Linalool nanocomposites and methods of making the same

Abstract

The application discloses a linalool nanocomposite and a preparation method thereof. The preparation method comprises the following steps: mixing solid lipids with liquid lipids, mixing the obtained solid-liquid lipids with linalool to prepare an oil phase; adding an aqueous phase containing a surfactant into the oil phase, stirring, ultrasonic dispersion, and filtering to obtain the linalool nanocomposite, and the temperature difference between the aqueous phase and the oil phase is within 3 DEG C. In the application, the linalool nanocomposite is prepared by using a melting emulsification-ultrasonic dispersion method, and the linalool nanocomposite with uniform particle size distribution, good dispersibility, high encapsulation rate and good stability can be obtained. When the linalool nanocomposite is used for preventing and treating water mold disease, the linalool nanocomposite exhibits very excellent antibacterial effect, has high application value and commercial value, and has important significance for promoting the wide application of linalool in the field of water mold disease prevention and treatment. The preparation method has the advantages of simple production process, short production period, low cost and the like, is suitable for large-scale preparation, and is beneficial to industrial application.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture and relates to a plant-derived fungicide for the prevention and control of water mold disease and its preparation method, specifically to a linalool nanocomposite material and its preparation method. Background Technology

[0002] Some species in the Saprolegniaceae family are the main pathogens of Saprolegnia diseases, such as the genera *Saprolegnia*, *Amycetes*, and *Hylocereus*, among which *Saprolegnia parasitica* (… Saprolegnia parasitica ), multi-seed water mold ( Saprolegnia ferax This fungus, widely found in freshwater and brackish waters and moist soil, has a significant impact on freshwater ecosystems. It infects a wide range of hosts, including fish, fish eggs, amphibians, and crustaceans. The main symptom of infection is the formation of a grayish-white, cotton-like covering on the body surface, hence the name "white hair disease." The fungal hyphae often appear grayish-white or pale yellow due to enveloping suspended matter or attaching to other organisms. Infected freshwater animals also experience decreased immunity, abnormal swimming, reduced appetite, and a large number of eggs die and deteriorate during incubation, reducing the hatching rate. This seriously endangers the healthy development of aquaculture.

[0003] Currently, the main methods for controlling saprolegniasis include chemical control, immunization, and ecological control, with chemical control remaining the primary measure, including antibiotics. However, the use of antibiotics is severely limited because most antibiotics are ineffective against saprolegniasis, and their residues have carcinogenic, teratogenic, and mutagenic effects on humans. Therefore, developing highly effective, low-toxicity, low-residue, and environmentally friendly plant-derived fungicides is a more realistic approach to addressing the urgent production needs.

[0004] Studies have shown that many plants possess properties that inhibit or kill fungi and other microorganisms. Currently, there is considerable research on the antibacterial mechanisms of plant essential oils, generally suggesting that the hydrophobic components of essential oils can directly act on the cell membranes of microorganisms, disrupting the cell membrane structure, causing important ions and genetic material to leak out of the cell, ultimately leading to cell death. Research reports that the essential oils of vanilla (Satureja tymbra L.) and oregano (Origanum onites L.), two natural traditional Chinese medicines, exhibit excellent antifungal effects against parasitic water molds. Rhein and aloe-emodin, isolated from the traditional Chinese medicine rhubarb, were tested for their in vitro inhibitory activity against the hyphal growth and spore germination of water molds, revealing good anti-parasitic activity against water molds. Furthermore, toxicity results indicate that the extracts are safe and non-toxic to aquatic organisms. Therefore, utilizing plant essential oils for the control of pathogenic fungi is currently a hot research topic. However, overall, the number of plant-derived fungicides actually available on the market is limited, requiring further research and development to create competitive plant-derived fungicides.

