A natural plant extract essential oil composition for antibiotic-free aquaculture and its application

Abstract

This invention provides a natural plant extract essential oil composition for antibiotic-free farming and its application. The natural plant extract essential oil composition is formulated from geranium oil, cedarwood leaf oil, citrus lemon peel oil, bergamot oil, and tea tree oil. Experiments have shown that this natural plant extract essential oil composition can effectively kill or inhibit various common pathogenic microorganisms in poultry farming, including bacteria, viruses, and fungi. Therefore, it can be used in the preparation of disinfectants for common pathogenic microorganisms in poultry farming. The raw materials of this invention are simple, readily available, non-toxic, harmless, and environmentally friendly, and the preparation method is simple to operate. The natural plant extract essential oil disinfectant is convenient, safe, and efficient in livestock and poultry farming, and can replace antibiotics to prevent and treat livestock and poultry diseases, improve the survival rate and meat yield of livestock and poultry, achieve green antibiotic-free farming, and ensure the safety and health of livestock and poultry meat.

Description

Technical Field

[0001] This invention belongs to the field of antibiotic-free aquaculture technology, specifically relating to a natural plant extract essential oil composition for antibiotic-free aquaculture and its application. It is a natural plant extract essential oil composition that has the effect of inhibiting pathogenic bacteria, fungi and viruses in aquaculture and its application in antibiotic-free aquaculture. Background Technology

[0002] With social development and improved economic levels, traditional free-range farming methods can no longer meet the meat demand of my country's 1.4 billion population. my country's farming model has rapidly shifted towards intensive and large-scale high-density farming. However, dense farming environments easily spread pathogenic bacteria, fungi, and viruses, leading to widespread disease and death among farmed animals and causing huge economic losses. To reduce disease in farmed animals, various broad-spectrum antibiotics with antibacterial, antifungal, and antiviral effects are widely used in high-density farming. Furthermore, antibiotics are widely added to animal feed as growth promoters, improving the economic efficiency of the farming industry. These factors have led to a serious dependence on antibiotics in the farming industry. In recent years, countries worldwide have recognized the risk of antibiotic overuse in the farming industry leading to the emergence of drug-resistant pathogens. At the same time, long-term sub-dose use of antibiotics reduces the immunity of farmed animals and easily leads to cross-infection of pathogens, posing a significant long-term risk to the farming industry. Even more seriously, antibiotic residues in 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 conditions of large-scale farming in my country, including density and disease prevention, directly discontinuing the use of antibiotics would significantly increase morbidity and mortality rates in livestock, reduce farming efficiency, and lead to greater economic losses. Currently, some antibiotic alternatives have emerged on the market, mainly feed additives, including acidifiers, microecological preparations, bioactive peptides, and traditional Chinese medicine preparations. Their mechanism of action is similar to antibiotics; they are added to feed and ingested by farmed animals, exhibiting certain antibacterial effects or enhancing their immunity. However, these products have high production costs, making large-scale promotion difficult, and they cannot fundamentally solve the problem of high morbidity rates in high-density farming. Furthermore, since these products are still added to feed, there is still a risk of residues in the animals' bodies.

[0004] Pathogenic microorganisms, including pathogenic bacteria, fungi, and viruses, are widely present and easily spread in the farming environment. When farmed animals are infected by these microorganisms, they quickly develop illnesses, leading to widespread outbreaks and deaths. Currently, the main pathogenic bacteria found in large-scale farming include *Rimeria anatipestifer*, *Salmonella pullorum*, *Escherichia coli*, *Staphylococcus aureus*, *Pasteurella multocida*, *Campylobacter jejuni*, *Clostridium perfringens*, *Haemophilus paragallinarum*, *Mycoplasma gallisepticum*, *Enterococcus*, and *Streptococcus*. Pathogenic fungi include *Candida albicans*, *Aspergillus fumigatus*, and *Aspergillus niger*. Pathogenic viruses include duck plague virus, Newcastle disease virus, avian influenza virus, fowlpox virus, avian adenovirus, duck hepatitis A virus, infectious bronchitis virus, infectious bursal disease virus, porcine coronavirus, parvovirus, Tembusu virus, and classical swine fever virus. The core issue of antibiotic-free farming is reducing the number of pathogenic microorganisms such as bacteria, fungi, and viruses in an antibiotic-free environment, blocking their transmission and infection of farmed animals, reducing morbidity, and improving economic efficiency. Natural plant essential oils, rich in active substances such as ketones, terpenes, aldehydes, and alcohols, possess immune-enhancing, antibacterial, and antiviral activities and are widely used in pharmaceuticals, antibacterial agents, and antiviral agents. Furthermore, natural plant essential oils are non-toxic, harmless, and aromatic, environmentally friendly, and pose no safety risks, making them widely used in air disinfection. Therefore, developing natural plant essential oil compositions to inhibit or kill pathogenic bacteria, fungi, and viruses in the farming environment can reduce morbidity and mortality in farmed animals, lower farming risks, and achieve antibiotic-free farming, possessing significant economic and social benefits. Summary of the Invention

[0005] The purpose of this invention is to provide a natural plant extract essential oil composition for antibiotic-free aquaculture, which is prepared by using natural plant extract essential oils in different proportions as raw materials. The raw materials used are simple, readily available, non-toxic, harmless, green and environmentally friendly. The preparation method is simple to operate. The resulting natural plant extract essential oil composition is convenient to use, has significant effects, and has no environmental pollution or safety hazards.

[0006] The natural plant extract essential oil composition used in this invention is a mixture of geranium oil, cedarwood leaf oil, citrus lemon peel oil, bergamot oil, and tea tree oil in specific proportions. 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 components are geranium oil, cedar leaf oil, citrus lemon peel oil, bergamot oil, and tea tree oil in a weight ratio of 4:8:3:3:2.

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

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

[0010] This invention also provides a method for preparing natural plant essential oils for antibiotic-free aquaculture: According to the mass ratio mentioned in the above formula, weigh the corresponding natural plant essential oils and add them sequentially to a sealed mixing vessel. Control the temperature at 15-45℃ and stir at 100-1000 rpm for 1-24 hours under magnetic stirring to ensure thorough mixing of the essential oil components. After ultrasonically dispersing the plant essential oil mixture obtained in the previous step for 0.1-1 hour, allow it to stand to obtain the stock solution of the natural plant essential oil composition. This stock solution or its dilution can inhibit or kill pathogenic microorganisms in the aquaculture environment and can also be directly used in aquaculture to replace antibiotics, achieving antibiotic-free aquaculture. The stock solution of the natural plant essential oil composition obtained by this method can be directly packaged, used, and sold.

[0011] In this invention, the temperature during the mixing process is controlled at 15-45℃, preferably 25-35℃; the stirring speed is 100-1000 rpm, preferably 300-800 rpm, and more preferably 500-600 rpm; 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] In this invention, all components of the natural plant essential oil compositions mentioned refer to those obtained by common methods. For example, geranium oil, also known as geranium leaf oil, is obtained by steam distillation of fresh geranium leaves; cedar leaf oil is obtained by steam distillation or solvent extraction of the leaves of cedar, red pine, or red cedar; citrus lemon peel oil is obtained by steam distillation of fresh lemon peel (Citrus medica var. sarcodactylis); bergamot oil is obtained by steam distillation of bergamot fruit (Citrus medica var. sarcodactylis); and tea tree oil is obtained by steam distillation of Melaleuca tiliaceae leaves (Melaleuca leucorrhiza).

[0013] Another object of the present invention is to provide the application of the aforementioned natural plant extract essential oil composition in the preparation of disinfectants for common pathogenic microorganisms in poultry farming. After being placed in livestock and poultry farming areas, the disinfectant rapidly disperses into the air through direct volatilization, exerting an inhibitory effect on bacteria, viruses, and fungi, and can replace the function of antibiotics.

