A compound essential oil preparation of kumquat for disinfection in aquaculture and its preparation method

Abstract

This invention relates to the field of aquaculture, specifically disclosing a kumquat compound essential oil preparation for disinfection in aquaculture and its preparation method. The kumquat compound essential oil preparation, by volume percentage of crude oil raw materials, comprises 30-50 parts kumquat essential oil, 20-40 parts clove essential oil, 5-15 parts myrrh essential oil, 5-15 parts costus root essential oil, and 5-15 parts frankincense essential oil. The preparation is obtained through the following steps: S1 Raw material pretreatment; S2 Extraction using low-boiling-point solvent subcritical circulation extraction technology to extract five crude oils from kumquat, clove, myrrh, costus root, and frankincense; S3 Mixing the crude oils; and S4 Separation and purification of the mixed crude oils. This kumquat compound essential oil preparation uses kumquat essential oil as its core component, incorporating clove, myrrh, costus root, and frankincense essential oils. It fully utilizes the complementary medicinal and antibacterial effects of the five essential oils, allowing them to exert a synergistic effect and effectively apply to bacterial disinfection in aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture, specifically relating to a kumquat compound essential oil preparation for disinfection in aquaculture and its preparation method. Background Technology

[0002] With the intensive development of aquaculture, high-density farming environments have exacerbated eutrophication of water bodies, leading to the proliferation of pathogenic microorganisms (such as Staphylococcus aureus and Escherichia coli), resulting in highly lethal diseases such as bacterial septicemia, gill rot, and enteritis.

[0003] The introduction of new disinfectant and antibacterial pesticide compounds has slowed, while resistance to older products has increased. Distributors are facing severe product homogenization and declining profits, creating an urgent need for new profit growth drivers. Against this backdrop, the aquaculture market needs a green, environmentally friendly, and safe disinfectant product to address these issues.

[0004] Furthermore, the continuous exposure of farmed aquatic animals to various external factors such as temperature, water physicochemical factors, stocking density, and transportation can suppress their immune function, leading to disease. Many intractable fish diseases are often the result of prolonged stress. Therefore, stress is a crucial issue that must be addressed in aquaculture. Summary of the Invention

[0005] In response to the current problems in the aquaculture industry, such as the increasing emergence of drug resistance due to the use of chemical antibiotics to prevent and control diseases in fish, and the cumulative residual effects of chemical antibiotics on farmed animals leading to quality issues in aquatic products, this invention provides a kumquat compound essential oil preparation, its preparation method, and its application.

[0006] Plant essential oils are secondary substances produced by plants. They are a general term for volatile, oily liquid substances composed of simple compounds with relatively small molecular weights and a certain odor.

[0007] This invention addresses the problems of chemical disinfectant resistance, ecotoxicity, and the narrow bactericidal spectrum of single essential oils by providing a broad-spectrum, low-toxicity, and non-resistant compound disinfectant made from plant essential oils.

[0008] The resulting kumquat compound essential oil preparation uses kumquat essential oil as its core ingredient, blended with clove, myrrh, costus root, and frankincense essential oils. It fully utilizes the complementary medicinal and antibacterial properties of these five essential oils, allowing them to exert a synergistic effect and more effectively kill bacteria in aquatic animals. This kumquat compound essential oil preparation contains no other fragrances. Its application in aquaculture will undoubtedly play a more significant role in the industry.

[0009] The present invention adopts the following technical solution to achieve the purpose of the invention.

[0010] First, this invention provides a kumquat compound essential oil preparation for disinfection in aquaculture.

[0011] The kumquat compound essential oil preparation comprises, by volume, 30-50 parts kumquat essential oil, 20-40 parts clove essential oil, 5-15 parts myrrh essential oil, 5-15 parts costus root essential oil, and 5-15 parts frankincense essential oil.

[0012] Plant essential oils are composed of a series of bioactive plant components and their derivatives, and have long been used as fragrances, preservatives, and medicinal materials. Plant essential oils are the core components of plant-derived extracts, and are mixtures of aromatic, volatile compounds. They are hydrophobic compounds, including alcohols, aldehydes, esters, ethers, ketones, phenols, terpenes, and other chemical components. The six-carbon ring in the essential oil molecule generates different biochemical components during plant growth and animal consumption, thus producing different biochemical functions.

[0013] The essential oils extracted from kumquat, clove, costus root, frankincense, and myrrh contain a variety of antibacterial components, providing a strong guarantee for the green and healthy aquaculture of modern aquatic animals.

[0014] Among them, kumquat essential oil has antibacterial function, as well as strong penetrability, good drying and spreading properties. Kumquat essential oil is an extract of natural kumquat, derived from nature, safe and environmentally friendly. It has strong compatibility, high safety in use, and can increase the absorption and utilization rate of other disinfectants.

[0015] Clove essential oil and costus root essential oil both have inhibitory effects on Escherichia coli, enterohemorrhagic Escherichia coli, and diarrheal Escherichia coli. Additionally, frankincense essential oil has antioxidant properties and can improve anxiety and insomnia; myrrh essential oil has significant bactericidal effects, containing abundant volatile components and monoterpenoids, which give it strong antibacterial activity and can effectively kill or inhibit the growth and reproduction of various bacteria, viruses, and fungi.

[0016] Preferably, the kumquat compound essential oil preparation comprises, by volume, 40 parts kumquat essential oil, 30 parts clove essential oil, 10 parts myrrh essential oil, 10 parts costus root essential oil, and 10 parts frankincense essential oil.

[0017] Secondly, the present invention also provides a method for preparing a compound essential oil preparation of kumquat.