[0005] Linalool is an unsaturated acyclic monoterpene alcohol found in the volatile oils of various natural aromatic plants. It possesses antibacterial activity and holds great promise for medicinal applications. However, its high volatility and poor water solubility limit its widespread use in freshwater systems, and it exhibits poor efficacy against water mold. Furthermore, some researchers have proposed a strategy to fabricate linalool into nanostructured lipid carriers. However, existing methods for preparing linalool nanostructured lipid carriers primarily rely on high-pressure homogenization, which yields products with low encapsulation efficiency, poor dispersibility, and uneven particle size. This hinders the improvement of the efficacy of linalool nanostructured lipid carriers against water mold, and the active ingredient's effect is short-lived and prone to degradation. Therefore, obtaining a linalool nanocomposite material with uniform particle size distribution, good dispersibility, high encapsulation efficiency, and good stability is crucial for the widespread application of linalool in the control of water mold. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a linalool nanocomposite material with uniform particle size distribution, good dispersibility, high encapsulation efficiency, and good stability, as well as a method for its preparation.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for preparing a linalool nanocomposite material includes the following steps: S1. Mix solid lipids and liquid lipids and stir to obtain solid-liquid lipids; S2. Mix the solid-liquid lipid obtained in step S1 with linalool and stir to obtain the oil phase; S3. Add the aqueous phase to the oil phase obtained in step S2, stir, sonicate, and filter to obtain linalool nanocomposite material; the aqueous phase is prepared by water and surfactant; the temperature difference between the aqueous phase and the oil phase is within 3°C.

[0008] In a further improvement to the above preparation method, in step S3, the temperature of the aqueous phase is 72℃~78℃.

[0009] In a further improvement to the above preparation method, step S3 includes the following steps: mixing water and a surfactant, stirring at 600 rpm for 40 min, and heating for 5 min until the temperature reaches 72℃~78℃; the mass ratio of water to surfactant is 18~18.9∶0.2~1; the surfactant is a mixture of Tween 80 and Span 80; and the HLB value of the surfactant is 8~15.

[0010] In a further improvement to the above preparation method, in step S3, the HLB value of the surfactant is 8.5 to 13.

[0011] In a further improvement to the above preparation method, in step S3, the temperature of the system is controlled at 75°C during the stirring process; the stirring time is 15 min; the ultrasonication time is 23 min; and a filter membrane with a pore size of 0.45 μm is used for filtration to remove large particulate impurities during the filtration process.

[0012] In a further improvement to the above preparation method, in step S1, the mass ratio of the solid lipid to the liquid lipid is 1:1 to 4; the solid lipid is glyceryl distearate; and the liquid lipid is oleic acid.

[0013] In a further improvement to the above preparation method, the stirring time in step S1 is 1 minute.

[0014] In a further improvement to the above preparation method, in step S2, the mass ratio of the solid-liquid lipid to linalool is 1 to 6:4.

[0015] In a further improvement to the above preparation method, the stirring time in step S2 is 1 min.

[0016] As a general technical concept, the present invention also provides a linalool nanocomposite material, which is prepared by the above-described preparation method; the linalool nanocomposite material is a nanoemulsion.

[0017] Compared with the prior art, the advantages of the present invention are as follows: (1) To address the shortcomings of existing methods in preparing linalool nanostructured lipid carriers, such as low encapsulation efficiency, poor dispersibility, and uneven particle size, resulting in poor control of water mold and short duration of efficacy, this invention creatively provides a method for preparing linalool nanocomposite materials. First, linalool is mixed with solid-liquid lipids to form an oil phase. Then, an aqueous phase containing surfactants is added to the oil phase and stirred. During this process, controlling the temperature difference between the aqueous and oil phases within 3°C facilitates the formation of a stable system and allows the lipids to effectively encapsulate linalool, forming a nanoparticle solution. The nanoparticles in the solution are then uniformly dispersed during ultrasonication. After filtering to remove large impurities, the linalool nanocomposite material is obtained. Specifically, directly mixing the aqueous and oil phases can easily lead to incomplete emulsification. Compared with traditional synthesis methods (such as high-pressure homogenization), this invention, for the first time, employs a melt emulsification-ultrasonic dispersion method to prepare linalool nanocomposites. This method yields linalool nanocomposites with uniform particle size distribution, good dispersibility, high encapsulation efficiency, and good stability. This is a linalool nanoemulsion that exhibits excellent antibacterial effects when used to control saprolegniasis, demonstrating high application and commercial value. It is of great significance for promoting the widespread application of linalool in the control of saprolegniasis. Furthermore, the preparation method of this invention has advantages such as simple production process, short production cycle, and low cost, making it suitable for large-scale preparation and conducive to industrial application.