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

[0015] Experiments show that when the natural plant extract concentrate obtained in this invention is diluted 100, 300, 1000, 3000, or 10000 times and mixed with *Riemerella anatipestifer*, or when *Riemerella anatipestifer* is exposed to the atmosphere of the natural plant extract concentrate of this formulation for 15, 30, or 60 minutes and then re-cultured in petri dishes, the results indicate that the natural plant extract concentrate of this invention has a significant killing or inhibitory effect on *Riemerella anatipestifer* under these two different treatment modes. This demonstrates that the natural plant extract concentrate of this 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 when the natural plant extract concentrate obtained in this invention is diluted 100, 300, 1000, 3000, or 10000 times and mixed with *Salmonella pullorum*, or when *Salmonella pullorum* is exposed to the atmosphere of the natural plant extract concentrate of this formulation for 15, 30, or 60 minutes and then re-cultured in petri dishes, the results indicate that the natural plant extract concentrate of this invention has a significant killing or inhibitory effect on *Salmonella pullorum* under these two different treatment modes. This demonstrates that the natural plant extract concentrate of this invention is effective and can be used in antibiotic-free farming to kill or inhibit the pathogenic bacterium *Salmonella pullorum* in farmed animals.

[0017] Experiments show that when the natural plant extract concentrate obtained in this invention is diluted 100, 300, 1000, or 3000 times and mixed with *E. coli*, or when *E. coli* is exposed to the atmosphere of the natural plant extract composition of this formulation for 15, 30, or 60 minutes and then re-cultured in petri dishes, the results indicate that the natural plant extract composition of this invention has a significant killing or inhibitory effect on *E. coli* under these two different treatment modes. This demonstrates that the natural plant extract composition of this invention is effective and can be used to kill or inhibit pathogenic bacteria *E. coli* in farmed animals in antibiotic-free aquaculture.

[0018] Experiments show that when the natural plant extract concentrate obtained in this invention is diluted 100, 300, 1000, 3000, or 10000 times and mixed with Staphylococcus aureus, or when Staphylococcus aureus is exposed to the atmosphere of the natural plant extract concentrate of this formulation for 15, 30, 60, or 120 minutes and then re-cultured in petri dishes, the results indicate that the natural plant extract concentrate of this invention has a significant killing or inhibitory effect on Staphylococcus aureus under these two different treatment modes. This demonstrates that the natural plant extract concentrate of this invention is effective and can be used to kill or inhibit Staphylococcus aureus, a pathogenic bacterium in farmed animals, in antibiotic-free aquaculture.

[0019] Experiments show that when the natural plant extract concentrate obtained in this invention is diluted 100, 300, 1000, 3000, or 10000 times and mixed with *Pasteurella multocida*, or when *Pasteurella multocida* is exposed to the atmosphere of the natural plant extract concentrate of this formulation for 15, 30, or 60 minutes and then re-cultured in petri dishes, the results indicate that the natural plant extract concentrate of this invention has a significant killing or inhibitory effect on *Pasteurella multocida* under these two different treatment modes. This demonstrates that the natural plant extract concentrate of this invention is effective and can be used to kill or inhibit *Pasteurella multocida*, a pathogenic bacterium in farmed animals, in antibiotic-free aquaculture.

[0020] Experiments show that when the natural plant extract concentrate obtained in this invention is diluted 100, 300, 1000, or 3000 times and then mixed with *Clostridium perfringens*, or when *Clostridium perfringens* is exposed to the atmosphere of the natural plant extract concentrate of this formulation for 15, 30, or 60 minutes and then re-cultured in petri dishes, the results indicate that the natural plant extract concentrate of this invention has a significant killing or inhibitory effect on *Clostridium perfringens* under these two different treatment modes. This demonstrates that the natural plant extract concentrate of this invention is effective and can be used to kill *Clostridium perfringens*, a pathogenic bacterium in farmed animals, in antibiotic-free aquaculture.

[0021] Experiments show that when the natural plant extract concentrate obtained in this invention is diluted 100, 300, 1000, 3000, or 10000 times and mixed with *Haemophilus paragallinarum*, or when *Haemophilus paragallinarum* is exposed to the atmosphere of the natural plant extract concentrate of this formulation for 15, 30, 60, or 120 minutes and then re-cultured in petri dishes, the results indicate that the natural plant extract concentrate of this invention has a significant killing or inhibitory effect on *Haemophilus paragallinarum* under these two different treatment modes. This demonstrates that the natural plant extract concentrate of this invention is effective and can be used to kill or inhibit *Haemophilus paragallinarum*, a pathogenic bacterium in farmed animals, in antibiotic-free farming.

[0022] Experiments show that when the natural plant extract concentrate obtained in this invention is diluted 100, 300, 1000, 3000, or 10000 times and mixed with Mycoplasma gallisepticum, or when Mycoplasma gallisepticum is exposed to the atmosphere of the natural plant extract concentrate of this formulation for 15, 30, or 60 minutes and then re-cultured in petri dishes, the results indicate that the natural plant extract concentrate of this invention has a significant killing or inhibitory effect on Mycoplasma gallisepticum under these two different treatment modes. This demonstrates that the natural plant extract concentrate of this invention is effective and can be used to kill or inhibit pathogenic Mycoplasma gallisepticum in farmed animals in antibiotic-free farming.

[0023] Experiments show that diluting the natural plant essential oil concentrate obtained in this invention by 100, 200, 400, or 1000 times and mixing it with Enterococci, followed by re-culturing in petri dishes, demonstrates that the natural plant essential oil composition of this invention has a significant killing or inhibitory effect on Enterococci. This indicates that the natural plant essential oil composition of this invention is effective and can be used to kill or inhibit pathogenic bacteria Enterococci in farmed animals in antibiotic-free aquaculture.

[0024] Experiments show that dilutions of the natural plant extract concentrate obtained in this invention (100, 200, 400, or 1000 times) mixed with Streptococcus bacteria and then re-cultured in petri dishes demonstrate that the natural plant extract composition of this invention has a significant killing or inhibitory effect on Streptococcus bacteria. This indicates that the natural plant extract composition of this invention is effective and can be used to kill or inhibit Streptococcus bacteria, a pathogenic bacterium in farmed animals, in antibiotic-free aquaculture.

[0025] Experiments show that diluting the natural plant extract concentrate obtained in this invention by 100, 200, 400, or 1000 times and mixing it with Aspergillus fumigatus, followed by re-culturing in petri dishes, demonstrates that the natural plant extract composition of this invention has a significant killing or inhibitory effect on Aspergillus fumigatus. This indicates that the natural plant extract composition of this invention is effective and can be used in antibiotic-free aquaculture to kill or inhibit Aspergillus fumigatus, a pathogenic fungus in farmed animals.

[0026] Experiments showed that after exposing *Aspergillus niger* to the atmosphere of the natural plant extract essential oil composition of this formulation for 4 or 24 hours, it was re-cultured in petri dishes. The results indicated that the natural plant extract essential oil composition of this invention had a significant killing or inhibitory effect on *Aspergillus niger*. This demonstrates that the natural plant extract essential oil composition of this invention is effective and can be used in antibiotic-free aquaculture to kill the pathogenic fungus *Aspergillus niger* in farmed animals.

[0027] Experiments show that after diluting the natural plant essential oil concentrate obtained in this invention by 100, 150, or 200 times and mixing it with Candida albicans, or after exposing Candida albicans to the atmosphere of the natural plant essential oil composition of this formulation for 4 and 24 hours, and then re-culturing it in petri dishes, the results indicate that the natural plant essential oil composition of this invention has a significant killing or inhibitory effect on Candida albicans under these two different treatment modes. This demonstrates that the natural plant essential oil composition of this invention is effective and can be used to kill or inhibit the pathogenic fungus Candida albicans in farmed animals in antibiotic-free aquaculture.

