pathogenic microorganisms in aquaculture, wherein the pathogenic microorganisms include bacteria, fungi, and viruses. Further, the natural plant extract essential oil composition of the present invention can kill or inhibit common pathogenic bacteria in aquaculture, including Riemerella anatipestifer, Salmonella pullorum, Escherichia coli, Staphylococcus aureus, Pasteurella multocida, Clostridium perfringens, Haemophilus paragallinarum, Mycoplasma gallisepticum, Enterococcus, and Streptococcus; common pathogenic fungi in aquaculture that can be killed or inhibited include Candida albicans, Aspergillus fumigatus, and Aspergillus niger; common pathogenic viruses in aquaculture that can be killed or inhibited include duck plague virus, Newcastle disease virus, avian influenza virus A, duck hepatitis A virus, infectious bronchitis virus, avian adenovirus, porcine coronavirus, goose parvovirus, duck Tembusu virus, and chicken infectious bursal disease virus.

[0015] Experiments show that after the natural plant essential oil stock solution obtained in the present invention is diluted 100, 300, 1000, 3000 or 10000 times and mixed with Riemerella anatipestifer, or Riemerella anatipestifer is exposed to the atmosphere of the natural plant essential oil composition of the formula for 15, 30, 60 minutes, it is re-cultured in a culture dish. The results show that under these two different modes of treatment, the natural plant essential oil composition of the present invention has a significant killing or inhibitory effect on Riemerella anatipestifer. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill or inhibit Riemerella anatipestifer, a pathogenic bacterium in farmed animals in antibiotic-free farming.

[0016] Experiments show that after the natural plant essential oil stock solution obtained in the present invention is diluted 100, 300, 1000, 3000 or 10000 times and mixed with Salmonella pullorum, or Salmonella pullorum is exposed to the atmosphere of the natural plant essential oil composition of the formula for 15, 30, 60 minutes, and then re-cultured in a culture dish. The results show that under these two different modes of treatment, the natural plant essential oil composition of the present invention has a significant killing or inhibitory effect on Salmonella pullorum. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill or inhibit Salmonella pullorum, a pathogenic bacterium of farmed animals in antibiotic-free farming.

[0017] The experiment shows that after the natural plant essential oil stock solution obtained in the present invention is diluted 100, 300, 1000 or 3000 times and mixed with Escherichia coli, or Escherichia coli is exposed to the atmosphere of the natural plant essential oil composition of the formula for 15, 30, 60 minutes, it is re-cultured in a culture dish. The results show that under these two different modes of treatment, the natural plant essential oil composition of the present invention has a significant killing or inhibitory effect on Escherichia coli. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill or inhibit pathogenic bacteria Escherichia coli in farmed animals in antibiotic-free farming.

[0018] The experiment shows that after the natural plant essential oil stock solution obtained in the present invention is diluted 100, 300, 1000, 3000 or 10000 times and mixed with Staphylococcus aureus, or Staphylococcus aureus is exposed to the atmosphere of the natural plant essential oil composition of the formula for 15, 30, 60, 120 minutes, it is re-cultured in a culture dish. The results show that under these two different modes of treatment, the natural plant essential oil composition of the present invention has a significant killing or inhibitory effect on Staphylococcus aureus. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill or inhibit Staphylococcus aureus, a pathogenic bacterium of farmed animals in antibiotic-free farming.

[0019] The experiment shows that after the natural plant essential oil stock solution obtained in the present invention is diluted 100, 300, 1000, 3000 or 10000 times and mixed with Pasteurella multocida, or Pasteurella multocida is exposed to the atmosphere of the natural plant essential oil composition of the formula for 15, 30, 60 minutes, it is re-cultured in a culture dish. The results show that under these two different modes of treatment, the natural plant essential oil composition of the present invention has a significant killing or inhibitory effect on Pasteurella multocida. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill or inhibit Pasteurella multocida, a pathogenic bacterium of farmed animals in antibiotic-free farming.

[0020] The experiment shows that after the natural plant essential oil stock solution obtained in the present invention is diluted 100, 300, 1000 or 3000 times and mixed with Clostridium perfringens, or Clostridium perfringens is exposed to the atmosphere of the natural plant essential oil composition of the formula for 15, 30, 60 minutes, it is re-cultured in a culture dish. The results show that under these two different modes of treatment, the natural plant essential oil composition of the present invention has a significant killing or inhibitory effect on Clostridium perfringens. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill Clostridium perfringens, a pathogenic bacterium of farmed animals in antibiotic-free farming.

[0021] Experiments show that after the natural plant essential oil stock solution obtained in the present invention is diluted 100, 300, 1000, 3000 or 10000 times and mixed with Haemophilus paragallinarum, or Haemophilus paragallinarum is exposed to the atmosphere of the natural plant essential oil composition of the formula for 15, 30, 60, 120 minutes, it is re-cultured in a culture dish. The results show that under these two different modes of treatment, the natural plant essential oil composition of the present invention has a significant killing or inhibitory effect on Haemophilus paragallinarum. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill or inhibit Haemophilus paragallinarum, a pathogenic bacterium of farmed animals in antibiotic-free farming.

[0022] Experiments show that after the natural plant essential oil stock solution obtained in the present invention is diluted 100, 300, 1000, 3000 or 10000 times and mixed with Mycoplasma gallisepticum, or Mycoplasma gallisepticum is exposed to the atmosphere of the natural plant essential oil composition of the formula for 15, 30, 60 minutes, it is re-cultured in a culture dish. The results show that under these two different modes of treatment, the natural plant essential oil composition of the present invention has a significant killing or inhibitory effect on Mycoplasma gallisepticum. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill or inhibit pathogenic Mycoplasma gallisepticum in farmed animals in antibiotic-free farming.

[0023] The experiment shows that the natural plant essential oil stock solution obtained in the present invention is diluted 100, 200, 400 or 1000 times and mixed with enterococci, and then cultured again in a culture dish. The results show that the natural plant essential oil composition of the present invention has a significant killing or inhibitory effect on enterococci. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill or inhibit pathogenic bacteria enterococci in farmed animals in antibiotic-free farming.

[0024] The experiment shows that the natural plant essential oil stock solution obtained in the present invention is diluted 100, 200, 400 or 1000 times and mixed with streptococci, and then cultured again in a culture dish. The results show that the natural plant essential oil composition of the present invention has a significant killing or inhibitory effect on streptococci. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill or inhibit pathogenic bacteria streptococci in farmed animals in antibiotic-free farming.

[0025] The experiment shows that the natural plant essential oil stock solution obtained in the present invention is diluted 100, 200, 400 or 1000 times and mixed with Aspergillus fumigatus, and then re-cultured in a culture dish. The results show that the natural plant essential oil composition of the present invention has a significant killing or inhibitory effect on Aspergillus fumigatus. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill or inhibit the pathogenic fungus Aspergillus fumigatus in antibiotic-free breeding.

[0026] The experiment showed that after Aspergillus niger was exposed to the atmosphere of the natural plant essential oil composition of the formula for 4 or 24 hours, it was re-cultured in a culture dish. The results showed that the natural plant essential oil composition of the present invention had a significant killing or inhibitory effect on Aspergillus niger. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill Aspergillus niger, a pathogenic fungus of farmed animals, in antibiotic-free farming.