[0018] The preparation method of this kumquat compound essential oil preparation includes the following steps: S1 raw material pretreatment; S2 crude oil extraction; S3 crude oil mixing; S4 separation and purification of the mixed crude oil to obtain kumquat compound essential oil.

[0019] Furthermore, the preparation method also includes an S5 emulsification step.

[0020] Further, the raw material pretreatment described in step S1 is specifically as follows: remove the stems from the fresh kumquats, wash and dry the fresh kumquats, remove the pulp, then pre-freeze the kumquat peels and dry them in a vacuum freeze dryer to remove moisture; at the same time, dry the cloves, myrrh, costus root, and frankincense to remove moisture; after drying, use a traditional Chinese medicine pulverizer to pulverize and sieve each herb for later use.

[0021] Further, the crude oil extraction in step S2 specifically involves using a low-boiling-point solvent subcritical circulation extraction technology to extract crude oil from kumquat, clove, myrrh, costus root, and frankincense, respectively.

[0022] Preferably, the low-boiling-point solvent is selected from CHClF2 (R22) or liquefied petroleum gas (propane:butane = 60:40, v / v).

[0023] Preferably, the subcritical cyclic extraction has a solid-liquid ratio of 1:(8-20), an extraction pressure of 0.8-1.6 MPa, an extraction temperature of 30℃-50℃, an extraction time of 0.5h-2.5h per extraction, and an extraction cycle of 3-12 times.

[0024] More preferably, the subcritical cyclic extraction is carried out at an extraction temperature of 40°C, with each extraction lasting 1 hour and the number of extraction cycles being 9.

[0025] Subcritical fluid extraction technology utilizes a subcritical fluid as the extractant. Within a closed, oxygen-free, low-pressure vessel, based on the principle of "like dissolves like" in organic compounds, the fat-soluble components in the solid material are transferred to the liquid extractant through molecular diffusion during the soaking process. The extractant is then separated from the target product through reduced pressure evaporation, ultimately yielding the target product. This novel extraction and separation technology offers numerous advantages over other separation methods: it is non-toxic, harmless, environmentally friendly, pollution-free, non-thermal processing, preserves the active ingredients of the extract without damage or oxidation, has high production capacity suitable for large-scale industrial production, is energy-efficient, has low operating costs, and is easy to separate from the product.

[0026] Further, the crude oil mixing in step S3 specifically involves mixing kumquat crude oil, clove crude oil, myrrh crude oil, costus root crude oil, and frankincense crude oil in proportions of 30-50, 20-40, 5-15, 5-15, and 5-15 parts respectively.

[0027] Further, the separation and purification in step S4 is as follows: the mixed crude oil is separated and purified by a POPE scraped membrane molecular distillation device; the conditions for the scraped membrane molecular distillation are: scraper gap 0.2-0.5mm, residence time ≤30s, vacuum degree ≤0.5mbar, evaporation temperature 40-60℃, and condensation temperature -15℃ to -5℃.

[0028] Preferably, the vacuum degree of the wiped-film molecular distillation is 0.1 mbar, the evaporation temperature is 50°C, and the condensation temperature is -10°C.

[0029] The POPE wiped-film molecular distillation apparatus used in this invention differs significantly from conventional distillation equipment in terms of separation principle, structural design, and operating conditions. The difference in separation principle lies in the fact that separation is achieved based on the difference in molecular free path, with lighter component molecules operating under high vacuum (<10⁻⁶ m / s). -2 Direct condensation at mbar (mbar) eliminates the need for component boiling point differences, enabling precise separation of heat-sensitive substances (such as monoterpenes and sesquiterpenes in essential oils). Conventional distillation, relying on gas-liquid phase equilibrium and requiring high-temperature vaporization, is prone to thermal decomposition. Optimized structural design: Forced film-forming system: A high-speed scraper (200-500 rpm) uniformly coats the material into an ultra-thin liquid film (thickness ≤0.5mm), improving mass transfer efficiency by more than 10 times compared to conventional distillation's reliance on gravity-fed film formation (thickness ≥5mm). Short-path condensation design: The distance between the condenser and the evaporation surface is ≤50mm, allowing direct condensation of light components within their molecular free path, avoiding secondary backmixing. Conventional distillation requires long condenser tubes, resulting in low separation efficiency. Heat sensitivity protection: The operating temperature is 50-80℃ lower than the material's boiling point, with a heating time ≤30 seconds (conventional distillation requires continuous heating for tens of minutes), effectively preserving volatile active ingredients in essential oils (e.g., limonene retention >95%). Technical effect: Through the above improvements, the present invention achieves efficient enrichment of antibacterial components (d-limonene, eugenol, etc.) in kumquat compound essential oil, and the total content of d-limonene and eugenol in the compound essential oil is ≥90wt%.

[0030] Furthermore, the S5 emulsification step specifically involves adding 0.5-5 wt% emulsifier to the compound essential oil obtained in step S4, followed by high-pressure homogenization at 60-80 MPa to form an O / W nanoemulsion with an average particle size ≤200 nm.

[0031] Preferably, the emulsifier is selected from at least one of Tween-80, sucrose fatty acid esters, and soybean lecithin.

[0032] The kumquat compound essential oil, when applied at a concentration of 100 mg / L for 120 minutes, showed a log kill value of 5.02 against Staphylococcus aureus, 5.25 against Escherichia coli, and 4.95 against Pseudomonas aeruginosa. The log kill values ​​against these three bacteria were all >4.90 and close to or exceeded 5.00, with no chemical residue. This demonstrates that in actual production scenarios, using a concentration of 100 ppm and ensuring a contact time of 120 minutes can meet the green disinfection requirements for aquaculture.