[0028] Experiments showed that porcine coronavirus applied to the surface of a carrier was exposed to the natural plant extract composition of this formulation for 15, 30, 45, and 60 minutes before being re-transfected into cells. The results indicate that the natural plant extract composition of this invention has a significant inhibitory effect on porcine coronavirus. This demonstrates that the natural plant extract composition of this invention is effective and can be used in antibiotic-free farming to kill or inhibit porcine coronavirus, a pathogenic virus in farmed animals.

[0029] Experiments showed that after 10, 20, and 30 minutes of exposure to the natural plant extract composition of this formulation, avian influenza A virus applied to the surface of a carrier was re-transfected into cells. The results indicate that the natural plant extract composition of this invention has a significant inhibitory or killing effect on avian influenza A virus. This demonstrates that the natural plant extract composition of this invention is effective and can be used in antibiotic-free aquaculture to kill or inhibit pathogenic avian influenza A virus in farmed animals.

[0030] Experiments showed that duck Tembusu virus applied to the carrier surface was exposed to the natural plant extract composition of this formulation for 15, 30, 45, and 60 minutes before being re-transfected into cells. The results indicate that the natural plant extract composition of this patent has a significant killing or inhibitory effect on duck Tembusu virus. This demonstrates that the natural plant extract composition of this patent is effective and can be used to kill duck Tembusu virus, a pathogenic virus in farmed animals, in antibiotic-free aquaculture.

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

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

[0033] The natural plant extract essential oil composition of this invention exhibits a significant killing or inhibitory effect on pathogenic microorganisms in aquaculture, solving a core problem in antibiotic-free aquaculture. It kills or reduces the number of pathogenic microorganisms such as bacteria, fungi, and viruses, blocking their spread and infection of farmed economic animals, reducing morbidity, and improving the economic benefits of aquaculture. One or more of the aforementioned pathogenic microorganisms are widely present in various farmed economic animals. Furthermore, the farmed economic animals are primarily livestock and poultry, with preferred livestock including pigs, cattle, sheep, horses, camels, rabbits, cats, and dogs, and preferred poultry including chickens, ducks, geese, pigeons, turkeys, and quails.

[0034] Experiments show that in broiler chicken farming, the natural plant extract essential oil composition of this invention, distributed in the breeding area using auxiliary equipment, effectively kills or inhibits pathogenic bacteria, fungi, and viruses in the breeding environment, replacing antibiotics. This results in higher survival rates and higher market weights for broilers compared to the control group using antibiotics. This demonstrates that the natural plant extract essential oil composition of this invention is effective and can replace antibiotics in chicken farming, improving breeding efficiency and achieving antibiotic-free chicken farming.

[0035] Experiments show that in Cherry Valley duck farming, the natural plant extract essential oil composition of this invention, distributed in the farming area using auxiliary equipment, effectively kills or inhibits pathogenic bacteria, fungi, and viruses in the farming environment, replacing antibiotics. This results in the survival rate and market weight of the ducks being consistent with the control group using antibiotics. This demonstrates that the natural plant extract essential oil composition of this invention is effective and can replace antibiotics in duck farming, improving farming efficiency and achieving antibiotic-free duck farming.

[0036] The experimental results of this invention confirm that the provided natural plant extract essential oil composition has a good killing or inhibitory effect on many pathogenic microorganisms present in the breeding process, such as pathogenic bacteria like *Rimeria anguillarum*, *Salmonella pullorum*, *Escherichia coli*, *Staphylococcus aureus*, *Pasteurella multocida*, *Clostridium perfringens*, *Haemophilus paragallinarum*, *Mycoplasma gallisepticum*, *Enterococcus*, and *Streptococcus*; pathogenic fungi like *Candida albicans*, *Aspergillus fumigatus*, and *Aspergillus niger*; and pathogenic viruses like duck plague virus, Newcastle disease virus, avian influenza A virus, duck hepatitis A virus, infectious bronchitis virus, avian adenovirus, swine coronavirus, goose parvovirus, duck Tembusu virus, and infectious bursal disease virus. It is worth noting that the natural plant extract essential oil composition of this invention also shows significant killing or inhibitory effects on the aforementioned pathogenic microorganisms in breeding under fumigation, which perfectly matches the application scenario in breeding facilities. Further antibiotic-free farming trials confirmed that in broiler chicken farming, the natural plant extract essential oil composition of this invention can effectively replace traditional antibiotics. The survival rate and average slaughter weight of the broilers in the experimental group were higher than those in the control group using antibiotics. In cherry valley duck farming, the natural plant extract essential oil composition of this invention can also effectively replace traditional antibiotics. The survival rate and average slaughter weight of the ducks in the experimental group were consistent with those in the control group using antibiotics. These experiments demonstrate that the natural plant extract essential oil composition of this invention is safe, non-toxic, harmless, and environmentally friendly. It also exhibits good killing or inhibitory effects on various pathogenic microorganisms in aquaculture, can replace the role of antibiotics, improve the survival rate and slaughter weight of farmed animals, increase farming efficiency, and achieve antibiotic-free farming.

[0037] The beneficial effects of this invention are as follows: using readily available natural plant essential oils as raw materials, a composition is prepared through a simple preparation process in a specific ratio. This composition 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 rate of farmed animals, reduce breeding risks and costs, and improve breeding efficiency. Furthermore, the natural plant essential oil composition of this invention uses readily available raw materials, has low preparation costs, is non-toxic and harmless, easily volatilizes and leaves no residue, and can be directly applied to antibiotic-free breeding of economic animals. Attached Figure Description

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

[0039] Figure 2 The fumigation method was used to determine the inhibitory effect of a natural plant extract essential oil composition on Riemerella anatipestifer.

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

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

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

[0043] Figure 6 The fumigation method was used to determine the inhibitory effect of a natural plant extract essential oil composition on Salmonella pullorum in chickens.

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

[0045] Figure 8 The inhibitory effect of natural plant essential oil compositions on Escherichia coli was determined by a mixed method.

[0046] Figure 9 The inhibitory effect of natural plant essential oil compositions on Escherichia coli was determined by a mixed method.

[0047] Figure 10 The fumigation method was used to determine the inhibitory effect of natural plant essential oil compositions on Escherichia coli.

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

[0049] Figure 12 The inhibitory effect of natural plant essential oil compositions on Staphylococcus aureus was determined by a mixed method.

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

[0051] Figure 14 The inhibitory effect of natural plant essential oil compositions on Pasteurella multocida was determined by a mixed method.

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

[0053] Figure 16 The inhibitory effect of a natural plant extract essential oil composition on Clostridium perfringens was determined by a mixed method.

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

[0055] Figure 18 The inhibitory effect of natural plant essential oil compositions on Enterococcus was determined by a mixed method.

[0056] Figure 19 The inhibitory effect of natural plant essential oil compositions on streptococci was determined by a mixed method.

[0057] Figure 20 The inhibitory effect of natural plant essential oil compositions on Aspergillus fumigatus was determined by a mixed method.

[0058] Figure 21 The inhibitory effect of natural plant extract essential oil composition on Aspergillus niger was determined by fumigation method.

[0059] Figure 22 The inhibitory effect of natural plant essential oil compositions on Candida albicans was determined by a mixed method.

[0060] Figure 23 The inhibitory effect of natural plant essential oil compositions on Candida albicans was determined by fumigation method.

[0061] Figure 24 The inhibitory effect of natural plant extract essential oil composition on porcine coronavirus was determined by fumigation method.

[0062] Figure 25 The fumigation method was used to determine the inhibitory effect of a natural plant extract essential oil composition on avian influenza A virus.