[0027] The experiment shows that after the natural plant essential oil stock solution obtained in the present invention is diluted 100, 150 or 200 times and mixed with Candida albicans, or Candida albicans is exposed to the atmosphere of the natural plant essential oil composition of the formula for 4 and 24 hours, it is re-cultured in a culture dish. The results show that under these two different modes of treatment, the natural plant essential oil composition of the present invention has a significant killing or inhibitory effect on Candida albicans. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill or inhibit the pathogenic fungus Candida albicans of farmed animals in antibiotic-free farming.

[0028] The experiment showed that the porcine coronavirus applied to the surface of the carrier was exposed to the natural plant essential oil composition of the formula for 15, 30, 45, and 60 minutes, and then re-transfected into the cells. The results show that the natural plant essential oil composition of the present invention has a significant inhibitory effect on porcine coronavirus. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill or inhibit the pathogenic virus porcine coronavirus in farmed animals in antibiotic-free farming.

[0029] The experiment showed that the avian influenza A virus applied to the surface of the carrier was exposed to the natural plant essential oil composition of the formula for 10, 20, and 30 minutes, and then re-transfected into the cells. The results show that the natural plant essential oil composition of the present invention has a significant inhibitory or killing effect on the avian influenza A virus. This shows that the natural plant essential oil composition of the present invention is effective and can be used to kill or inhibit the avian influenza A virus, which is a pathogenic virus of farmed animals in antibiotic-free farming.

[0030] The experiment showed that the duck Tembusu virus applied to the surface of the carrier was exposed to the natural plant essential oil composition of the formula for 15, 30, 45, and 60 minutes, and then re-transfected into the cells. The results show that the natural plant essential oil composition in this patent has a significant killing or inhibitory effect on duck Tembusu virus. This shows that the natural plant essential oil composition in this patent is effective and can be used to kill duck Tembusu virus, a pathogenic virus of farmed animals, in antibiotic-free farming.

[0031] The experiment showed that the chicken infectious bursal disease virus applied to the surface of the carrier was exposed to the natural plant extract essential oil composition of the formula for 1, 2, 3, 4, 5, and 6 hours, and then re-transfected into the cells. The results show that the natural plant extract essential oil composition in this patent has a significant killing or inhibitory effect on chicken infectious bursal disease virus. This shows that the natural plant extract essential oil composition in this patent is effective and can be used to kill chicken infectious bursal disease virus, a pathogenic virus of farmed animals, in antibiotic-free farming.

[0032] The experiment showed that the goose parvovirus applied on the surface of the carrier was exposed to the natural plant extract essential oil composition of the formula for 1, 2, 3, 4, 5, and 6 hours, and then re-transfected into the cells. The results show that the natural plant extract essential oil composition in this patent has a significant killing or inhibitory effect on goose parvovirus. This shows that the natural plant extract essential oil composition in this patent is effective and can be used to kill goose parvovirus, a pathogenic virus of farmed animals, in antibiotic-free farming.

[0033] The significant killing or inhibiting effect of the natural plant essential oil composition on pathogenic microorganisms in aquaculture in the present invention can solve the core problem in antibiotic-free aquaculture, that is, to kill or reduce the number of pathogenic microorganisms such as bacteria, fungi and viruses, block their spread and infection of aquaculture economic animals, reduce the incidence of disease, and improve the economic benefits of aquaculture. One or more of the pathogenic microorganisms are widely present in different aquaculture economic animals. Further, the aquaculture economic animals are mainly livestock and poultry, wherein the livestock are preferably pigs, cattle, sheep, horses, camels, rabbits, cats, dogs, and the poultry are preferably chickens, ducks, geese, pigeons, turkeys, and quails.

[0034] Experiments show that in white-feathered chicken farming, the natural plant essential oil composition of the present invention is distributed in the breeding site through auxiliary equipment, and its effect of killing or inhibiting pathogenic bacteria, fungi and viruses in the breeding environment can replace antibiotics, so that the survival rate and market weight of broiler chickens are higher than those of the control group using antibiotics. This shows that the natural plant essential oil composition of the present invention is effective and can replace antibiotics in chicken farming, improve farming efficiency, and realize antibiotic-free chicken farming.

[0035] The experiment showed that in Cherry Valley duck farming, the natural plant essential oil composition of the present invention was distributed in the breeding site through auxiliary equipment, and its effect of killing or inhibiting pathogenic bacteria, fungi and viruses in the breeding environment can replace antibiotics, so that the survival rate and market weight of meat ducks are consistent with those of the control group using antibiotics. This shows that the natural plant essential oil composition of the present invention is effective and can replace antibiotics in duck farming, improve farming efficiency, and realize antibiotic-free duck farming.

[0036] The experimental results of the present invention confirm that the provided natural plant extract essential oil composition has a good killing or inhibiting effect on many pathogenic microorganisms present in the breeding process, such as pathogenic bacteria Riemerella anatipestifer, Salmonella pullorum, Escherichia coli, Staphylococcus aureus, Pasteurella multocida, Clostridium perfringens, Haemophilus paragallinarum, Mycoplasma gallisepticum, Enterococcus, Streptococcus, etc., pathogenic fungi such as Candida albicans, Aspergillus fumigatus and Aspergillus niger, pathogenic viruses such as duck plague virus, Newcastle disease virus, avian influenza A virus, duck hepatitis A virus, infectious bronchitis virus, avian adenovirus, porcine coronavirus, goose parvovirus, duck Tembusu virus, chicken infectious bursal disease virus. It is worth noting that the natural plant extract essential oil composition of the present invention has a significant killing or inhibiting effect on the pathogenic microorganisms in the breeding under the fumigation mode, which is in line with the application scenario in the breeding site. Further antibiotic-free breeding test results confirmed that in broiler white-feathered chicken breeding, the natural plant essential oil composition of the present invention can effectively replace traditional antibiotics, and the survival rate and average market weight of broiler chickens in the experimental group were higher than those of the control group using antibiotics. In the breeding of meat ducks and Cherry Valley ducks, the natural plant essential oil composition of the present invention can also effectively replace traditional antibiotics, and the survival rate and average market weight of meat ducks in the experimental group are consistent with those of the control group using antibiotics. These experiments have proved that the natural plant essential oil composition of the present invention is safe, non-toxic, harmless, and environmentally friendly, and has a good killing or inhibitory effect on pathogenic microorganisms in various breedings, can replace the effect of antibiotics and increase the survival rate and market weight of farmed animals, improve breeding efficiency and achieve antibiotic-free breeding.

[0037] The beneficial effects of the present invention are: using readily available natural plant essential oil as a raw material, a composition is prepared by a simple preparation process at a specific ratio, which has a good killing or inhibitory effect on pathogenic microorganisms present in the breeding environment, and can replace antibiotics to reduce the morbidity or mortality of farmed animals, reduce breeding risks and costs, and improve breeding benefits. In addition, the natural plant essential oil composition of the present invention has readily available raw materials, low preparation costs, is non-toxic and harmless, is easily volatile and has no residue, and can be directly applied to antibiotic-free breeding of economic animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : The inhibitory effect of natural plant essential oil composition on Riemerella anatipestifer was determined by mixing method.

[0039] Figure 2 : The inhibitory effect of natural plant essential oil composition on Riemerella anatipestifer was determined by fumigation method.

[0040] Figure 3 : The inhibitory effect of natural plant essential oil composition on Riemerella anatipestifer was determined by fumigation method.

[0041] Figure 4: The inhibitory effect of natural plant essential oil composition on Salmonella pullorum was determined by mixing method.