[0033] In addition, this invention also provides the application of kumquat compound essential oil preparations in the disinfection of bacteria in aquaculture.

[0034] The kumquat compound essential oil preparation can be applied to bacterial disinfection in aquaculture by mixing it into feed and / or spraying it into water.

[0035] The antibacterial activity of plant essential oils is not a unique mechanism of action, but rather involves a series of complex bacterial cellular responses; these characteristics are known as the "universality of essential oils." Aquatic animals primarily rely on their sensitive olfactory and gustatory systems for feeding, especially fish, whose taste buds are almost ubiquitous throughout their bodies. Adding this kumquat compound essential oil preparation to aquaculture feed in a specific ratio not only imparts a rich aroma to the feed but also infects the surrounding aquatic environment, stimulating the aquatic animals' olfactory organs and inducing them to feed. After feeding, it further stimulates the taste organs, promoting food intake and exciting the gastrointestinal tract, stimulating the secretion of bile, digestive mucus, and digestive enzymes to improve nutrient absorption.

[0036] A certain amount of kumquat compound essential oil preparation can also be sprayed into the water in a specific ratio. The main factors causing stress in aquatic animals during aquaculture include inadequate water quality management, improper use of medications, and the use of substandard feed. It has been proven that adding a certain amount of plant essential oils to the water can improve the stress resistance of farmed animals. Adding certain amounts of essential oil disinfectants and essential oil disease-preventing agents to the water can also enhance the stress resistance of aquatic animals, effectively reducing the mortality rate of farmed animals caused by stress.

[0037] In aquaculture, the use of this kumquat compound essential oil preparation in mixed feed and / or spraying it on the water can achieve antibacterial effects, improve the body's digestive capacity, and promote the body's growth and development, providing a strong guarantee for the green and healthy aquaculture of modern aquatic animals.

[0038] Beneficial effects:

[0039] (1) The kumquat compound essential oil preparation and its application provided by this invention fill the gap in the existing technology for using plant essential oils as disinfection and sterilization agents in the aquaculture industry. This kumquat compound essential oil preparation can be mixed with aquaculture feed for feeding or sprayed directly into the water. It is convenient to use and has a significant antibacterial effect.

[0040] (2) The kumquat compound essential oil provided by this invention is mainly composed of kumquat essential oil, and is supplemented with four other essential oils: clove essential oil, myrrh essential oil, costus root essential oil, and frankincense essential oil. The five plant essential oils produce a synergistic effect of complementary functions, which enhances the antibacterial, disinfectant, and anti-inflammatory effects of the compound plant essential oil, making the antibacterial, disinfectant, and anti-inflammatory effects more significant. Moreover, this compound plant essential oil does not contain any other chemical components or preservatives, which is easy to protect the environment, and is safe and non-toxic to the growth of aquatic animals.

[0041] (3) The present invention utilizes subcritical circulating extraction technology of low boiling point solvent to extract crude oil from kumquat, clove, myrrh, costus root and frankincense respectively. It has the characteristics of ultra-low temperature extraction, no destruction of active ingredients and high extraction yield.

[0042] (4) This invention uses a POPE scraped-film molecular distillation device to separate and purify mixed crude oil to obtain a kumquat compound essential oil preparation. Compared with conventional distillation equipment, the POPE scraped-film molecular distillation device has significant differences in separation principle, structural design and operating conditions. Therefore, using this device can: 1) accurately separate heat-sensitive substances; 2) increase mass transfer efficiency by more than 10 times; and 3) effectively retain volatile active ingredients in the essential oil. This effectively ensures the efficient enrichment of antibacterial components (such as d-limonene, eugenol, etc.) in the kumquat compound essential oil. The total content of d-limonene and eugenol in the compound essential oil is ≥90wt%, and there are no chemical residues, meeting the green disinfection requirements of aquaculture. Attached Figure Description

[0043] Figure 1 This is a simplified flow chart of the extraction process for preparing compound essential oils using subcritical extraction. Wherein: 1 is the solvent tank; 2 is the extraction tank; 3 is the first buffer tank; 4 is the second buffer tank; 5 is the third buffer tank; 6 is the compressor; V1-V7 are the first to seventh valves. Detailed Implementation

[0044] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the raw materials are all available from publicly available commercial sources.

[0045] Example 1: Effect of different extraction temperatures on the extraction rate of crude oil from kumquats

[0046] S1. Pre-treatment of kumquat raw materials: Remove the stems from the fresh kumquats, wash and dry them, remove the pulp, then pre-freeze the kumquat peels and dry them in a vacuum freeze dryer to remove moisture. After drying, use a traditional Chinese medicine pulverizer to pulverize and sieve the pre-frozen kumquat peels for later use.

[0047] S2. Crude oil extraction: Follow the instructions in the appendix... Figure 1 As shown in the simplified process flow diagram, crude oil is extracted from kumquats using subcritical circulation extraction technology with the low-boiling-point solvent CHCIF2(R22). The specific operations are as follows:

[0048] CHCIF2(R22) was stored in solvent tank 1. 1000g of kumquat peel pretreated with S1 was placed in extraction tank 2 and sealed. Solvent was injected into extraction tank 2 through solvent tank 1. The injection was stopped when the pressure in extraction tank 2 reached 1MPa. At this pressure, the solvent volume in the extraction tank was 15L. The extraction temperatures were set to 30℃, 35℃, 40℃, 45℃, and 50℃, with each extraction time being 1.0h, and the extraction cycle was repeated 6 times.