[0063] Figure 26 The inhibitory effect of a natural plant extract essential oil composition on duck Tembusu virus was determined by fumigation method. Detailed Implementation

[0064] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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 in antibiotic-free aquaculture. Furthermore, simply extending the natural plant essential oils of 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, disease prevention reagents, and instruments and equipment mentioned in this description are all commercially available conventional products. Therefore, the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0065] Example 1:

[0066] A natural plant-based essential oil composition is formulated by combining the ingredients in the following weight ratios: geranium oil: cedarwood leaf oil: citrus lemon peel oil: bergamot oil: tea tree oil in a ratio of 4:8:3:3:2. 100g of geranium oil, 200g of cedarwood leaf oil, 75g of citrus lemon peel oil, 75g of bergamot oil, and 50g of tea tree oil are sequentially added to a mixing vessel. The mixture is stirred at 550 rpm for 0.5 hours under magnetic stirring to ensure thorough mixing. The resulting plant-based essential oil mixture is then ultrasonically dispersed for 0.5 hours and allowed to stand to obtain the stock solution of the plant-based essential oil composition. The above-mentioned stock solution of the natural plant-based essential oil composition, or diluted solutions obtained by diluting with different solvents such as water, alcohol, and disinfectant in different proportions, can be directly used for antibiotic-free aquaculture.

[0067] Example 2:

[0068] A natural plant-based essential oil composition is formulated by combining the ingredients in the following weight ratios: geranium oil: cedarwood leaf oil: citrus lemon peel oil: bergamot oil: tea tree oil in a ratio of 6:6:1:4:3. 150g of geranium oil, 150g of cedarwood leaf oil, 25g of citrus lemon peel oil, 100g of bergamot oil, and 75g of tea tree oil are sequentially added to a mixing vessel. The mixture is stirred at 550 rpm for 0.5 hours under magnetic stirring to ensure thorough mixing. The resulting plant-based essential oil mixture is then ultrasonically dispersed for 0.5 hours and allowed to stand to obtain the stock solution of the plant-based essential oil composition. The above-mentioned stock solution of the natural plant-based essential oil composition, or diluted solutions obtained by diluting with different solvents such as water, alcohol, and disinfectant in different proportions, can be directly used for antibiotic-free aquaculture.

[0069] Example 3:

[0070] A natural plant-based essential oil composition is formulated by combining the ingredients in the following weight ratios: geranium oil: cedarwood leaf oil: citrus lemon peel oil: bergamot oil: tea tree oil in a ratio of 2:10:5:2:1. 50g of geranium oil, 250g of cedarwood leaf oil, 125g of citrus lemon peel oil, 50g of bergamot oil, and 25g of tea tree oil are sequentially added to a mixing vessel. The mixture is stirred at 550 rpm for 0.5 hours under magnetic stirring to ensure thorough mixing. The resulting plant-based essential oil mixture is then ultrasonically dispersed for 0.5 hours and allowed to stand to obtain the stock solution of the plant-based essential oil composition. The above-mentioned stock solution of the natural plant-based essential oil composition, or diluted solutions obtained by diluting with different solvents such as water, alcohol, and disinfectant in different proportions, can be directly used for antibiotic-free aquaculture.

[0071] Example 4: Determination of the inhibitory effect of plant extract essential oil composition on Riemerella anatipestifer using a mixed method

[0072] The original liquid of the plant extract essential oil composition in Example 1 of this invention was diluted with dimethyl sulfoxide at times of 100, 300, 1000, 3000, and 10000, respectively, and then mixed with *Riemerella anatipestifer* (1.5 × 10⁻⁶). 3 The sample (CFU / mL) was incubated at 22°C in a shaker at 220 rpm for 60 minutes. Then, it was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 200 μL of physiological saline was added and mixed thoroughly using a pipette. 100 μL of this mixture was injected into a sterile culture dish containing TSA and 5% defibrinated sheep blood. The dish was incubated at 37°C for 48 hours, and the samples were counted. All experiments were repeated three times in parallel, with a blank control provided by the original solution of the natural plant extract essential oil composition or any dilution. The experimental results are attached. Figure 1 .

[0073] Experimental results showed that, compared with the blank control group, the natural plant extract essential oil composition of this formula, when mixed with Riemerella anatipestifer, showed an inhibition rate of over 98% against Riemerella anatipestifer when diluted 100 times, and the undiluted solution diluted 300-10000 times still had a good inhibitory effect on Riemerella anatipestifer.

[0074] Example 5: Determination of the inhibitory effect of plant extract essential oil composition on Riemerella anatipestifer by fumigation method

[0075] 30 μL of Riemerella anatipestifer (1.5 × 10⁻⁶) was smeared onto the surface of a glass slide. 3 The bacterial culture (CFU / mL) was exposed to the volatile atmosphere of the natural plant extract composition or rapeseed oil (control group) in Example 1 for 15, 30, and 60 minutes. The slides were repeatedly rinsed with 200 μL of physiological saline. The washed bacterial culture was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 100 μL of physiological saline was added, the culture was resuspended, and 30 μL was injected into a petri dish containing TSA and 5% defibrinated sheep blood. The petri dish was incubated at 37°C for 48 hours, and colony counting was performed. All experiments were repeated in triplicate. The results are shown in the attached figure. Figure 2 and attached Figure 3 .

[0076] Experimental results show that the natural plant extract essential oil composition of this formula can inhibit Riemerella anatipestifer by more than 99% after 30 minutes of fumigation, and kill Riemerella anatipestifer after 60 minutes of fumigation.

[0077] Example 6: Determination of the inhibitory effect of plant extract essential oil composition on Salmonella pullorum in chickens using a mixed method

[0078] The stock solution of the natural plant extract essential oil composition in Example 2 of this invention was diluted with dimethyl sulfoxide at times of 100, 300, 1000, 3000, and 10000, respectively, and then mixed with Salmonella pullorum (1.5 × 10⁻⁶). 3 The sample (CFU / mL) was incubated at 22°C in a shaker at 220 rpm for 60 minutes. Then, it was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 200 μL of physiological saline was added and mixed thoroughly using a pipette. 100 μL was injected into LB agar plates, and the plates were incubated at 37°C for 48 hours before counting. All experiments were repeated three times, with a blank control provided by the absence of any natural plant extract essential oil composition stock solution or dilution. The experimental results are attached. Figure 4 and attached Figure 5 .

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

[0080] Example 7: Determination of the inhibitory effect of plant extract essential oil composition on Salmonella pullorum in chickens using fumigation method

[0081] 30 μL of Salmonella pullorum (1.5 × 10⁻⁶) was smeared onto the surface of a glass slide. 3 The bacterial culture (CFU / mL) was exposed to the volatile atmosphere of the natural plant extract composition or rapeseed oil (control group) in Example 2 for 15, 30, and 60 minutes. The slides were repeatedly rinsed with 200 μL of physiological saline. The washed bacterial culture was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 100 μL of physiological saline was added, the culture was resuspended, and 30 μL was injected into LB agar plates. The plates were incubated at 37°C for 48 hours, and colony counting was performed. All experiments were repeated three times in parallel. The results are shown in the attached figure. Figure 6 and attached Figure 7 .

[0082] Experimental results show that fumigation treatment with the natural plant extract essential oil composition of this formula can kill Salmonella pullorum after 60 minutes.

[0083] Example 8: Determination of the inhibitory effect of plant extract essential oil composition on Escherichia coli using the mixed method

[0084] The plant extract essential oil composition stock solution from Example 3 of this invention was diluted with dimethyl sulfoxide at times of 100, 300, 1000, and 3000, respectively, and then mixed with Escherichia coli (1.5 × 10⁻⁶).3 The sample (CFU / mL) was incubated at 22°C in a shaker at 220 rpm for 60 minutes. Then, it was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 200 μL of physiological saline was added and mixed thoroughly using a pipette. 100 μL was injected into LB agar plates, and the plates were incubated at 37°C for 48 hours before counting. All experiments were repeated three times, with a blank control provided by the absence of any natural plant extract essential oil composition stock solution or dilution. The experimental results are attached. Figure 8 and attached Figure 9 .