[0042] Figure 5 : The inhibitory effect of natural plant essential oil composition on Salmonella pullorum was determined by mixing method.

[0043] Figure 6 : The inhibitory effect of natural plant essential oil composition on Salmonella pullorum was determined by fumigation method.

[0044] Figure 7 : The inhibitory effect of natural plant essential oil composition on Salmonella pullorum was determined by fumigation method.

[0045] Figure 8 : The inhibitory effect of natural plant essential oil composition on Escherichia coli was determined by mixing method.

[0046] Fig. 9 : The inhibitory effect of natural plant essential oil composition on Escherichia coli was determined by mixing method.

[0047] Fig.10 : The inhibitory effect of natural plant essential oil composition on Escherichia coli was determined by fumigation method.

[0048] Fig.11 : The inhibitory effect of natural plant essential oil composition on Escherichia coli was determined by fumigation method.

[0049] Fig.12 : The inhibitory effect of natural plant essential oil composition on Staphylococcus aureus was determined by mixing method.

[0050] Fig.13 : The inhibitory effect of natural plant essential oil composition on Staphylococcus aureus was determined by fumigation method.

[0051] Fig.14 : The inhibitory effect of natural plant essential oil composition on Pasteurella multocida was determined by mixing method.

[0052] Fig.15 : The inhibitory effect of natural plant essential oil composition on Pasteurella multocida was determined by fumigation method.

[0053] Fig.16 : The inhibitory effect of natural plant essential oil composition on Clostridium perfringens was determined by mixing method.

[0054] Fig.17 : The inhibitory effect of natural plant essential oil composition on Clostridium perfringens was determined by fumigation method.

[0059] Fig.18 : The inhibitory effect of natural plant essential oil composition on enterococci was determined by mixing method.

[0060] Fig.19 : The inhibitory effect of natural plant essential oil composition on Streptococcus was determined by mixing method.

[0061] Fig. 20 : The inhibitory effect of natural plant essential oil composition on Aspergillus fumigatus was determined by mixing method.

[0062] Fig.21 : The inhibitory effect of natural plant essential oil composition on Aspergillus niger was determined by fumigation method.

[0063] Fig. 22 : The inhibitory effect of natural plant essential oil composition on Candida albicans was determined by mixing method.

[0064] Fig.23 : The inhibitory effect of natural plant essential oil composition on Candida albicans was determined by fumigation method.

[0065] Fig.24 : The inhibitory effect of natural plant essential oil composition on porcine coronavirus was determined by fumigation method.

[0066] Fig.25 : The inhibitory effect of natural plant essential oil composition on avian influenza A virus was determined by fumigation method.

[0067] Fig.26 : The inhibitory effect of natural plant essential oil composition on duck Tembusu virus was determined by fumigation method. DETAILED DESCRIPTION

[0070] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. The natural plant essential oils mentioned in the present invention, alone or in any combination, also have the effect of inhibiting or killing pathogenic microorganisms in aquaculture, and can also be used for antibiotic-free aquaculture. In addition, simply promoting the natural plant essential oils in the present invention, alone or in any combination, to antibiotic-free aquaculture of different economic animals also falls within the scope of protection of the present invention. The feed, epidemic prevention reagents, and instruments and equipment mentioned in this description in the aquaculture process are all commercially available conventional products. Therefore, the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0071] Embodiment 1: A natural plant essential oil composition is composed of the ingredients in the formula according to the corresponding weight ratio of geranium oil: cedar leaf oil: citrus lemon peel oil: bergamot oil: tea tree oil of 4:8:3:3:2. 100 grams of geranium oil, 200 grams of cedar leaf oil, 75 grams of citrus lemon peel oil, 75 grams of bergamot oil, and 50 grams of tea tree oil are added to a mixing kettle in sequence, stirred and mixed at a speed of 550 rpm for 0.5 hours under magnetic stirring, and the prepared plant essential oil mixed liquid is ultrasonically dispersed for 0.5 hours and then allowed to stand to obtain a plant essential oil composition stock solution. The above-mentioned natural plant essential oil composition stock solution, or the diluted solution after being diluted with different solvents such as water, alcohol, disinfectant water, etc. in different proportions, can be directly used for antibiotic-free breeding.

[0072] Embodiment 2: A natural plant essential oil composition, which is composed of the ingredients in the formula according to the corresponding weight ratio of geranium oil: cedar leaf oil: citrus lemon peel oil: bergamot oil: tea tree oil of 6:6:1:4:3, 150 grams of geranium oil, 150 grams of cedar leaf oil, 25 grams of citrus lemon peel oil, 100 grams of bergamot oil, and 75 grams of tea tree oil are added to a mixing kettle in sequence, stirred and mixed at a speed of 550 rpm for 0.5 hours under magnetic stirring, and after each mixture is fully mixed, the prepared plant essential oil mixture is ultrasonically dispersed for 0.5 hours and then allowed to stand to obtain a plant essential oil composition stock solution. The above-mentioned natural plant essential oil composition stock solution, or the diluted solution after being diluted with different solvents such as water, alcohol, disinfectant water, etc. in different proportions, can be directly used for antibiotic-free breeding.

[0073] Embodiment 3: A natural plant essential oil composition is composed of the ingredients in the formula according to the corresponding weight ratio of geranium oil: cedar leaf oil: citrus lemon peel oil: bergamot oil: tea tree oil of 2:10:5:2:1. 50 grams of geranium oil, 250 grams of cedar leaf oil, 125 grams of citrus lemon peel oil, 50 grams of bergamot oil, and 25 grams of tea tree oil are added to a mixing kettle in sequence, stirred and mixed at a speed of 550 rpm for 0.5 hours under magnetic stirring, and after each mixture is fully mixed, the prepared plant essential oil mixture is ultrasonically dispersed for 0.5 hours and then allowed to stand to obtain a plant essential oil composition stock solution. The above-mentioned natural plant essential oil composition stock solution, or the diluted solution after being diluted with different solvents such as water, alcohol, disinfectant water, etc. in different proportions, can be directly used for antibiotic-free breeding.

[0074] Example 4: Determination of the inhibitory effect of plant essential oil composition on Riemerella anatipestifer by mixing method The plant extract essential oil composition in Example 1 of the present invention was diluted with dimethyl sulfoxide by 100, 300, 1000, 3000, and 10000 times, respectively, and mixed with Riemerella anatipestifer (1.5×10 3CFU / mL) was cultured at 22°C in a 220rpm shaker and incubated for 60 minutes, then centrifuged at 8°C and 10,000rpm for 2 minutes in a centrifuge and the supernatant was discarded. 200 μL of normal saline was added and mixed by pipetting with a pipette. 100 μL was injected into a sterile culture dish containing TSA and 5% defibrinated sheep blood, and the culture dish was placed at 37°C for 48 hours before counting. All experiments were repeated three times in parallel, with no natural plant extract essential oil composition solution or any diluent added as a blank control. The experimental results are shown in the attached Figure 1 .

[0075] The experimental results show that compared with the blank control group, the natural plant essential oil composition of this formula was mixed with Riemerella anatipestifer, and the inhibition rate of the original solution diluted 100 times on Riemerella anatipestifer exceeded 98%, and the original solution diluted 300-10,000 times still had a good inhibitory effect on Riemerella anatipestifer.