[0049] After extraction at a certain temperature for a period of time, the solvent and extract are discharged from the bottom of the extraction tank. After passing through the first buffer tank 3 and the second buffer tank 4, the mixture is depressurized, achieving gas-liquid separation. The solvent vaporizes and flows to the third buffer tank 5, where it is pressurized, cooled, and liquefied by the compressor 6, and then refilled into the extraction tank 2 for repeated extraction. After a certain number of cycles, the solvent is slowly released from the top of the third buffer tank 5. Once the solvent is exhausted, the mixed crude oil is released from the bottom of the buffer tank. Both the extraction tank and the buffer tank have a volume of 200L. The extraction tank is jacketed, and circulating hot water is used in the jacket to control the extraction temperature.

[0050] 1000g of pretreated kumquat peel was used to extract crude oil from kumquats according to the above-mentioned process parameters. The volume of crude oil obtained and its extraction yield are shown in Table 1 below:

[0051] Table 1 Extraction rates at different extraction temperatures

[0052]

[0053] The results showed that the extraction rate increased with increasing extraction temperature, reaching a maximum of 2.23% at 45℃. However, the extraction rate decreased when the extraction temperature exceeded 45℃. During the extraction process, the extraction pressure increased along with the extraction temperature, the diffusion rate accelerated, and the density of R22 also increased, thus enhancing the solubility of the crude kumquat oil components, which was beneficial for the extraction of crude kumquat oil. However, excessively high extraction temperatures led to increased R22 vaporization and decreased liquid solvent, resulting in a significant increase in system pressure. The reduced solvent volume was detrimental to solute diffusion, hindering the dissolution of crude oil and ultimately causing a decrease in the extraction rate. Therefore, considering factors such as system safety and overall extraction efficiency, 40℃ was selected as the optimal temperature for the extraction of crude kumquat oil.

[0054] Example 2: Effect of different extraction times on the extraction rate of crude oil from kumquat

[0055] S1. Pretreatment of kumquat raw materials: The operation steps are the same as in Example 1.

[0056] S2. Extraction of crude oil from kumquats: The extraction time was set to 0.5h, 1.0h, 1.5h, 2.0h, and 2.5h for each extraction, and the extraction temperature was set to 40℃ for each extraction. The extraction cycle was repeated 6 times for each extraction. Other operating steps were the same as in Example 1.

[0057] 1000g of kumquat peels pretreated with S1 were used to extract crude oil from the kumquats according to the above-mentioned process parameters. The volume of crude oil obtained and its extraction yield are shown in Table 2 below:

[0058] Table 2 Extraction rates at different extraction times

[0059]

[0060] The results showed that extraction time had a significant impact on the extraction rate of kumquat crude oil. The extraction rate gradually increased with prolonged extraction time, but after exceeding 1.0 h / extraction, the change in extraction rate became insignificant, as the solubility of the kumquat crude oil components in the extraction solvent approached saturation, and the extraction rate essentially ceased to increase. While extending the extraction time per extraction could increase the extraction rate to some extent, it also increased energy consumption, resulting in a decrease in overall extraction efficiency. Therefore, 1.0 h / extraction was selected as the optimal extraction time for kumquat crude oil.

[0061] Example 3: Effect of different extraction times on the extraction rate of crude oil from kumquat

[0062] S1. Pretreatment of kumquat raw materials: The operation steps are the same as in Example 1.

[0063] S2. Extraction of crude oil from kumquats: The number of extraction cycles was set to 3, 6, 9, 12, and 15, respectively. The extraction temperature was 40℃ for each cycle, and the extraction time was 1.0 h for each cycle. Other operating steps were the same as in Example 1.

[0064] 1000g of kumquat peels pretreated with S1 were used to extract crude oil from the kumquats according to the above-mentioned process parameters. The volume of crude oil obtained and its extraction yield are shown in Table 3 below:

[0065] Table 3 Extraction rates at different extraction times

[0066]

[0067] The results showed that the number of extractions had the most significant impact on the extraction rate of kumquat crude oil. The extraction rate increased with the number of extractions. When the number of extractions reached 12, the extraction rate reached 2.35%. Further increasing the number of extractions did not significantly improve the extraction rate, indicating that the kumquat crude oil was almost completely extracted. Continuing to increase the number of extractions solely to improve the extraction rate would only increase equipment wear and tear, reducing overall economic benefits and extraction efficiency. Therefore, 12 extractions were considered the optimal number of extractions for kumquat crude oil. However, considering factors such as extraction efficiency and cost, the final experiment selected 9 extractions as the preferred number.

[0068] Conclusion: The use of low-boiling-point solvents and a circulating extraction process can effectively extract crude kumquat oil. The R22 solvent can reach subcritical conditions at relatively low temperatures; for example, the pressure can reach 1.44 MPa at 40°C. The solvent's solubility for crude kumquat oil is significantly enhanced, but the pressure remains low, enabling large-scale, low-cost industrial production. The solvent is chemically stable, inexpensive, and readily available. It rapidly separates from the extract without residue at room temperature and pressure, allowing for recycling and saving costs while being environmentally friendly.

[0069] The preferred process parameters for extracting crude oil from kumquats are: temperature 40℃, time 1.0h / cycle, and number of cycles 9. Under these preferred conditions, the extraction rate of crude oil from kumquats can reach 2.25%.

[0070] Example 4: Preparation of Kumquat Compound Essential Oil Formulation A

[0071] S1. Raw Material Pretreatment: Remove the stems from fresh kumquats, wash and dry them, remove the pulp, then pre-freeze the kumquat peels and dry them in a vacuum freeze dryer to remove moisture. Simultaneously, dry the cloves, myrrh, costus root, and frankincense to remove moisture. After drying, use a traditional Chinese medicine pulverizer to pulverize each herb and sieve it through a standard sieve to separate particles of different sizes for later use.