[0085] Experimental results showed that, compared with the blank control group, the natural plant extract essential oil composition of this formula, when mixed with Escherichia coli, had a bactericidal effect on Escherichia coli when diluted 100 times and 300 times, and the 1000-fold diluted undiluted solution still had an inhibition rate of over 99% on Escherichia coli.

[0086] Example 9: Determination of the inhibitory effect of plant extract essential oil composition on Escherichia coli by fumigation method

[0087] 30 μL of E. coli (1.5 × 10⁻⁶) was smeared onto the surface of a glass slide. 3 The bacterial culture (CFU / mL) was exposed to the volatile atmosphere of the natural plant extract composition or rapeseed oil (control group) in Example 3 for 15, 30, and 60 minutes. The slides were repeatedly rinsed with 200 μL of physiological saline. The washed bacterial culture was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 100 μL of physiological saline was added, the culture was resuspended, and 30 μL was injected into LB agar plates. The plates were incubated at 37°C for 48 hours, and colony counting was performed. All experiments were repeated in triplicate. The results are shown in the attached figure. Figure 10 and attached Figure 11 .

[0088] Experimental results show that the natural plant extract essential oil composition of this formula can kill Escherichia coli after 60 minutes of fumigation treatment.

[0089] Example 10: Determination of the inhibitory effect of plant extract essential oil composition on Staphylococcus aureus using a mixed method

[0090] The stock solution of the natural plant extract essential oil composition in Example 1 of this invention was diluted with dimethyl sulfoxide at times of 100, 300, 1000, 3000, and 10000, respectively, and then mixed with Staphylococcus aureus (1.5 × 10⁻⁶). 3The sample (CFU / mL) was incubated at 22°C in a shaker at 220 rpm for 60 minutes. Then, it was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 200 μL of physiological saline was added and mixed thoroughly using a pipette. 100 μL was injected into LB agar plates, and the plates were incubated at 37°C for 48 hours before counting. All experiments were repeated three times, with a blank control provided by the absence of any natural plant extract essential oil composition stock solution or dilution. The experimental results are attached. Figure 12 .

[0091] Experimental results show that, compared with the blank control group, the natural plant extract essential oil composition of this formula, when mixed with Staphylococcus aureus, and diluted 100 to 10,000 times, all showed a certain inhibitory effect on Staphylococcus aureus.

[0092] Example 11: Determination of the inhibitory effect of plant extract essential oil composition on Staphylococcus aureus by fumigation method

[0093] 30 μL of Staphylococcus aureus (1.5 × 10⁻⁶) was smeared onto the surface of a glass slide. 3 CFU / mL of the bacterial culture was exposed to the volatile atmosphere of the natural plant extract composition or rapeseed oil (control group) in Example 1 for 15, 30, 60, and 120 minutes. The slides were repeatedly rinsed with 200 μL of physiological saline. The washed bacterial culture was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 100 μL of physiological saline was added, the culture was resuspended, and 30 μL was injected into LB agar plates. The plates were incubated at 37°C for 48 hours, and colony counting was performed. All experiments were repeated in triplicate. The results are shown in the attached figure. Figure 13 .

[0094] Experimental results show that the natural plant extract 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 treatment.

[0095] Example 12: Determination of the inhibitory effect of plant extract essential oil composition on Pasteurella multocida by the mixed method

[0096] The stock solution of the natural plant extract essential oil composition in Example 1 of this invention was diluted with dimethyl sulfoxide at times of 100, 300, 1000, 3000, and 10000, respectively, and then mixed with Pasteurella multocida (1.5 × 10⁻⁶). 3The sample (CFU / mL) was incubated at 22°C in a shaker at 220 rpm for 60 minutes. Then, it was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 200 μL of physiological saline was added and mixed thoroughly by pipetting. 100 μL of the mixture was injected into a culture dish containing BHI and 5% defibrinated sheep blood. The dishes were incubated at 37°C for 48 hours before counting. All experiments were repeated three times in parallel, with a blank control provided by the original solution of the natural plant extract essential oil composition or any dilution. The experimental results are attached. Figure 14 .

[0097] Experimental results show that, compared with the blank control group, the natural plant extract essential oil composition of this formula, when mixed with Pasteurella multocida, has a killing effect on Pasteurella multocida when diluted 100 times and 300 times, and the undiluted solution diluted 1000 times still has a certain inhibitory effect on Pasteurella multocida.

[0098] Example 13: Determination of the inhibitory effect of plant extract essential oil composition on Pasteurella multocida by fumigation method

[0099] 30 μL of Pasteurella multocida (1.5 × 10⁻⁶) was applied to the surface of a glass slide. 3 The bacterial culture (CFU / mL) was exposed to the volatile atmosphere of the natural plant extract composition or rapeseed oil (control group) in Example 1 for 15, 30, and 60 minutes. The slides were repeatedly rinsed with 200 μL of physiological saline. The washed bacterial culture was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 100 μL of physiological saline was added, the culture was resuspended, and 30 μL was injected into BHI and 5% defibrinated sheep blood agar plates. The plates were incubated at 37°C for 48 hours, and colony counting was performed. All experiments were repeated in triplicate. The results are shown in the attached figure. Figure 15 .

[0100] Experimental results show that the natural plant extract 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 treatment.

[0101] Example 14: Determination of the inhibitory effect of plant extract essential oil composition on Clostridium perfringens using a mixed method

[0102] The stock solution of the natural plant extract essential oil composition in Example 1 of this invention was diluted with dimethyl sulfoxide at times of 100, 300, 1000, and 3000, respectively, and then mixed with Clostridium perfringens (1.5 × 10⁻⁶). 3The sample (CFU / mL) was incubated at 22°C in a shaker at 220 rpm for 60 minutes. Then, it was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 200 μL of physiological saline was added and mixed thoroughly using a pipette. 100 μL was injected into LB agar plates, and the plates were incubated at 37°C for 48 hours before counting. All experiments were repeated three times, with a blank control provided by the absence of any natural plant extract essential oil composition stock solution or dilution. The experimental results are attached. Figure 16 .

[0103] Experimental results show that, compared with the blank control group, the natural plant extract essential oil composition of this formula, when mixed with Clostridium perfringens, has a killing effect on Clostridium perfringens when diluted 100 and 300 times, and the undiluted solution diluted 1000 times still has a good inhibitory effect on Clostridium perfringens.

[0104] Example 15: Determination of the inhibitory effect of plant extract essential oil composition on Clostridium perfringens by fumigation method

[0105] 30 μL of Clostridium perfringens (1.5 × 10⁻⁶) was applied to the surface of a glass slide. 3 The bacterial culture (CFU / mL) was exposed to the volatile atmosphere of the natural plant extract composition or rapeseed oil (control group) in Example 1 for 15, 30, and 60 minutes. The slides were repeatedly rinsed with 200 μL of physiological saline. The washed bacterial culture was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 100 μL of physiological saline was added, the culture was resuspended, and 30 μL was injected into LB agar plates. The plates were incubated at 37°C for 48 hours, and colony counting was performed. All experiments were repeated in triplicate. The results are shown in the attached figure. Figure 17 .

[0106] Experimental results show that the natural plant extract essential oil composition of this formula has a significant inhibitory effect on Clostridium perfringens after fumigation treatment for 30 and 60 minutes.

[0107] Example 16: Determination of the inhibitory effect of the plant extract essential oil composition on Haemophilus paragallinarum using the mixed method. The original solution of the natural plant extract essential oil composition in Example 1 of this invention was diluted with dimethyl sulfoxide at 100, 300, 1000, 3000 or 10000 times, respectively, and then mixed with Haemophilus paragallinarum (1.5 × 10⁻⁶). 3The mixture (CFU / mL) was incubated at 22°C in a shaker at 220 rpm for 60 minutes. Then, it was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 100 μL of physiological saline was added and mixed using a pipette. 50 μL of the mixture was injected into BHI plates, 5% defibrinated sheep blood plates, and 4% chicken serum plates. The plates were incubated at 37°C for 48 hours before counting. All experiments were repeated three times in parallel, with a blank control consisting of the undiluted natural plant extract composition or any dilution.