[0076] Example 5: Fumigation method to determine the inhibitory effect of plant essential oil composition on Riemerella anatipestifer 30 μL of Riemerella anatipestifer (1.5×10 3 CFU / mL) were exposed to the volatile atmosphere of the natural plant essential oil composition or rapeseed oil (control group) in Example 1 for 15, 30 and 60 minutes, and the slides were repeatedly rinsed with 200 μL of saline. The washed bacterial solution was centrifuged at 10000 rpm at 8°C for 2 minutes and the supernatant was discarded. 100 μL of saline was added to resuspend by shaking and 30 μL was injected into a plate culture dish containing TSA and 5% defibrinated sheep blood. The culture dish was placed at 37°C and cultured for 48 hours before colony counting. All experiments were repeated three times in parallel. The experimental results are shown in the attached Figure 2 and attached Figure 3 .

[0077] The experimental results show that the natural plant essential oil composition of the formula can inhibit Riemerella anatipestifer by fumigation treatment, and the inhibition rate of Riemerella anatipestifer exceeds 99% after 30 minutes of action, and Riemerella anatipestifer can be killed after 60 minutes of action.

[0078] Example 6: Determination of the inhibitory effect of plant essential oil composition on Salmonella pullorum by mixing method The original solution of the natural plant essential oil composition in Example 2 of the present invention was diluted with dimethyl sulfoxide by 100, 300, 1000, 3000, and 10000 times, respectively, and then mixed with Salmonella pullorum (1.5×10 3CFU / mL) was cultured at 22°C in a 220rpm shaker and incubated for 60 minutes, then centrifuged at 8°C and 10,000rpm in a centrifuge for 2 minutes and the supernatant was discarded. 200 μL of physiological saline was added and mixed by pipetting with a pipette. 100 μL was injected into an LB plate culture dish, and the culture dish was placed at 37°C for 48 hours before counting. All experiments were repeated three times in parallel, with no natural plant extract essential oil composition solution or any diluent added as a blank control. The experimental results are shown in the attached Figure 4 and attached Figure 5 .

[0079] The experimental results show that compared with the blank control group, the natural plant essential oil composition of this formula was mixed with Salmonella pullorum, and the original solution diluted 100 times had a killing effect on Salmonella pullorum, and the inhibition rate of the original solution diluted 300 times on Salmonella pullorum exceeded 99%. The original solution diluted 1000-3000 times still had a good inhibitory effect on Salmonella pullorum.

[0080] Example 7: Determination of the inhibitory effect of plant essential oil composition on Salmonella pullorum by fumigation method 30 μL of Salmonella pullorum (1.5×10 3 CFU / mL) were exposed to the volatile atmosphere of the natural plant essential oil composition or rapeseed oil (control group) in Example 2 for 15, 30 and 60 minutes, and the slides were repeatedly rinsed with 200 μL of saline. The washed bacterial solution was centrifuged at 10000 rpm at 8°C for 2 minutes and the supernatant was discarded. 100 μL of saline was added to resuspend by shaking and 30 μL was injected into the LB plate culture dish. The culture dish was placed at 37°C and cultured for 48 hours before the colony count was performed. All experiments were repeated three times in parallel. The experimental results are shown in the attached figure. Figure 6 and attached Figure 7 .

[0081] Experimental results show that the natural plant essential oil composition of the formula can kill the Salmonella pullorum after fumigation treatment of the Salmonella pullorum for 60 minutes.

[0082] Example 8: Determination of the inhibitory effect of plant essential oil composition on Escherichia coli by mixing method The plant extract essential oil composition solution in Example 3 of the present invention was diluted 100, 300, 1000, and 3000 times with dimethyl sulfoxide and mixed with Escherichia coli (1.5×10 3CFU / mL) was cultured at 22°C in a 220rpm shaker and incubated for 60 minutes, then centrifuged at 8°C and 10,000rpm in a centrifuge for 2 minutes and the supernatant was discarded. 200 μL of physiological saline was added and mixed by pipetting with a pipette. 100 μL was injected into an LB plate culture dish, and the culture dish was placed at 37°C for 48 hours before counting. All experiments were repeated three times in parallel, with no natural plant extract essential oil composition solution or any diluent added as a blank control. The experimental results are shown in the attached Figure 8 and attached Fig. 9 .

[0083] The experimental results show that compared with the blank control group, the natural plant essential oil composition of this formula mixed with Escherichia coli has a killing effect on Escherichia coli when diluted 100 and 300 times, and the inhibition rate of the original solution diluted 1000 times on Escherichia coli is still more than 99%.

[0084] Example 9: Determination of the inhibitory effect of plant essential oil composition on Escherichia coli by fumigation method 30 μL of Escherichia coli (1.5×10 3 CFU / mL) were exposed to the volatile atmosphere of the natural plant essential oil composition or rapeseed oil (control group) in Example 3 for 15, 30 and 60 minutes, and the slides were repeatedly rinsed with 200 μL of saline. The washed bacterial solution was centrifuged at 10000 rpm at 8°C for 2 minutes and the supernatant was discarded. 100 μL of saline was added to resuspend by shaking and 30 μL was injected into the LB plate culture dish. The culture dish was placed at 37°C and cultured for 48 hours before the colony count was performed. All experiments were repeated three times in parallel. The experimental results are shown in the attached figure. Fig.10 and attached Fig.11 .

[0085] The experimental results show that the natural plant essential oil composition of the formula can kill Escherichia coli after fumigation treatment for 60 minutes.

[0086] Example 10: Determination of the inhibitory effect of plant essential oil composition on Staphylococcus aureus by mixing method The natural plant essential oil composition solution of Example 1 of the present invention was diluted 100, 300, 1000, 3000, and 10000 times with dimethyl sulfoxide and mixed with Staphylococcus aureus (1.5×10 3CFU / mL) was cultured at 22°C in a 220rpm shaker and incubated for 60 minutes, then centrifuged at 8°C and 10,000rpm in a centrifuge for 2 minutes and the supernatant was discarded. 200 μL of physiological saline was added and mixed by pipetting with a pipette. 100 μL was injected into an LB plate culture dish, and the culture dish was placed at 37°C for 48 hours before counting. All experiments were repeated three times in parallel, with no natural plant extract essential oil composition solution or any diluent added as a blank control. The experimental results are shown in the attached Fig.12 .

[0087] The experimental results show that compared with the blank control group, the natural plant essential oil composition of this formula mixed with Staphylococcus aureus, the original solution diluted 100-10000 times has a certain inhibitory effect on Staphylococcus aureus.

[0088] Example 11: Determination of the inhibitory effect of plant essential oil composition on Staphylococcus aureus by fumigation method 30 μL of Staphylococcus aureus (1.5×10 3 CFU / mL) were exposed to the volatile atmosphere of the natural plant essential oil composition or rapeseed oil (control group) in Example 1 for 15, 30, 60 and 120 minutes, and the slides were repeatedly rinsed with 200 μL of saline. The washed bacterial solution was centrifuged at 10000 rpm at 8°C for 2 minutes and the supernatant was discarded. 100 μL of saline was added to resuspend by shaking and 30 μL was injected into the LB plate culture dish. The culture dish was placed at 37°C and cultured for 48 hours before the colony count was performed. All experiments were repeated three times in parallel. The experimental results are shown in the attached figure. Fig.13 .

[0089] The experimental results show that the natural plant essential oil composition of this formula has a good inhibitory effect on Staphylococcus aureus after 60 minutes of fumigation treatment, and can kill Staphylococcus aureus after 120 minutes of action.