[0072] S2. Crude oil extraction: According to the appendix Figure 1 As shown in the simplified process flow diagram, crude oils were extracted from kumquat, clove, myrrh, costus root, and frankincense using subcritical circulating extraction technology with the low-boiling-point solvent CHCIF2(R22). The specific operations are as follows:

[0073] CHCIF2(R22) was stored in solvent tank 1. 10000g of kumquat peel pretreated with S1 was placed in extraction tank 2 and sealed. Solvent was injected into extraction tank 2 through solvent tank 1. The injection was stopped when the pressure in extraction tank 2 reached 1MPa. At this pressure, the solvent volume in the extraction tank was 150L. The extraction temperature was set at 40℃, the extraction time was 1.0h per extraction, and the number of extraction cycles was 9.

[0074] After extraction at a certain temperature for a period of time, the solvent and extract are discharged from the bottom of the extraction tank. After passing through the first buffer tank 3 and the second buffer tank 4, the mixture is depressurized, achieving gas-liquid separation. The solvent vaporizes and flows to the third buffer tank 5, where it is pressurized, cooled, and liquefied by the compressor 6, and then refilled into the extraction tank 2 for repeated extraction. After a certain number of cycles, the solvent is slowly released from the top of the third buffer tank 5. Once the solvent is exhausted, the mixed crude oil is released from the bottom of the buffer tank. Both the extraction tank and the buffer tank have a volume of 200L. The extraction tank is jacketed, and circulating hot water is used in the jacket to control the extraction temperature.

[0075] 10,000g of kumquat peel pretreated with S1 was extracted by subcritical circulation using the above-mentioned low-boiling-point solvent to obtain 224.5ml of crude kumquat oil.

[0076] Following the extraction steps described above, 10,000 g each of pretreated cloves, myrrh, costus root, and frankincense were sequentially subjected to subcritical cyclic extraction with low-boiling-point solvents to obtain 245.6 ml of clove crude oil, 147.1 ml of myrrh crude oil, 222.4 ml of costus root crude oil, and 176.5 ml of frankincense crude oil.

[0077] S3. Mixing crude oils: Take portions of the crude oils obtained in step S2 (kumquat, clove, myrrh, costus root, and frankincense) and mix them in a volume ratio of 40:30:10:10:10 to obtain 100ml of mixed crude oil.

[0078] S4. Separation and purification of the mixed crude oil: Molecular distillation was carried out using a POPE scraped-film molecular distillation apparatus under the following conditions: scraper gap 0.2-0.5 mm, residence time ≤30 s, vacuum degree 0.1 mbar, evaporation temperature 50℃, condensation temperature -10℃, and scraper rotation speed 300 rpm. 100 ml of the mixed crude oil obtained in step S3 was separated and purified to obtain 85.0 ml of kumquat compound essential oil A.

[0079] S5 Emulsification: Take 85ml of the above compound essential oil A, add 2.0wt% Tween-80 and 1.00wt% soybean lecithin, stir and dissolve at 60℃, and homogenize twice under high pressure of 70 MPa to obtain kumquat compound essential oil nanoemulsion preparation A with an average particle size of 152±18nm (PDI=0.15) and a Zeta potential of -38 mV.

[0080] Example 5: Preparation of Kumquat Compound Essential Oil Formulation B

[0081] S1. Raw material pretreatment: Same as in Example 4.

[0082] S2. Crude oil extraction: Same as in Example 4.

[0083] S3. Mixing crude oils: Mix the crude oils of kumquat, clove, myrrh, costus root, and frankincense obtained in step S2 in a volume ratio of 30:40:15:5:10 to obtain 100ml of mixed crude oil.

[0084] S4. Separation and purification of the mixed crude oil: Using a POPE scraped membrane molecular distillation device, 100ml of the mixed crude oil obtained in step S3 was separated and purified to obtain 85.0ml of kumquat compound essential oil B.

[0085] S5 Emulsification: The operation steps are the same as in Example 4, to obtain Kumquat Compound Essential Oil Nanoemulsion Formulation B.

[0086] Example 6: Preparation of Kumquat Compound Essential Oil Preparation C

[0087] S1. Raw material pretreatment: Same as in Example 4.

[0088] S2. Crude oil extraction: Same as in Example 4.

[0089] S3. Mixing crude oils: Mix the crude oils of kumquat, clove, myrrh, costus root, and frankincense obtained in step S2 in a volume ratio of 50:20:5:15:10 to obtain 100ml of mixed crude oil.

[0090] S4. Separation and purification of the mixed crude oil: Using a POPE scraped membrane molecular distillation device, 100 ml of the mixed crude oil obtained in step S3 was separated and purified to obtain 85.0 ml of kumquat compound essential oil C.

[0091] S5 Emulsification: The operation steps are the same as in Example 4, to obtain the kumquat compound essential oil nanoemulsion preparation C.

[0092] Comparative Example 1: Preparation of compound essential oil formulation (clove + myrrh + costus root + frankincense, without kumquat)

[0093] S1. Pretreatment of raw materials (clove, myrrh, costus root, frankincense): The operation steps are the same as in Example 4.

[0094] S2. Extraction of crude oil (clove, myrrh, costus root, frankincense): The operation steps are the same as in Example 4.

[0095] S3. Mixing crude oils: According to the volume ratio of clove crude oil 30, myrrh crude oil 10, costus root crude oil 10, frankincense crude oil 10, 30 ml of mixed crude oil is obtained.