[0108] Experimental results show that, compared with the blank control group, the natural plant extract essential oil composition of this formula, when mixed with Haemophilus paragallinarum, and diluted 100 to 10000 times, all showed a certain inhibitory effect on Haemophilus paragallinarum.

[0109] Example 17: Determination of the inhibitory effect of plant extract essential oil composition on Haemophilus paragallinarum by fumigation method. 30 μL of Haemophilus paragallinarum (1.5 × 10⁻⁶) was applied to the surface of a glass slide. 3 The bacterial culture (CFU / mL) was exposed to the volatile atmosphere of the natural plant extract composition or rapeseed oil (control group) in Example 1 for 15, 30, 60, and 120 minutes. The slides were repeatedly rinsed with 100 μL of physiological saline. The washed bacterial culture was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. The culture was resuspended in 100 μL of physiological saline, and 30 μL was injected into BHI plates, 5% defibrinated sheep blood plates, and 4% chicken serum plates. The plates were incubated at 37°C for 48 hours, and colony counting was performed. All experiments were repeated in triplicate.

[0110] Experimental results show that the natural plant extract essential oil composition of this formula has a significant inhibitory effect on Haemophilus paragallinarum after 30 and 60 minutes of fumigation treatment, and can kill Haemophilus paragallinarum after 120 minutes of treatment.

[0111] Example 18: Determination of the inhibitory effect of the plant extract essential oil composition on Mycoplasma gallisepticum using the mixed method. The original solution of the natural plant extract essential oil composition in Example 1 of this invention was diluted with dimethyl sulfoxide at 100, 300, 1000, 3000, or 10000 times, respectively, and mixed with Mycoplasma gallisepticum (1.5 × 10⁻⁶). 6The mixture (CFU / mL) was incubated at 22°C in a shaker at 220 rpm for 60 minutes, followed by centrifugation at 10,000 rpm for 2 minutes at 8°C. The supernatant was discarded, and 100 μL of physiological saline was added and mixed by pipetting. 50 μL of the mixture was injected into modified Frey agar plates, 10% inactivated porcine serum, and 0.1% ampicillin plates. The plates were incubated at 37°C for 4 days, and the concentrations were then counted. All experiments were repeated in triplicate, with a blank control consisting of the stock solution of the natural plant extract essential oil composition or any dilution.

[0112] Experimental results show that, compared with the blank control group, the natural plant extract essential oil composition of this formula, when mixed with Mycoplasma gallisepticum, has a killing effect on Mycoplasma gallisepticum when diluted 100 times and 300 times, and the undiluted solution diluted 1000 times still has a good inhibitory effect on Mycoplasma gallisepticum.

[0113] Example 19: Determination of the inhibitory effect of plant extract essential oil composition on Mycoplasma gallisepticum by fumigation method. 40 μL of Mycoplasma gallisepticum (2.37 × 10⁻⁶) was applied to the surface of a glass slide. 8 The bacterial culture (CFU / mL) was exposed to the volatile atmosphere of the natural plant extract composition or rapeseed oil (control group) in Example 1 for 15, 30, and 60 minutes. The slides were repeatedly rinsed with 100 μL of physiological saline. The washed bacterial culture was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 100 μL of physiological saline was added, the culture was resuspended, and 50 μL was injected into LB agar plates. The plates were incubated at 37°C for 4 days, and colony counting was performed. All experiments were repeated in triplicate.

[0114] Experimental results show that fumigation treatment with the natural plant extract essential oil composition of this formula has a significant inhibitory effect on Mycoplasma gallisepticum after 15 and 30 minutes of treatment, and can kill Mycoplasma gallisepticum after 60 minutes of treatment.

[0115] Example 20: Determination of the inhibitory effect of plant essential oil composition on Enterococci using a mixed method

[0116] The original extract of the plant essential oil composition in Example 1 of this invention was diluted with dimethyl sulfoxide to a concentration of 100, 200, 400, and 1000 times, respectively, and then mixed with Enterococcus (1.5 × 10⁻⁶). 3The sample (CFU / mL) was incubated at 22°C in a shaker at 220 rpm for 60 minutes. Then, it was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 200 μL of physiological saline was added and mixed thoroughly using a pipette. 100 μL was injected into LB agar plates, and the plates were incubated at 37°C for 48 hours before counting. All experiments were repeated three times, with a blank control provided by the absence of any natural plant extract essential oil composition stock solution or dilution. The experimental results are attached. Figure 18 .

[0117] Experimental results showed that, compared with the blank control group, the natural plant extract essential oil composition of this formula, when mixed with Enterococcus, showed an inhibition rate of over 99% against Enterococcus even when diluted 100 times, and the original solution diluted 100-1000 times still had a good inhibitory effect on Enterococcus.

[0118] Example 21: Determination of the inhibitory effect of plant essential oil composition on streptococci using a mixed method

[0119] The stock solution of the natural plant extract essential oil composition in Example 1 of this invention was diluted with dimethyl sulfoxide at times of 100, 200, 400, and 1000 times, respectively, and then mixed with streptococci (1.5 × 10⁻⁶). 3 The sample (CFU / mL) was incubated at 22°C in a shaker at 220 rpm for 60 minutes. Then, it was centrifuged at 8°C and 10000 rpm for 2 minutes, and the supernatant was discarded. 200 μL of physiological saline was added and mixed thoroughly using a pipette. 100 μL of the mixture was injected into blood agar plates, and the plates were incubated at 37°C for 48 hours before counting. All experiments were repeated three times, with a blank control provided by the absence of any natural plant extract essential oil composition stock solution or dilution. The experimental results are attached. Figure 19 .

[0120] Experimental results showed that, compared with the blank control group, the natural plant extract essential oil composition of this formula inhibited Streptococcus bacteria by more than 99% when diluted 100-400 times, and the inhibition rate of Streptococcus bacteria was still more than 99% when diluted 1000 times.

[0121] Example 22: Determination of the inhibitory effect of plant extract essential oil composition on Aspergillus fumigatus using the mixed method

[0122] The original extract of the plant essential oil composition in Example 1 of this invention was diluted with dimethyl sulfoxide at times of 100, 200, 400, and 1000 times, respectively, and then mixed with Aspergillus fumigatus (1.5 × 10⁻⁶). 3The extract (CFU / mL) was cultured at 22°C in a shaker at 220 rpm for 60 minutes. Then, it was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 200 μL of physiological saline was added and mixed thoroughly by pipetting. 100 μL was injected into a Sabouraud agar plate and incubated at 28°C for 72 hours before counting. All experiments were repeated three times. A blank control group was used without the undiluted plant extract essential oil composition or any dilution. The experimental results are attached. Figure 20 .

[0123] Experimental results showed that, compared with the blank control group, the natural plant extract essential oil composition of this formula inhibited Aspergillus fumigatus by more than 99% when diluted 100 to 1000 times.

[0124] Example 23: Determination of the inhibitory effect of plant extract essential oil composition on Aspergillus niger by fumigation method

[0125] 30 μL of Aspergillus niger (1.5 × 10⁻⁶) was applied to the surface of a glass slide. 3 The bacterial culture (CFU / mL) was exposed to the volatile atmosphere of the natural plant extract composition or rapeseed oil (control group) in Example 1 for 4 and 24 hours. The slides were repeatedly rinsed with 200 μL of physiological saline. The washed bacterial culture was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 100 μL of physiological saline was added, the culture was resuspended, and 30 μL was injected into modified Martin medium. The culture dishes were incubated at 37°C for 48 hours, and colony counting was performed. All experiments were repeated in triplicate. The results are shown in the attached figure. Figure 21 .