[0090] Example 12: Determination of the inhibitory effect of plant essential oil composition on Pasteurella multocida by mixing method The natural plant essential oil composition solution of Example 1 of the present invention was diluted 100, 300, 1000, 3000, and 10000 times with dimethyl sulfoxide and mixed with Pasteurella multocida (1.5×10 3CFU / mL) was cultured at 22°C in a 220rpm shaker and incubated for 60 minutes, then centrifuged at 8°C and 10,000rpm in a centrifuge for 2 minutes and the supernatant was discarded. 200 μL of normal saline was added and mixed by pipetting with a pipette. 100 μL was injected into a BHI and 5% defibrinated sheep blood plate culture dish, and the culture dish was placed at 37°C for 48 hours before counting. All experiments were repeated three times in parallel, with no natural plant extract essential oil composition solution or any diluent added as a blank control. The experimental results are shown in the attached Fig.14 .

[0091] The experimental results show that compared with the blank control group, the natural plant essential oil composition of this formula mixed with Pasteurella multocida has a killing effect on Pasteurella multocida when diluted 100 and 300 times, and the original solution diluted 1000 times still has a certain inhibitory effect on Pasteurella multocida.

[0092] Example 13: Determination of the inhibitory effect of plant essential oil composition on Pasteurella multocida by fumigation method

[0093] 30 μL of Pasteurella multocida (1.5×10 3 CFU / mL) were exposed to the volatile atmosphere of the natural plant essential oil composition or rapeseed oil (control group) in Example 1 for 15, 30 and 60 minutes, and the slides were repeatedly rinsed with 200 μL of saline. The washed bacterial solution was centrifuged at 10000 rpm at 8°C for 2 minutes and the supernatant was discarded. 100 μL of saline was added to resuspend by shaking and 30 μL was injected into BHI and 5% defibrinated sheep blood plate culture dishes. The culture dishes were placed at 37°C and cultured for 48 hours before colony counting. All experiments were repeated three times in parallel. The experimental results are shown in the attached Fig.15 .

[0094] The experimental results show that the natural plant essential oil composition of this formula has an inhibitory effect on Pasteurella multocida after 15 and 30 minutes of fumigation treatment, and the inhibition rate of Pasteurella multocida exceeds 95% after 60 minutes of action.

[0095] Example 14: Determination of the inhibitory effect of plant essential oil composition on Clostridium perfringens by mixing method The original solution of the natural plant essential oil composition in Example 1 of the present invention was diluted 100, 300, 1000, and 3000 times with dimethyl sulfoxide and mixed with Clostridium perfringens (1.5×10 3CFU / mL) was cultured at 22°C in a 220rpm shaker and incubated for 60 minutes, then centrifuged at 8°C and 10,000rpm in a centrifuge for 2 minutes and the supernatant was discarded. 200 μL of physiological saline was added and mixed by pipetting with a pipette. 100 μL was injected into an LB plate culture dish, and the culture dish was placed at 37°C for 48 hours before counting. All experiments were repeated three times in parallel, with no natural plant extract essential oil composition solution or any diluent added as a blank control. The experimental results are shown in the attached Fig.16 .

[0096] The experimental results show that compared with the blank control group, the natural plant essential oil composition of this formula mixed with Clostridium perfringens has a killing effect on Clostridium perfringens when diluted 100 and 300 times, and the original solution diluted 1000 times still has a good inhibitory effect on Clostridium perfringens.

[0097] Example 15: Determination of the inhibitory effect of plant essential oil composition on Clostridium perfringens by fumigation method 30 μL of Clostridium perfringens (1.5×10 3 CFU / mL) were exposed to the volatile atmosphere of the natural plant essential oil composition or rapeseed oil (control group) in Example 1 for 15, 30 and 60 minutes, and the slides were repeatedly rinsed with 200 μL of saline. The washed bacterial solution was centrifuged at 10000 rpm at 8°C for 2 minutes and the supernatant was discarded. 100 μL of saline was added to resuspend by shaking and 30 μL was injected into the LB plate culture dish. The culture dish was placed at 37°C and cultured for 48 hours before the colony count was performed. All experiments were repeated three times in parallel. The experimental results are shown in the attached figure. Fig.17 .

[0098] The experimental results show that the natural plant essential oil composition of the formula has a significant inhibitory effect on Clostridium perfringens after fumigation treatment of Clostridium perfringens for 30 and 60 minutes.

[0099] Example 16: Determination of the inhibitory effect of plant essential oil composition on Haemophilus paragallinarum by mixing method The natural plant essential oil composition solution of Example 1 of the present invention was diluted 100, 300, 1000, 3000 or 10000 times with dimethyl sulfoxide and mixed with Haemophilus paragallinarum (1.5×10 3CFU / mL) was cultured and incubated together at 22°C in a 220rpm shaker for 60 minutes, then centrifuged at 10,000rpm for 2 minutes at 8°C in a centrifuge and the supernatant was discarded, 100 μL of physiological saline was added and mixed by pipetting with a pipette, 50 μL was injected into a BHI plate, 5% defibrinated sheep blood and 4% chicken serum plate culture dish, and the culture dish was placed at 37°C for 48 hours for counting. All experiments were repeated three times in parallel, with no natural plant extract essential oil composition solution or any diluent added as a blank control.

[0100] The experimental results show that compared with the blank control group, the natural plant essential oil composition of this formula mixed with Haemophilus paragallinarum, the original solution diluted 100-10000 times has a certain inhibitory effect on Haemophilus paragallinarum.

[0101] Example 17: Determination of the inhibitory effect of plant essential oil composition on Haemophilus paragallinarum by fumigation method 30 μL of Haemophilus paragallinarum (1.5×10 3 CFU / mL) were exposed to the volatile atmosphere of the natural plant essential oil composition or rapeseed oil (control group) in Example 1 for 15, 30, 60, and 120 minutes, and the slides were repeatedly rinsed with 100 μL of physiological saline. The washed bacterial solution was centrifuged at 10000 rpm at 8°C for 2 minutes and the supernatant was discarded. 100 μL of physiological saline was added to oscillate and resuspend, and 30 μL was injected into BHI plates, 5% defibrinated sheep blood and 4% chicken serum plate culture dishes, and the culture dishes were placed at 37°C for 48 hours for colony counting. All experiments were repeated three times in parallel.

[0102] The experimental results show that the natural plant essential oil composition of this formula has a significant inhibitory effect on Haemophilus paragallinarum after fumigation treatment for 30 and 60 minutes, and can kill Haemophilus paragallinarum after 120 minutes.

[0103] Example 18: Determination of the inhibitory effect of plant essential oil composition on Mycoplasma gallisepticum by mixing method The natural plant essential oil composition solution of Example 1 of the present invention was diluted 100, 300, 1000, 3000, or 10000 times with dimethyl sulfoxide and mixed with Mycoplasma gallisepticum (1.5×10 6CFU / mL) was cultured and incubated together at 22°C in a 220rpm shaker for 60 minutes, then centrifuged at 10,000rpm for 2 minutes at 8°C in a centrifuge and the supernatant was discarded, 100 μL of physiological saline was added and mixed by pipetting with a pipette, 50 μL was injected into a modified Frey agar plate, 10% inactivated porcine serum and 0.1% ampicillin plate culture dish, and the culture dish was placed at 37°C for 4 days before counting. All experiments were repeated three times in parallel, with no addition of the original solution of the natural plant extract essential oil composition or any diluent as a blank control.

[0104] The experimental results show that compared with the blank control group, the natural plant essential oil composition of this formula mixed with Mycoplasma gallisepticum, the original solution diluted 100 and 300 times has a killing effect on Mycoplasma gallisepticum, and the original solution diluted 1000 times still has a good inhibitory effect on Mycoplasma gallisepticum.