[0096] S4. Separate and purify the mixed crude oil: The operation steps are the same as in Example 4, and 25.5 ml of kumquat compound essential oil is obtained.

[0097] S5 Emulsification: The operation steps are the same as in Example 4.

[0098] Comparative Example 2: Preparation of Kumquat Essential Oil Preparation (only kumquat essential oil, no other essential oils)

[0099] S1. Pre-treatment of kumquat raw materials: The operation steps are the same as in Example 4.

[0100] S2. Extraction of crude oil from kumquats: The operation steps are the same as in Example 4.

[0101] S3. This kumquat essential oil preparation contains only kumquat essential oil and does not contain any blend of clove crude oil, myrrh crude oil, costus root crude oil, or frankincense crude oil.

[0102] S4. Separate and purify the crude kumquat oil to obtain kumquat essential oil: The operation steps are the same as in Example 4. Separate and purify 30ml of crude kumquat oil to obtain 25.5ml of kumquat essential oil.

[0103] S5 Emulsification: The operation steps are the same as in Example 4.

[0104] Comparative Example 3: Preparation of Compound Essential Oil Preparation (Kumquat + Clove)

[0105] S1. Raw material (kumquat, clove) pretreatment: The operation steps are the same as in Example 4.

[0106] S2. Extraction of crude oil (kumquat, clove): The operation steps are the same as in Example 4.

[0107] S3. Mixing crude oils: According to the volume ratio of 40 parts kumquat crude oil and 30 parts clove crude oil, 35 ml of mixed crude oil is obtained.

[0108] S4. Separate and purify the mixed crude oil: The operation steps are the same as in Example 4, and 29.7 ml of kumquat compound essential oil is obtained.

[0109] S5 Emulsification: The operation steps are the same as in Example 4.

[0110] Comparative Example 4: Preparation of Compound Essential Oil Preparation (Kumquat + Clove + Myrrh)

[0111] S1. Pretreatment of raw materials (kumquat, clove, myrrh): The operation steps are the same as in Example 4.

[0112] S2. Extraction of crude oil (kumquat, clove, myrrh): The operation steps are the same as in Example 4.

[0113] S3. Mixing crude oils: According to the volume ratio of 40 parts kumquat crude oil, 30 parts clove crude oil, and 10 parts myrrh crude oil, 40 ml of mixed crude oil is obtained.

[0114] S4. Separate and purify the mixed crude oil: The operation steps are the same as in Example 4, and 34.0 ml of kumquat compound essential oil is obtained.

[0115] S5 Emulsification: The operation steps are the same as in Example 4.

[0116] Comparative Example 5: Preparation of Compound Essential Oil Preparation (Kumquat + Clove + Myrrh + Costus Root)

[0117] S1. Pretreatment of raw materials (kumquat, clove, myrrh, costus root): The operation steps are the same as in Example 4.

[0118] S2. Extraction of crude oil (kumquat, clove, myrrh, costus root): The operation steps are the same as in Example 4.

[0119] S3. Mixing crude oils: According to the volume ratio of 40 parts kumquat crude oil, 30 parts clove crude oil, 10 parts myrrh crude oil, and 10 parts costus root crude oil, 45 ml of mixed crude oil is obtained.

[0120] S4. Separate and purify the mixed crude oil: The operation steps are the same as in Example 4, and 38.2 ml of kumquat compound essential oil is obtained.

[0121] S5 Emulsification: The operation steps are the same as in Example 4.

[0122] Experiment Example 1: The antibacterial effect of kumquat compound essential oil

[0123] The kumquat compound essential oil nanoemulsion preparations obtained in Examples 4-6 and Comparative Examples 1-5 of this invention were subjected to bactericidal effect tests. The composition ratios are shown in Table 4 below:

[0124] Table 4 Composition and Proportioning of Kumquat Compound Essential Oil Nanoemulsion Formulation

[0125]

[0126] The sterilization test method is as follows:

[0127] (1) The test strains were Escherichia coli (8099), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 15442), Candida albicans (ATCC 10231), Aspergillus niger (ATCC 16404) and Mycobacterium guilloché (ATCC 93326).

[0128] (2) Reagents used for culturing bacteria include ordinary nutrient agar medium, Saburg dextrose agar medium and malt extract agar (MEA). The neutralizing agent is composed of D / E broth containing 2 g / L histidine, 20 g / L soap base, 80 g / L lecithin and 100 g / L Tween 80, which is sterilized and ready for use.

[0129] (3) Preparation of bacterial suspension: Single typical colonies of each bacterial vegetative body, after purification and culture, were inoculated onto nutrient agar slant culture and cultured for 24 h. Fresh slant cultures were washed with tryptone saline (TPS) to prepare bacterial suspensions for later use. Aspergillus niger was inoculated onto MEM slant culture, and enrichment broth was inoculated into Roche flasks. The cultures were cultured at 30℃ for 7–9 days. Conidia were washed off with Tween 80 saline solution, mycelia were removed by filtration, and the bacterial suspension was collected and centrifuged to prepare test bacterial suspensions. Mycobacterium guilloché subsp. abscessus was prepared by slant culture of Mycobacterium guilloché for 72 h, washed off with TPS, and added to sterile conical centrifuge tubes containing glass beads. Bacterial suspensions were then prepared. During the experiment, all bacterial suspensions were diluted with 3% bovine serum albumin (BSA) for bactericidal tests.