[0018] (2) In this invention, the temperature of the oil phase and the water phase during the stirring process is 72℃~78℃, which is more conducive to the formation of a stable system, thereby improving the emulsification effect.

[0019] (3) In this invention, by optimizing the HLB value of the surfactant to 8-15, it is not only beneficial to obtain nanoparticles with small particle size, uniform distribution and good dispersibility, but also beneficial to improve the encapsulation effect of linalool, so that more linalool is encapsulated inside the particles, thereby making the linalool nanocomposite material have a better inhibitory effect on Saprolegnia and a longer-lasting effect. At the same time, as a preferred embodiment, in this invention, a mixture of Tween 80 and Span 80 is used as the surfactant, which is more beneficial to improve the stability of the system, thereby ensuring that a linalool nanocomposite material with uniform particle size distribution, good dispersibility, high encapsulation rate and good stability is obtained.

[0020] (4) In this invention, by optimizing the mass ratio of solid-liquid lipids to linalool to 1-6:4, it is not only beneficial to obtain nanoparticles with smaller particle sizes, but also beneficial to improve the encapsulation effect of linalool, so that more linalool is encapsulated inside the particles, thereby making the linalool nanocomposite material have a better inhibitory effect on Saprolegnia and a longer-lasting efficacy. At the same time, as a preferred embodiment, in this invention, using distearate as the solid lipid and oleic acid as the liquid lipid is more beneficial to obtaining nanoparticles with small particle size and good dispersibility, thereby ensuring that the linalool nanocomposite material with uniform particle size distribution, good dispersibility, high encapsulation rate, and good stability is obtained. For example, when tristearate or triglycerides are used as solid lipids, the prepared material has the defect of high particle size and PDI. Similarly, when propylene glycol dioctanoate, medium-chain glycerides, and isopropyl myristate are used as liquid lipids, the defect of high particle size and PDI also exists. Attached Figure Description

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0022] Figure 1 This is a plate inhibition diagram of saprolegnia against water mold by the linalool nanocomposite material prepared in Example 1 of the present invention.

[0023] Figure 2 This is a comparison chart showing the effect of different lipid dosages on the particle size and polydispersity index of linalool nanocomposites in Example 2 of the present invention.

[0024] Figure 3 This is a comparison graph showing the effect of different lipid dosages on the encapsulation efficiency of linalool nanocomposites in Example 2 of the present invention.

[0025] Figure 4 This is a comparison chart showing the effect of HLB values ​​of different surfactants on the particle size and polydispersity index of linalool nanocomposites in Example 3 of the present invention.

[0026] Figure 5 This is a comparison chart showing the effect of HLB values ​​of different surfactants on the encapsulation efficiency of linalool nanocomposites in Example 3 of the present invention. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0028] The materials and instruments used in the following examples are all commercially available.

[0029] Example 1: A method for preparing a linalool nanocomposite material includes the following steps: S1. Mix distearate and oleic acid in a mass ratio of 1:4 and stir for 1 min to obtain a solid-liquid lipid mixture.

[0030] S2. According to the mass ratio of solid-liquid lipid to linalool of 3.084:4, mix 0.3084 g of solid-liquid lipid obtained in step S1 with 0.4 g of linalool and stir for 1 min to obtain the oil phase.

[0031] S3. Add the aqueous phase at the same temperature (75℃) to the oil phase obtained in step S2, stir and emulsify for 15 min, sonicate for 23 min in an ultrasonic crusher at 70% power, cool to room temperature, and filter with a filter membrane with a pore size of 0.4 μm to remove large particulate impurities, and obtain linalool nanocomposite material.