[0105] Example 19: Determination of the inhibitory effect of plant essential oil composition on Mycoplasma gallisepticum by fumigation method 40 μL of Mycoplasma gallisepticum bacteria (2.37×10 8 CFU / mL) were exposed to the volatile atmosphere of the natural plant essential oil composition or rapeseed oil (control group) in Example 1 for 15, 30 and 60 minutes, and the slides were repeatedly rinsed with 100 μL of saline. The washed bacterial solution was centrifuged at 10000 rpm at 8°C for 2 minutes and the supernatant was discarded. 100 μL of saline was added to resuspend by shaking and 50 μL was injected into the LB plate culture dish, and the culture dish was placed at 37°C for 4 days before the colony count. All experiments were repeated three times in parallel.

[0106] The experimental results show that the natural plant essential oil composition of this formula has a significant inhibitory effect on Mycoplasma gallisepticum after fumigation treatment for 15 and 30 minutes, and can kill Mycoplasma gallisepticum after 60 minutes.

[0107] Example 20: Determination of the inhibitory effect of plant essential oil composition on enterococci by mixing method The plant extract essential oil composition solution of Example 1 of the present invention was diluted 100, 200, 400, and 1000 times with dimethyl sulfoxide and then mixed with enterococci (1.5×10 3 CFU / mL) was cultured at 22°C in a 220rpm shaker and incubated for 60 minutes, then centrifuged at 8°C and 10,000rpm in a centrifuge for 2 minutes and the supernatant was discarded. 200 μL of physiological saline was added and mixed by pipetting with a pipette. 100 μL was injected into an LB plate culture dish, and the culture dish was placed at 37°C for 48 hours before counting. All experiments were repeated three times in parallel, with no natural plant extract essential oil composition solution or any diluent added as a blank control. The experimental results are shown in the attached Fig.18.

[0108] The experimental results show that compared with the blank control group, the inhibition rate of enterococci by the original solution diluted 100 times was more than 99% when mixed with enterococci, and the original solution diluted 100-1000 times still had a good inhibitory effect on enterococci.

[0109] Example 21: Determination of the inhibitory effect of plant essential oil composition on Streptococcus by mixing method The natural plant essential oil composition solution of Example 1 of the present invention was diluted 100, 200, 400, and 1000 times with dimethyl sulfoxide and mixed with Streptococcus (1.5×10 3 CFU / mL) was cultured at 22°C in a 220rpm shaker and incubated for 60 minutes, then centrifuged at 8°C and 10,000rpm in a centrifuge for 2 minutes and the supernatant was discarded. 200 μL of normal saline was added and mixed by pipetting with a pipette. 100 μL was injected into a blood agar plate culture dish, and the culture dish was placed at 37°C for 48 hours for counting. All experiments were repeated three times in parallel, with no natural plant essential oil composition solution or any diluent added as a blank control. The experimental results are shown in the attached Fig.19 .

[0110] The experimental results show that compared with the blank control group, the inhibition rate of Streptococcus treated with the natural plant essential oil composition of this formula is more than 99% when the original solution is diluted 100-400 times, and the inhibition rate of Streptococcus treated with the original solution diluted 1000 times is still more than 99%.

[0111] Example 22: Determination of the inhibitory effect of plant essential oil composition on Aspergillus fumigatus by mixing method The plant extract essential oil composition solution of Example 1 of the present invention was diluted 100, 200, 400, and 1000 times with dimethyl sulfoxide and mixed with Aspergillus fumigatus (1.5×10 3 CFU / mL) was cultured at 22°C in a 220rpm shaker and incubated for 60 minutes, then centrifuged at 8°C and 10,000rpm in a centrifuge for 2 minutes and the supernatant was discarded. 200 μL of normal saline was added and mixed by pipetting with a pipette. 100 μL was injected into a Sabouraud agar plate and cultured in a culture dish. The culture dish was placed at 28°C and cultured for 72 hours before counting. All experiments were repeated three times in parallel, with the blank control group without adding the original solution of the plant extract essential oil composition or any diluent. The experimental results are shown in the attached Fig. 20 .

[0112] The experimental results show that compared with the blank control group, the inhibition rate of Aspergillus fumigatus treated with the natural plant essential oil composition of this formula on Aspergillus fumigatus diluted 100-1000 times was more than 99%.

[0113] Example 23: Determination of the inhibitory effect of plant essential oil composition on Aspergillus niger by fumigation method 30 μL of Aspergillus niger (1.5×10 3 CFU / mL) were exposed to the volatile atmosphere of the natural plant essential oil composition or rapeseed oil (control group) in Example 1 for 4 and 24 hours, and the slides were repeatedly rinsed with 200 μL of saline. The washed bacterial solution was centrifuged at 10000 rpm at 8°C for 2 minutes and the supernatant was discarded. 100 μL of saline was added to resuspend by shaking and 30 μL was injected into the modified Martin medium. The culture dish was placed at 37°C and cultured for 48 hours before colony counting. All experiments were repeated three times in parallel. The experimental results are shown in the attached figure. Fig.21 .

[0114] The experimental results show that after 4 hours of fumigation treatment with the natural plant essential oil composition of this formula, the inhibition rate of Aspergillus niger exceeds 99%, and after 24 hours of action, it still has a significant inhibitory effect on Aspergillus niger.

[0115] Example 24: Determination of the inhibitory effect of plant essential oil composition on Candida albicans by mixing method The plant extract essential oil composition obtained in Example 1 of the present invention was diluted 100, 150 and 200 times with dimethyl sulfoxide to obtain the dilutions and mixed with Candida albicans (1.5×10 3 CFU / mL) was cultured at 22°C in a 220rpm shaker and incubated for 60 minutes, then centrifuged at 8°C and 10,000rpm in a centrifuge for 2 minutes and the supernatant was discarded. 200 μL of normal saline was added and mixed by pipetting with a pipette. 100 μL was injected into a Sabouraud agar plate and cultured in a culture dish. The culture dish was placed at 28°C and cultured for 24 hours before counting. All experiments were repeated three times in parallel, with the blank control group without adding the natural plant extract essential oil composition stock solution dilution. The experimental results are shown in the attached Fig. 22 .

[0116] The experimental results show that compared with the blank control group, the natural plant essential oil composition of this formula mixed with Candida albicans has a killing effect on Candida albicans when diluted 100-150 times, and the inhibition rate of Candida albicans when diluted 200 times exceeds 99%.

[0117] Example 25: Determination of the inhibitory effect of plant essential oil composition on Candida albicans by fumigation method 30 μL of Candida albicans (1.5×10 3CFU / mL) were exposed to the volatile atmosphere of the natural plant essential oil composition or rapeseed oil (control group) in Example 1 for 4 and 24 hours, and the slides were repeatedly rinsed with 200 μL of saline. The washed bacterial solution was centrifuged at 10000 rpm at 8°C for 2 minutes and the supernatant was discarded. 100 μL of saline was added to vortex and resuspend, and 30 μL was injected into the culture dish on the Sabouraud agar plate. The culture dish was placed at 37°C and cultured for 48 hours before colony counting. All experiments were repeated three times in parallel. The experimental results are shown in the attached figure. Fig.23 .

[0118] The experimental results show that the natural plant essential oil composition of this formula has a significant inhibitory effect on Candida albicans after 4 hours of fumigation, and can kill Candida albicans after 24 hours of action.