[0130] (4) Quantitative bactericidal test of suspension: The test was conducted in a 20℃ water bath. Before the test, each bacterial suspension and the disinfectant solution of the test concentration were kept at a constant temperature for more than 5 minutes. 1.0 mL of bacterial suspension and 4.0 mL of disinfectant solution (TPS for the control group) were added to a sterile test tube, mixed well, and the time was set. After the predetermined time of action, 0.5 mL of the bacterial-drug mixture was added to a test tube containing 4.5 mL of neutralizing agent and neutralized for 10 minutes. 1.0 mL of the sample solution was inoculated into a sterile Petri dish, in duplicate. Melted nutrient agar was poured in and mixed well. After solidification, the mixture was incubated for the set time, and viable bacteria were counted to calculate the kill log value. Each group of tests was repeated 3 times.

[0131] (5) Experimental results:

[0132] ① The bactericidal effects of different dilution ratios (10x, 100x, 1000x, and 10000x dilutions) of kumquat compound essential oil nanoemulsion preparation A (Example 4) are shown in Table 5 below:

[0133] Table 5. Antibacterial effect of kumquat compound essential oil A (Example 4) at different dilution ratios

[0134]

[0135] The analysis and comparison of the kill logarithmic values ​​of formulation A in Example 4 at different treatment times and dilution ratios show that:

[0136] When the compound essential oil of kumquat was diluted 100 times and applied for 20 minutes, the kill log value of Staphylococcus aureus (log kill number 5.05), Escherichia coli (log kill number 5.49), and Pseudomonas aeruginosa (log kill number 5.78) all exceeded 5.00, which met the disinfection requirements.

[0137] When the compound essential oil of kumquat was diluted 1000 times and applied for 60 minutes, the log kill value of Staphylococcus aureus (log kill value 5.12), Escherichia coli (log kill value 5.33), and Pseudomonas aeruginosa (log kill value 5.25) all exceeded 5.00, which met the disinfection requirements.

[0138] A 10,000-fold dilution of the kumquat compound essential oil (corresponding to an actual application concentration of 100 ppm) was applied for 120 minutes. The log kill values ​​against Staphylococcus aureus (log kill value 5.02), Escherichia coli (log kill value 5.25), and Pseudomonas aeruginosa (log kill value 4.95) were all >4.90 and close to or exceeded 5.00. This proves that in actual production scenarios, using a concentration of 100 ppm and ensuring a contact time of 120 minutes can effectively meet the disinfection requirements.

[0139] ② The sterilization results of the kumquat compound essential oil nanoemulsion preparations A (Example 4), B (Example 5), and C (Example 6), all diluted 1000 times, and Comparative Examples 1-5, after 60 minutes of contact, are shown in Table 6 below:

[0140] Table 6. Bactericidal effects of kumquat compound essential oil nanoemulsion formulations A / B / C and comparative examples 1-5 (1000x dilution)

[0141]

[0142] Analysis and comparison of the bacterial count logarithmic values ​​in the above experiments showed that the bactericidal effects of kumquat compound essential oil nanoemulsion preparations A / B / C at a 1000-fold dilution for 60 minutes were generally better than those of comparative examples 1-5. The bactericidal effects, ranked from highest to lowest, are as follows: Compound Essential Oil A > Compound Essential Oil C > Compound Essential Oil B > Comparative Example 5 > Comparative Example 4 > Comparative Example 3 > Comparative Example 2 > Comparative Example 1. Among them, Example 4 (the ratio of the five essential oils was kumquat: clove: myrrh: costus root: frankincense = 4:3; 1:1:1) had the best bactericidal effect, while Comparative Example 1 (which contained no kumquat essential oil, only clove, myrrh, costus root, and frankincense essential oils) had the worst bactericidal effect.

[0143] The above experimental results show that:

[0144] The kumquat compound essential oil A / B / C (Examples 4-6) provided by this invention is based on kumquat essential oil and combined with four other essential oils: clove essential oil, myrrh essential oil, costus root essential oil, and frankincense essential oil. The five plant essential oils produce a synergistic effect of complementary functions, which significantly enhances the antibacterial and disinfection efficacy of the compound plant essential oil at relevant concentrations (e.g., 1000 times dilution, approximately 1000 ppm) and action time (60 min) in actual applications.

[0145] Of particular importance is that when Compound Essential Oil A is diluted 10,000 times (100 ppm) and acted for 120 minutes, its killing effect on major pathogens can approach or reach the disinfection standard (kill log value ≥ 5). This provides a solid experimental basis for disinfection at an economically feasible low concentration (100 ppm) in large-scale aquaculture (see application effect in Experiment Example 2).

[0146] This kumquat compound essential oil contains no other chemical ingredients or preservatives, making it easy to protect the environment. It is also safe and non-toxic to the growth of aquatic animals, making it a green, safe, and environmentally friendly bactericidal and disinfectant preparation that will undoubtedly play an important role in aquaculture.

[0147] Experimental Example 2: Application Effect of Kumquat Compound Essential Oil Preparation A (Example 4) in Bacterial Disinfection of Aquaculture

[0148] (1) Aquaculture species: Red loach, Taiwan loach, mandarin fish, turbot

[0149] (2) Time and location of use: Time of use: This experiment was conducted in 2024, specifically in the spring, summer and autumn seasons. Bacterial disinfection was carried out once a month, and data was continuously observed and recorded. Location of use: Aquaculture base of Ruichang Xingxiang Company in Jiujiang.

[0150] (3) How to use

[0151] Preparation of formulation: Kumquat compound essential oil nanoemulsion formulation A was prepared according to Example 4.

[0152] After pond clearing, the initial dosage and concentration for use is 100g / m³. 3 (i.e., 100 ppm). This concentration was determined based on the results in Table 5 of Experimental Example 1, corresponding to the 10,000-fold dilution in the small-scale test, and demonstrated in the small-scale test that it could effectively kill the main pathogens after 120 min of action (the log killing value was close to or greater than 5).