[0032] In this embodiment, the aqueous phase is prepared by water and surfactant. Specifically, 18.4414g of water and 0.8502g of surfactant are mixed, stirred at 600 rpm for 40 min, and heated for 5 min until the temperature reaches 75°C to obtain the aqueous phase. The surfactant is a mixture of Tween 80 and Span 80 with an HLB value of 11.986.

[0033] Table 1 compares the particle size, polydispersity index (PDI), and encapsulation efficiency (EE) of linalool nanocomposites prepared by different synthesis methods, using response surface methodology (DSM) to predict the optimal solution with the smallest particle size. As shown in Table 1, when the amount of lipid was 1.542% and the HLB value was 11.986, the particle size, PDI, and EE% of the prepared linalool nanocomposites were 70.86%, 0.217%, and 95.41%, respectively, which are significantly better than those prepared by conventional synthesis methods (high-pressure homogenization) for linalool nanostructure lipid carriers. Therefore, compared with conventional synthesis methods, the melt emulsification-ultrasonic dispersion method used in this invention can obtain linalool nanocomposites with uniform particle size distribution, good dispersibility, high encapsulation efficiency, and good stability.

[0034] Table 1. Comparison of particle size, polydispersity index (PDI), and encapsulation efficiency (EE) of linalool nanocomposites prepared by different synthesis methods.

[0035] The linalool nanocomposite material prepared in Example 1 was subjected to a water mold susceptibility test. The water mold used was... Saprolegnia parasitica ATCC200013, Result Figure 1 As shown.

[0036] Figure 1 This is a plate inhibition diagram of the linalool nanocomposite material prepared in Example 1 of the present invention against Saprolegnia. Figure 1 It can be seen that the linalool nanocomposite material prepared by the present invention has a good inhibitory effect on water mold.

[0037] Example 2 In this embodiment, the effects of different lipid dosages on particle size, polydispersity index (PDI), and linalool encapsulation efficiency (EE) were also investigated. Figure 2 and Figure 3 As shown. In this embodiment, when the mass percentage of lipids in the system is 3%, 2.5%, 2%, 1.5%, 1%, and 0.5%, the corresponding mass ratios of solid-liquid lipids to linalool are 3:2, 5:4, 1:1, 3:4, 1:2, and 1:4, respectively. Other preparation conditions are the same as in Example 1.

[0038] Figure 2 This is a comparison chart showing the effect of different lipid dosages on the particle size and polydispersity index of linalool nanocomposites in Example 2 of the present invention.

[0039] Figure 3 This is a comparison graph showing the effect of different lipid dosages on the encapsulation efficiency of linalool nanocomposites in Example 2 of the present invention.

[0040] Depend on Figure 2 and Figure 3 It is known that by optimizing the lipid mass percentage in the system to 0.5%-3%, the solid-liquid lipid to linalool mass ratio is 1-6:4. This not only helps to obtain smaller nanoparticles, but also improves the encapsulation effect of linalool, allowing more linalool to be encapsulated inside the particles. In particular, when the lipid mass percentage in the system is 2.5%, the linalool nanocomposite has the lowest PDI, and the particle size is relatively small, resulting in better encapsulation and higher stability. Consequently, the linalool nanocomposite has a better inhibitory effect on Saprolegnia and a longer-lasting efficacy.

[0041] In addition, the test results show that the linalool nanocomposites prepared under different lipid dosage conditions can effectively inhibit Saprolegnia. Moreover, compared with conventional linalool, the linalool nanocomposites prepared in this invention have a longer antibacterial time and can achieve long-term inhibition of Saprolegnia.

[0042] Example 3 In this embodiment, the effects of HLB values ​​of different surfactants on particle size, polydispersity index (PDI), and encapsulation efficiency (EE) of linalool were also investigated. Figure 4 and Figure 5As shown. In this embodiment, the HLB values ​​of the surfactants used were 8, 9, 10, 11, 12, 13, 14, and 15, respectively, and other preparation conditions were the same as in Example 1.

[0043] Figure 4 This is a comparison chart showing the effect of HLB values ​​of different surfactants on the particle size and polydispersity index of linalool nanocomposites in Example 3 of the present invention.