[0119] Example 26: Determination of the inhibitory effect of plant essential oil composition on porcine coronavirus by fumigation method 10 μL of porcine coronavirus (1.0×10 7 TCID 50 / mL) were exposed to the volatile atmosphere of the natural plant essential oil composition or rapeseed oil (control group) in Example 1 for 0, 15, 30, 45 and 60 minutes, the slides were repeatedly rinsed with 1000 μL of physiological saline, the viruses were resuspended and mixed, the resuspended viruses were diluted by 10^1 to 8 times, and then added to the cultured Vero cells, and each dilution was repeated in 4 wells. Five days after infection, the cytopathic effect of each well was observed under an inverted microscope, and the TCID of samples with different treatment times was calculated by the Reed-Muench method. 50 All experiments were repeated three times in parallel. The experimental results are shown in the attached Fig.24 .

[0120] The experimental results show that the natural plant essential oil composition of this formula has a significant inhibitory effect on porcine coronavirus after 30 minutes of fumigation treatment of porcine coronavirus, and the inhibition rate of porcine coronavirus exceeds 99.99% after 45 and 60 minutes of action.

[0121] Example 27: Determination of the inhibitory effect of plant essential oil composition on avian influenza A virus by fumigation method 10 μL of avian influenza A virus (5×10 4PFU / mL) were exposed to the natural plant essential oil composition or rapeseed oil volatilization atmosphere in Example 1 for 10, 20 and 30 minutes, and the slides were repeatedly rinsed with 1000 μL of saline to resuspend the virus and mix well, and then added to the cultured A549 cells. After the virus adsorbed for 1 hour, a layer of melted semi-solid nutrient agar was covered to allow the virus to diffuse in the monolayer cell culture, and the plaques were counted to calculate the titer. All experiments were repeated three times in parallel, and the experimental results are shown in the attached Fig.25 .

[0122] The experimental results show that the natural plant essential oil composition of this formula can inhibit the type A avian influenza virus by fumigation treatment, and the inhibition rate of the type A avian influenza virus exceeds 99% after 10 and 20 minutes of action, and has a killing effect on the type A avian influenza virus after 30 minutes of action.

[0123] Example 28: Determination of the inhibitory effect of plant essential oil composition on duck Tembusu virus by fumigation method 10 μL duck Tembusu virus (1.0×10 7 TCID 50 / mL) were exposed to the volatile atmosphere of the natural plant essential oil composition or rapeseed oil (control group) in Example 1 for 10, 30 and 45 minutes, and the slides were repeatedly rinsed with 1000 μL of physiological saline to resuspend the virus and mix well. The resuspended virus was diluted by 10^1 to 8 times and added to the cultured DF-1 cells respectively. Each dilution was repeated in 4 wells. Five days after infection, the cytopathic effect of each well was observed under an inverted microscope, and the TCID of samples with different treatment times was calculated by the Reed-Muench method. 50 All experiments were repeated three times in parallel. The experimental results are shown in the attached Fig.26 .

[0124] The experimental results show that the natural plant essential oil composition of this formula can inhibit duck Tembusu virus by fumigation treatment, and the inhibition rate of duck Tembusu virus reaches 90% after 30 and 45 minutes of action, and the inhibition rate of duck Tembusu virus exceeds 95% after 60 minutes of action.

[0125] Example 29: Determination of the inhibitory effect of plant essential oil composition on goose parvovirus by fumigation method 10 μL goose parvovirus (1.0×10 5 TCID 50 / mL) were exposed to the volatile atmosphere of the natural plant essential oil composition or rapeseed oil (control group) in Example 1 for 1, 2, 3, 4, 5, and 6 hours, and the slides were repeatedly rinsed with 1000 μL of physiological saline to resuspend the virus and mix well. The resuspended virus was diluted by 10^1 to 8 times and added to the cultured GEF cells respectively. Each dilution was repeated in 4 wells. Five days after infection, the cells were stained by indirect immunofluorescence. The cytopathic effect of each well was observed under an inverted microscope, and the TCID of samples with different treatment times was calculated by the Reed-Muench method. 50 All experiments were repeated three times in parallel.

[0126] The experimental results show that the natural plant essential oil composition of this formula can inhibit goose parvovirus by fumigation treatment. After 2 hours of action, the inhibition rate of goose parvovirus reaches 96.8%, and after 6 hours of action, the inhibition rate of goose parvovirus exceeds 99%.

[0127] Example 30: Determination of the inhibitory effect of plant essential oil composition on infectious bursal disease virus by fumigation method. 10 μL of infectious bursal disease virus (1.0×10 7 TCID 50 / mL) were exposed to the volatile atmosphere of the natural plant essential oil composition or rapeseed oil (control group) in Example 1 for 1, 2, 3, 4, 5, and 6 hours, and the slides were repeatedly rinsed with 1000 μL of physiological saline to resuspend the virus and mix well. The resuspended virus was diluted by 10^1 to 8 times and added to the cultured DF-1 cells respectively. Each dilution was repeated in 4 wells. Five days after infection, the cytopathic effect of each well was observed under an inverted microscope, and the TCID of samples with different treatment times was calculated by the Reed-Muench method. 50 All experiments were repeated three times in parallel.

[0128] The experimental results show that after fumigation treatment with the natural plant essential oil composition of this formula on the infectious bursal disease virus of chickens, the inhibition rate of the infectious bursal disease virus of chickens exceeds 99.9% after 3 hours of action, and has a killing effect on the infectious bursal disease virus of chickens after 4 hours of action.

[0129] Example 31: Antibiotic-free breeding experiment of broiler chickens 15,000 one-day-old broiler white-feathered chicken mixed chicks (Shandong Yisheng Breeding Livestock and Poultry Co., Ltd.) were selected, and the natural plant extract essential oil composition in Example 1 of the present invention was used instead of antibiotics for breeding as the experimental group; another 15,000 one-day-old broiler white-feathered chicken mixed chicks (Shandong Yisheng Breeding Livestock and Poultry Co., Ltd.) were taken, and antibiotics were used during the breeding process as a conventional breeding control group. The effective breeding area of ​​the experimental group and the control group was 780 square meters, and the breeding density was 19 per square meter. After the experimental group and the control group were thoroughly disinfected by spraying and fumigating with potassium permanganate before the chicks entered the pens, the experimental group used a blast equipment to fully distribute the natural plant extract essential oil composition solution in Example 1 of the present invention in the chicken house before the chicks entered the pens, and the control group was ventilated and used. Before the chicks entered the pens, the temperature and humidity in the chicken house were maintained at 32-35°C and 55-70%, respectively.

[0130] The chicks in the experimental group and the control group were allowed to drink water and eat freely during the period of feeding after entering the pen, and were managed according to the conventional feeding procedures for white-feathered broilers. The immunization was carried out according to the conventional immunization procedures: 0.15 mL / bird of infectious bursal vaccine was injected subcutaneously in the neck at 1 day old; Newcastle disease and infectious bronchitis two-in-one live vaccine was administered by nose drops and eye drops at 7 days old; Newcastle disease live vaccine was administered by nose drops and eye drops at 21 days old. 1-12 days is the seedling stage, using a small feed bucket, adding feed (Dongguan Haida Feed Co., Ltd.) 4-6 times a day, and the indoor temperature is basically maintained at 34-30℃; 13-28 days is the rearing period, switching to feeding troughs, automatically adding feed 1-2 times a day, mixing manually, and the indoor temperature should be gradually reduced to about 26℃; after 28 days, it is the fattening period, adding feed 3-5 times a day, and the indoor temperature is reduced to about 24℃ until the chickens are marketed at 40 days old.