[0126] Experimental results show that the natural plant extract essential oil composition of this formula, when used for fumigation treatment of Aspergillus niger, has an inhibition rate of over 99% after 4 hours of treatment, and still has a significant inhibitory effect on Aspergillus niger after 24 hours of treatment.

[0127] Example 24: Determination of the inhibitory effect of plant essential oil composition on Candida albicans using a mixed method

[0128] The plant extract essential oil composition stock solution obtained in Example 1 of this invention was diluted with dimethyl sulfoxide by 100, 150, and 200 times, respectively. The resulting diluted solutions were then mixed with Candida albicans (1.5 × 10⁻⁶). 3The sample (CFU / mL) was incubated at 22°C in a shaker at 220 rpm for 60 minutes. Then, it was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 200 μL of physiological saline was added and mixed thoroughly using a pipette. 100 μL of the mixture was injected into a Sabouraud agar plate and incubated at 28°C for 24 hours before counting. All experiments were repeated three times, with a blank control group (without the added natural plant extract essential oil composition dilution). The experimental results are attached. Figure 22 .

[0129] Experimental results showed that, compared with the blank control group, the natural plant extract essential oil composition of this formula, when mixed with Candida albicans, had a killing effect on Candida albicans when diluted 100-150 times, and the undiluted solution diluted 200 times had an inhibition rate of over 99% on Candida albicans.

[0130] Example 25: Determination of the inhibitory effect of plant extract essential oil composition on Candida albicans by fumigation method

[0131] 30 μL of Candida albicans (1.5 × 10⁻⁶) was smeared onto the surface of a glass slide. 3 The bacterial culture (CFU / mL) was exposed to the volatile atmosphere of the natural plant extract composition or rapeseed oil (control group) in Example 1 for 4 and 24 hours. The slides were repeatedly rinsed with 200 μL of physiological saline. The washed bacterial culture was centrifuged at 10,000 rpm for 2 minutes at 8°C, and the supernatant was discarded. 100 μL of physiological saline was added, the culture was resuspended, and 30 μL was injected into a Sabouraud agar plate. The plates were incubated at 37°C for 48 hours, and colony counting was performed. All experiments were repeated three times in parallel. The results are shown in the attached figure. Figure 23 .

[0132] Experimental results show that the natural plant extract essential oil composition of this formula has a significant inhibitory effect on Candida albicans after 4 hours of fumigation treatment, and can kill Candida albicans after 24 hours of treatment.

[0133] Example 26: Determination of the inhibitory effect of plant extract essential oil composition on porcine coronavirus by fumigation method

[0134] 10 μL of porcine coronavirus (1.0 × 10⁻⁶) was smeared onto the surface of a glass slide. 7 TCID 50The virus ( / mL) was exposed to the volatile atmosphere of the natural plant extract 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 to resuspend the virus and mix thoroughly. The resuspended virus was diluted to a power of 10^1 to 8 and added to cultured Vero cells, with each dilution replicated in 4 wells. Five days post-infection, the cytopathic effect was observed in each well under an inverted microscope, and the TCID of samples treated at different times was calculated using the Reed-Muench method. 50 All experiments were repeated three times in parallel, and the results are attached. Figure 24 .

[0135] Experimental results showed that the natural plant extract essential oil composition of this formula had a significant inhibitory effect on porcine coronavirus fumigation treatment after 30 minutes of treatment, and the inhibition rate of porcine coronavirus exceeded 99.99% after 45 and 60 minutes of treatment.

[0136] Example 27: Determination of the inhibitory effect of plant extract essential oil composition on avian influenza A virus by fumigation method

[0137] 10 μL of avian influenza A virus (5 × 10⁻⁶) was smeared onto the surface of a glass slide. 4 The virus (PFU / mL) was exposed to the natural plant extract composition or rapeseed oil volatile atmosphere of Example 1 for 10, 20, and 30 minutes. The slides were repeatedly rinsed with 1000 μL of physiological saline, resuspended, and mixed. The mixture was then added to cultured A549 cells. After virus adsorption for 1 hour, a layer of melted semi-solid nutrient agar was added to allow limited virus diffusion in the monolayer cell culture. Plaques were counted to calculate the titer. All experiments were repeated in triplicate. The results are attached. Figure 25 .

[0138] Experimental results show that the natural plant extract essential oil composition of this formula, when used for fumigation treatment of avian influenza A virus, has an inhibition rate of over 99% after 10 and 20 minutes of treatment, and has a killing effect on avian influenza A virus after 30 minutes of treatment.

[0139] Example 28: Determination of the inhibitory effect of plant extract essential oil composition on duck Tembusu virus by fumigation method

[0140] 10 μL of duck Tembusu virus (1.0 × 10⁻⁶) was smeared onto the surface of a glass slide. 7 TCID 50The virus ( / mL) was exposed to the volatile atmosphere of the natural plant extract composition or rapeseed oil (control group) in Example 1 for 10, 30, and 45 minutes. The slides were repeatedly rinsed with 1000 μL of physiological saline to resuspend the virus and mix thoroughly. The resuspended virus was diluted to the power of 10^1 to 8 and added to cultured DF-1 cells, with each dilution replicated in 4 wells. Five days post-infection, the cytopathic effect was observed in each well under an inverted microscope, and the TCID of samples treated at different times was calculated using the Reed-Muench method. 50 All experiments were repeated three times in parallel, and the results are attached. Figure 26 .

[0141] Experimental results showed that the natural plant extract essential oil composition of this formula, when used for fumigation treatment of duck Tembusu virus, achieved an inhibition rate of 90% after 30 and 45 minutes of treatment, and an inhibition rate of over 95% after 60 minutes of treatment.

[0142] Example 29: Determination of the inhibitory effect of plant extract essential oil composition on goose parvovirus by fumigation method. 10 μL of goose parvovirus (1.0 × 10⁻⁶) was applied to the surface of a glass slide. 5 TCID 50 The virus ( / mL) was exposed to the volatile atmosphere of the natural plant extract composition or rapeseed oil (control group) in Example 1 for 1, 2, 3, 4, 5, and 6 hours. The slides were repeatedly rinsed with 1000 μL of physiological saline to resuspend the virus and mix thoroughly. The resuspended virus was diluted to a power of 10^1 to 8 and added to cultured GEF cells, with each dilution replicated in 4 wells. Five days post-infection, indirect immunofluorescence staining was used to observe the cytopathic effects in each well under an inverted microscope. The TCID of samples treated at different times was calculated using the Reed-Muench method. 50 All experiments were repeated three times in parallel.

[0143] Experimental results showed that the natural plant extract essential oil composition of this formula, when used for fumigation treatment of goose parvovirus, achieved an inhibition rate of 96.8% after 2 hours and over 99% after 6 hours.

[0144] Example 30: Determination of the inhibitory effect of plant extract essential oil composition on infectious bursal disease virus in chickens by fumigation method. 10 μL of infectious bursal disease virus (1.0 × 10⁻⁶) was applied to the surface of a glass slide. 7 TCID 50The virus ( / mL) was exposed to the volatile atmosphere of the natural plant extract composition or rapeseed oil (control group) in Example 1 for 1, 2, 3, 4, 5, and 6 hours. The slides were repeatedly rinsed with 1000 μL of physiological saline to resuspend the virus and mix thoroughly. The resuspended virus was diluted to a power of 10^1 to 8 and added to cultured DF-1 cells, with each dilution replicated in 4 wells. Five days post-infection, the cytopathic effect was observed in each well under an inverted microscope, and the TCID of samples treated at different times was calculated using the Reed-Muench method. 50 All experiments were repeated three times in parallel.

[0145] Experimental results show that the natural plant extract essential oil composition of this formula, when used for fumigation treatment of infectious bursal disease virus in chickens, has an inhibition rate of over 99.9% after 3 hours of treatment and a killing effect on infectious bursal disease virus after 4 hours of treatment.