[0153] During the aquaculture process, depending on the tolerance of the farmed species, the proliferation of bacteria and parasites in aquatic animals can be prevented by reducing the concentration and extending the duration of action. Specific dosage, concentration, and duration of action can be adjusted as needed based on actual circumstances.

[0154] Specific operations:

[0155] ① Calculate the volume of water and weigh essential oil nanoemulsion preparation A.

[0156] ② After pre-diluting with an appropriate amount of warm water (35-40℃), sprinkle evenly throughout the pool.

[0157] ③ After spraying, depending on the tolerance of the aquaculture species, the oxygenation equipment can be turned on to maintain water flow, but avoid excessive stirring to prevent the essential oil from evaporating too quickly.

[0158] Frequency of use: Perform bacterial disinfection once a month. Ensure adequate aeration of the water before treatment, and closely observe and record the fish's reactions and growth after treatment.

[0159] (4) Application effect

[0160] ① Reduce mortality rate:

[0161] Compared with previous years, the mortality rate of red loach using Kumquat Compound Essential Oil Preparation A for bacterial disinfection decreased by about 20%, that of Taiwan loach by about 18%, that of mandarin fish by about 14%, and that of turbot by about 8-12%.

[0162] This significant reduction in mortality indicates that kumquat compound essential oil preparation A has a significant antibacterial effect, which can effectively reduce bacterial diseases in aquaculture and improve the survival rate of aquaculture.

[0163] ② Improved growth performance:

[0164] During the experiment, the growth rates of all farmed species improved to varying degrees. Among them, the weight growth rate of red loach and Taiwan loach increased by about 8% and 10% respectively, while the weight growth rate of mandarin fish and turbot increased by about 9% and 7% respectively. This improvement in growth performance may be related to the fact that the kumquat compound essential oil preparation A promotes intestinal health and improves nutrient absorption efficiency.

[0165] ③ Improved gut health:

[0166] Microscopic observation revealed that fish treated with kumquat compound essential oil preparation A had more intact intestinal villi and healthier intestinal walls.

[0167] This indicates that kumquat compound essential oil preparation A can improve the balance of intestinal microecology, reduce the adhesion of pathogens, and enhance the permeability of the intestinal barrier, thereby improving intestinal health.

[0168] ④ Water quality improvement:

[0169] During the experiment, water quality indicators (such as dissolved oxygen, ammonia nitrogen, nitrite, etc.) were tested regularly. It was found that the water quality indicators using kumquat compound essential oil preparation A were more stable and had a smaller fluctuation range.

[0170] This indicates that the kumquat compound essential oil preparation A has a certain regulatory effect on water quality, can maintain water quality stability, and provide a good growth environment for fish.

[0171] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention are covered within the scope of the present invention.

Claims

1. A kumquat compound essential oil preparation for disinfection in aquaculture, characterized in that: The kumquat compound essential oil preparation comprises, by volume, 30-50 parts kumquat essential oil, 20-40 parts clove essential oil, 5-15 parts myrrh essential oil, 5-15 parts costus root essential oil, and 5-15 parts frankincense essential oil. The preparation method of the kumquat compound essential oil preparation includes the following steps: S1 raw material pretreatment; S2 extraction of crude oil from each raw material separately; S3 mixing of crude oils; S4 separation and purification of the mixed crude oil to obtain the kumquat compound essential oil. The crude oil extraction in step S2 is performed using a subcritical circulation extraction technology with a low-boiling-point solvent to extract crude oil from kumquat, clove, myrrh, costus root, and frankincense respectively. The low-boiling-point solvent is selected from CHClF2. The separation and purification in step S4 is performed using a POPE scraped-film molecular distillation device to separate and purify the mixed crude oil.

2. The kumquat compound essential oil preparation for disinfection in aquaculture according to claim 1, characterized in that: The kumquat compound essential oil preparation comprises, by volume, 40 parts kumquat essential oil, 30 parts clove essential oil, 10 parts myrrh essential oil, 10 parts costus root essential oil, and 10 parts frankincense essential oil.

3. The kumquat compound essential oil preparation according to claim 1, characterized in that: The preparation method further includes an S5 emulsification step.

4. The kumquat compound essential oil preparation according to claim 1, characterized in that, The raw material pretreatment described in step S1 is as follows: remove the stems from the fresh kumquats, wash and dry the fresh kumquats, remove the pulp, pre-freeze the kumquat peels and then put them into a vacuum freeze dryer to dry them to remove moisture; at the same time, dry the cloves, myrrh, costus root, and frankincense to remove moisture; after drying, use a traditional Chinese medicine pulverizer to pulverize and sieve each herb for later use.

5. The kumquat compound essential oil preparation according to claim 1, characterized in that, The crude oil mixing in step S3 specifically involves mixing kumquat crude oil, clove crude oil, myrrh crude oil, costus root crude oil, and frankincense crude oil in a volume ratio of 30-50:20-40:5-15:5-15:5-15.

6. The kumquat compound essential oil preparation according to claim 1, characterized in that, The subcritical cyclic extraction described in step S2 has a solid-liquid ratio of 1:(8-20), an extraction pressure of 0.8-1.6 MPa, an extraction temperature of 30℃-50℃, an extraction time of 0.5h-2.5h per extraction, and a number of cyclic extractions of 3-12.

7. The kumquat compound essential oil preparation according to any one of claims 1-2 is used to prepare a disinfectant for aquaculture bacteria.

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