[0131] The experimental group used a blast device to assist in uniformly volatilizing the natural plant essential oil composition solution in Example 1 of the present invention into the chicken house, and the amount used was 400 grams per day. No antibiotics were used during the feeding process of the experimental group. The control group used enrofloxacin once (0.003g / hen), amoxicillin once (0.003g / hen), and spectinomycin once (0.003g / hen) in the seedling stage, tilmicosin twice (0.003g / hen), decoquinate once (0.003g / hen) in the breeding period, and amoxicillin once (0.006g / hen) in the fattening period, all of which were added to the feed for consumption.

[0132] After the experiment, the survival rate and average body weight of the chickens in the experimental group and the control group were recorded. The results are shown in Table 1.

[0133] Table 1 Effect of natural plant essential oil composition in antibiotic-free breeding of broiler white-feathered chickens Survival rate Average weight Experimental group (natural plant essential oil composition) 92.6％ 2.8kg Control group (antibiotics) 90％ 2.6kg

[0134] The experimental results show that compared with the control group using antibiotics, the survival rate and average body weight of broilers in the antibiotic-free breeding group treated with the natural plant essential oil composition of this formula were improved, confirming that the natural plant essential oil composition of the present invention can be used to replace antibiotics for antibiotic-free breeding of chickens.

[0135] Example 32: Antibiotic-free breeding experiment of meat ducks 7000 one-day-old meat ducks Cherry Valley ducks (Shandong Yisheng Breeding Livestock and Poultry Co., Ltd.) were selected, and the natural plant extract essential oil composition in Example 2 of the present invention was used instead of antibiotics for breeding as the experimental group; another 7000 one-day-old meat ducks Cherry Valley ducks (Shandong Yisheng Breeding Livestock and Poultry Co., Ltd.) were selected, and antibiotics were used during the breeding process as the conventional breeding control group. The effective breeding area of ​​the experimental group and the control group is 1000 square meters, and the breeding density is 7 per square meter. After the experimental group and the control group were thoroughly disinfected by spraying and fumigating with potassium permanganate before the ducklings entered the pen, the experimental group used a blast equipment to fully distribute the original solution of the natural plant extract essential oil composition in Example 2 of the present invention in the duck house before the ducklings entered the pen, and the control group was ventilated and used. Before the ducklings enter the pen, the temperature and humidity in the duck house are maintained at 33°C and 60%, respectively.

[0136] The experimental and control ducklings were allowed to eat and drink freely during the feeding period after entering the pen, and were managed according to the conventional feeding and management procedures of Cherry Valley ducks. The immunization was carried out according to the conventional immunization procedures: 1 day old, subcutaneous injection of goose plague live vaccine 0.5mL / bird; 7 days old, subcutaneous injection of goose plague live vaccine 1mL / bird again; 10 days old, subcutaneous injection of goose paramyxovirus goose influenza bivalent inactivated vaccine. 1-12 days is the brooding period, the daily feed is gradually increased from 100 kg to 400 kg, the indoor temperature should be gradually reduced to about 20℃, and the humidity is basically maintained at 60%; 13-42 days is the growth period, the daily feed is gradually increased from 600 kg to 2000 kg, the indoor temperature is maintained at 20℃, and the humidity is maintained at 50%.

[0137] The experimental group used a blast device to evenly volatilize the natural plant essential oil composition in Example 2 of the present invention into the duck house, and the amount used was 500 g / day. No antibiotics were used during the feeding process of the experimental group. The control group used three kinds of drugs during the growth period, namely florfenicol, Feibao and colistin sulfate, all of which were added to the feed for consumption.

[0138] After the experiment, the survival rate and average body weight of ducks in the experimental and control groups were recorded. The results are shown in Table 2.

[0139] Table 2 Effect of plant essential oil composition in antibiotic-free breeding of meat ducks Cherry Valley ducks Survival rate Average weight Experimental group (natural plant essential oil composition) 95.26％ 3.4kg Control group (antibiotics) 97.14％ 3.4kg

[0140] The experimental results show that compared with the control group using antibiotics, the survival rate and average weight of the ducks in the antibiotic-free farming group treated with the natural plant essential oil composition of this formula did not change significantly, confirming that the natural plant essential oil composition of the present invention can be used to replace antibiotics for antibiotic-free farming of ducks.

Claims

1. A natural plant essential oil composition and application for antibiotic-free farming, characterized in that: The composition comprises geranium oil, cedar leaf oil, citrus lemon peel oil, bergamot oil and tea tree oil. The weight ratio of geranium oil: cedar leaf oil: citrus lemon peel oil: bergamot oil: tea tree oil in the natural plant essential oil composition is 2-6: 5-11: 1-5: 2-4: 1-3.

2. The natural plant essential oil composition according to claim 1, characterized in that: The weight ratio of geranium oil: cedar leaf oil: citrus lemon peel oil: bergamot oil: tea tree oil in the natural plant essential oil composition is 4:8:3:3:

2.

3. The natural plant essential oil composition according to claim 1, characterized in that: The weight ratio of geranium oil: cedar leaf oil: citrus lemon peel oil: bergamot oil: tea tree oil in the natural plant essential oil composition is 6:6:1:4:

3.

4. The natural plant essential oil composition according to claim 1, characterized in that: The weight ratio of geranium oil: cedar leaf oil: citrus lemon peel oil: bergamot oil: tea tree oil in the natural plant essential oil composition is 2:10:5:2:

1.

5. Use of the natural plant essential oil composition according to any one of claims 1 to 4 in the preparation of a disinfectant for livestock and poultry breeding, which kills or inhibits pathogenic bacteria, fungi, and viruses in breeding sites, replaces the effect of antibiotics, and achieves antibiotic-free breeding.

6. The use according to claim 5, characterized in that: The pathogenic bacteria are further preferably: Riemerella anatipestifer, Salmonella pullorum, Escherichia coli, Staphylococcus aureus, Pasteurella multocida, Clostridium perfringens, Haemophilus paragallinarum, Mycoplasma gallisepticum, Enterococcus, Streptococcus; the pathogenic fungi are further preferably: Candida albicans, Aspergillus fumigatus, Aspergillus niger; the viruses are further preferably: duck plague virus, Newcastle disease virus, avian influenza virus, duck hepatitis A virus, infectious bronchitis virus, avian adenovirus, porcine coronavirus, goose parvovirus, duck Tembusu virus.

7. The use according to claim 5, characterized in that: The livestock and poultry are pigs, cattle, sheep, horses, camels, rabbits, cats, dogs, chickens, ducks, geese, pigeons, turkeys or quails.

8. The use according to claim 5, characterized in that: The livestock and poultry are preferably pigs, cattle, sheep, chickens, ducks, geese or quails.

9. The use according to claim 5, characterized in that: The livestock and poultry are more preferably chickens, ducks, and geese.

10. The use according to claim 9, characterized in that: Among the livestock and poultry, chickens are more preferably white-feathered chickens, and among the livestock and poultry, ducks are more preferably cherry valley ducks.

11. The use according to claim 5, characterized in that: The method of using the disinfectant is to dilute the disinfectant concentrate directly or with water or other solvents in different volume ratios, and distribute the diluted solution in the breeding site by placing volatilization, spraying, mixing or matching equipment.

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