[0146] Example 31: Antibiotic-free breeding experiment of broilers

[0147] 15,000 one-day-old mixed-breed broiler chicks (Shandong Yisheng Livestock and Poultry Co., Ltd.) were selected as the experimental group and fed with the natural plant extract essential oil composition from Example 1 of this invention instead of antibiotics. Another 15,000 one-day-old mixed-breed broiler chicks (Shandong Yisheng Livestock and Poultry Co., Ltd.) were used as the control group, fed with antibiotics during the rearing process. The effective rearing area for both the experimental and control groups was 780 square meters, with a rearing density of 19 chicks / square meter. Before the chicks entered the coop, both groups underwent thorough disinfection by spraying and fumigating with potassium permanganate. For the experimental group, a blower was used to fully distribute the undiluted natural plant extract essential oil composition from Example 1 of this invention in the coop before the chicks entered, while the control group was ventilated before use. The temperature and humidity inside the coop were maintained at 32-35°C and 55-70%, respectively, before the chicks entered the coop.

[0148] Both the experimental and control groups of chicks had free access to water and feed after entering the pen and were managed according to the standard broiler chicken rearing procedure. Immunization followed the standard procedure: at 1 day old, 0.15 mL / chick of attenuated infectious bursal disease vaccine was injected subcutaneously in the neck; at 7 days old, Newcastle disease and infectious bronchitis bivalent live vaccine was administered via nasal and ocular drops; at 21 days old, Newcastle disease live vaccine was administered via nasal and ocular drops. From 1 to 12 days was the chick stage, using small feed troughs and adding feed (Dongguan Haida Feed Co., Ltd.) 4-6 times daily, maintaining an indoor temperature of approximately 34-30℃; from 13 to 28 days was the growing stage, switching to feed troughs and adding feed automatically 1-2 times daily, mixing manually, and gradually reducing the indoor temperature to around 26℃; after 28 days was the fattening stage, adding feed 3-5 times daily, reducing the indoor temperature to around 24℃, until slaughter at 40 days old.

[0149] In the experimental group, a blower was used to evenly disperse the natural plant extract essential oil composition from Example 1 of this invention into the chicken coop. The dosage was 400 grams per day. No antibiotics were used in the experimental group during the feeding process. In the control group, enrofloxacin was administered once (0.003 g / bird), amoxicillin once (0.003 g / bird), and spectinomycin once (0.003 g / bird) during the seedling stage; tilmicosin twice (0.003 g / bird) and decaoxyquin ester once (0.003 g / bird) during the growing stage; and amoxicillin once (0.006 g / bird) during the fattening stage. All treatments were added to the feed.

[0150] After the experiment, the survival rate and average weight at slaughter of chickens in the experimental group and the control group were recorded. The results are shown in Table 1.

[0151] Table 1. Effects of natural plant extract essential oil compositions on antibiotic-free broiler chicken farming.

[0152] Survival rate average weight Experimental group (natural plant essential oil composition) 92.6％ 2.8kg Control group (antibiotics) 90％ 2.6kg

[0153] Experimental results show that, compared with the control group using antibiotics, the survival rate and average weight of broilers treated with the natural plant extract essential oil composition of this formula are improved, confirming that the natural plant extract essential oil composition of this invention can be used to replace antibiotics in antibiotic-free chicken farming.

[0154] Example 32: Antibiotic-free breeding experiment of meat ducks

[0155] 7000 one-day-old Cherry Valley ducks (Shandong Yisheng Livestock and Poultry Co., Ltd.) were selected as the experimental group and fed with the natural plant extract essential oil composition from Example 2 of this invention instead of antibiotics. Another 7000 one-day-old Cherry Valley ducks (Shandong Yisheng Livestock and Poultry Co., Ltd.) were used as the control group, fed with antibiotics during the breeding process. The effective breeding area for both the experimental and control groups was 1000 square meters, with a breeding density of 7 ducks / square meter. Before the ducklings entered the pen, both the experimental and control groups underwent thorough disinfection of the duck house by spraying and fumigating with potassium permanganate. Before the ducklings entered the pen, the experimental group used a blower to fully distribute the undiluted natural plant extract essential oil composition from Example 2 of this invention in the duck house, while the control group was ventilated before use. The temperature and humidity inside the duck house were maintained at 33°C and 60%, respectively, before the ducklings entered the pen.

[0156] Both the experimental and control groups of ducklings had free access to feed and water after entering the pen and were managed according to the standard Cherry Valley duck rearing and management procedures. Immunization was carried out according to the standard immunization program: at 1 day old, 0.5 mL / bird of gosling plague live vaccine was injected subcutaneously in the neck; at 7 days old, 1 mL / bird of gosling plague live vaccine was injected subcutaneously in the neck; at 10 days old, a bivalent inactivated vaccine of goose paramyxovirus and goose influenza was injected subcutaneously in the neck. During the brooding period (days 1-12), the daily feed intake was gradually increased from 100 kg to 400 kg, and the indoor temperature was gradually reduced to around 20℃, with humidity maintained at approximately 60%. During the growth period (days 13-42), the daily feed intake was gradually increased from 600 kg to 2000 kg, with the indoor temperature maintained at 20℃ and humidity maintained at 50%.

[0157] In the experimental group, a blower was used to evenly disperse the natural plant extract essential oil composition from Example 2 of this invention into the duck house, at a dosage of 500 grams per day. No antibiotics were used in the experimental group during the feeding process. The control group received three medications during the growth period: florfenicol, fenpropathrin, and colistin sulfate, all of which were added to the feed.

[0158] After the experiment, the survival rate and average weight at slaughter of ducks in the experimental group and the control group were recorded. The results are shown in Table 2.

[0159] Table 2. Effects of plant extract essential oil compositions on antibiotic-free farming of Cherry Valley ducks.

[0160] Survival rate average weight Experimental group (natural plant essential oil composition) 95.26％ 3.4kg Control group (antibiotics) 97.14％ 3.4kg

[0161] Experimental results show that, compared with the control group using antibiotics, the survival rate and average weight of ducks treated with the natural plant extract essential oil composition of this formula did not change significantly, confirming that the natural plant extract essential oil composition of this invention can be used to replace antibiotics in antibiotic-free duck farming.

Claims

1. A natural plant extract essential oil composition for antibiotic-free aquaculture, characterized in that, The composition includes geranium oil, cedarwood leaf oil, citrus lemon peel oil, bergamot oil, and tea tree oil. The weight ratio of geranium oil: cedarwood 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 extract essential oil composition according to claim 1, characterized in that, The weight ratio of geranium oil, cedarwood leaf oil, citrus lemon peel oil, bergamot oil, and tea tree oil in the natural plant essential oil composition is 4:8:3:3:

2.

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

3.

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

1.

5. The application of the natural plant extract essential oil composition according to any one of claims 1-4 in the preparation of livestock and poultry breeding disinfectants, by killing or inhibiting pathogenic bacteria, fungi and viruses in breeding sites, replaces the role of antibiotics and achieves antibiotic-free breeding.

6. The application 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*, and *Streptococcus*; the pathogenic fungi are further preferably: *Candida albicans*, *Aspergillus fumigatus*, and *Aspergillus niger*; the viruses are further preferably: duck plague virus, avian influenza virus, duck hepatitis A virus, avian adenovirus, swine coronavirus, goose parvovirus, duck Tembusu virus, and chicken infectious diseases virus. Infectious bursal virus .

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

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

9. The application according to claim 5, characterized in that, The livestock and poultry are further preferably chickens, ducks, or geese.

10. The application according to claim 9, characterized in that, The poultry raised from chickens are further preferably white-feathered chickens, and the poultry raised from ducks are further preferably Cherry Valley ducks.

11. The application according to claim 5, characterized in that, The method of using disinfectant is to dilute the disinfectant concentrate directly or with water or other solvents in different volume ratios, and then distribute the diluted solution to the breeding area through evaporation, spraying, mixing or other equipment.

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