[0044] Figure 5 This is a comparison chart showing the effect of HLB values ​​of different surfactants on the encapsulation efficiency of linalool nanocomposites in Example 3 of the present invention.

[0045] Depend on Figure 4 and Figure 5 It is understood that in this invention, by optimizing the HLB value of the surfactant to 8-15, it is not only beneficial to obtain nanoparticles with small particle size, uniform distribution and good dispersibility, but also beneficial to improve the encapsulation effect of linalool, so that more linalool is encapsulated inside the particles, thereby making the linalool nanocomposite material have a better inhibitory effect on water mold and a longer-lasting effect.

[0046] As shown in the above results, in the preparation method of this invention, linalool is first mixed with solid-liquid lipids to form an oil phase, and then an aqueous phase containing surfactants is added to the oil phase and stirred. During this process, controlling the temperature difference between the aqueous and oil phases to within 3°C is beneficial for forming a stable system and enabling the lipids to effectively encapsulate linalool and form a nanoparticle solution. Then, during ultrasonication, the nanoparticles in the solution are uniformly dispersed. After filtration to remove large particle impurities, linalool nanocomposite materials are obtained. In particular, directly mixing the aqueous and oil phases can easily lead to incomplete emulsification. Compared with traditional synthesis methods (such as high-pressure homogenization), this invention is the first to use a melt emulsification-ultrasonic dispersion method to prepare linalool nanocomposite materials, which can obtain linalool nanocomposite materials with uniform particle size distribution, good dispersibility, high encapsulation efficiency, and good stability. This is a linalool nanoemulsion that exhibits excellent antibacterial effects when used to control saprolegniasis, possessing high application and commercial value. It is of great significance for promoting the widespread application of linalool in the control of saprolegniasis. In addition, the preparation method of the present invention has the advantages of simple production process, short production cycle and low cost, which are suitable for large-scale preparation and conducive to industrial application.

[0047] The above embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing linalool nanocomposite materials, characterized in that, Includes the following steps: S1. Solid lipids and liquid lipids are mixed and stirred to obtain solid-liquid lipids; the mass ratio of solid lipids to liquid lipids is 1:1 to 4; the solid lipid is glyceryl distearate; the liquid lipid is oleic acid; S2. Mix the solid-liquid lipid obtained in step S1 with linalool and stir to obtain an oil phase; the mass ratio of the solid-liquid lipid to linalool is 1 to 6:

4. S3. Add the aqueous phase to the oil phase obtained in step S2, stir, sonicate, and filter to obtain linalool nanocomposite material; the aqueous phase is prepared by water and surfactant; the HLB value of the surfactant is 8-15; the temperature difference between the aqueous phase and the oil phase is within 3℃.

2. The preparation method according to claim 1, characterized in that, In step S3, the temperature of the aqueous phase is 72℃~78℃.

3. The preparation method according to claim 2, characterized in that, In step S3, the method for preparing the aqueous phase includes the following steps: mixing water with a surfactant, stirring at 600 rpm for 40 min, heating for 5 min until the temperature reaches 72℃~78℃; the mass ratio of water to surfactant is 18~18.9∶0.2~1; the surfactant is a mixture of Tween 80 and Span 80.

4. The preparation method according to claim 3, characterized in that, In step S3, the HLB value of the surfactant is 8.5 to 13.

5. The preparation method according to claim 1, characterized in that, In step S3, the temperature of the system is controlled at 75°C during the stirring process; the stirring time is 15 min, and the ultrasonic time is 23 min; during the filtration process, a filter membrane with a pore size of 0.45 μm is used to remove large particulate impurities.

6. The preparation method according to any one of claims 1 to 5, characterized in that, In step S1, the stirring time is 1 minute.

7. The preparation method according to any one of claims 1 to 5, characterized in that, In step S2, the stirring time is 1 minute.

8. A linalool nanocomposite material, characterized in that, The linalool nanocomposite material is prepared by the preparation method according to any one of claims 1 to 7; the linalool nanocomposite material is a nanoemulsion.

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