A Litsea cubeba essential oil preparation and its application

Abstract

This invention discloses a Litsea cubeba essential oil preparation, which is prepared by mixing a 1% (w / w) chitosan quaternary ammonium salt solution with a Litsea cubeba essential oil ethanol mixture at a volume ratio of 4:1; the volume ratio of Litsea cubeba essential oil to anhydrous ethanol in the Litsea cubeba essential oil ethanol mixture is 2:3. The Litsea cubeba essential oil preparation prepared by this invention exhibits good dispersibility and stability, achieving 100% short-term and long-term antibacterial effect against Aspergillus flavus. This is significantly superior to using Litsea cubeba essential oil or chitosan quaternary ammonium salt alone, as well as other chitosan quaternary ammonium salt-Litsea cubeba essential oil preparations, and even more superior to commonly available antifungal agents. Its preparation method is simple, non-toxic, harmless, and does not pollute the environment. It has low production costs and can be used to prevent or inhibit the growth of Aspergillus flavus, reducing its damage to wooden household products.

Description

Technical Field

[0001] This application relates to the field of plant product preservation technology, and more specifically, to a Litsea cubeba essential oil preparation and its application. Background Technology

[0002] Litsea cubeba, a perennial shrub or small tree belonging to the genus Litsea in the family Lauraceae, is mainly distributed in some provinces and regions south of the Yangtze River in my country. The fruit of Litsea cubeba is rich in oil, with the pericarp containing 3%–5% essential oil. Litsea cubeba essential oil is an active secondary metabolite of the fruit. Processed from fresh Litsea cubeba fruit, it is a unique natural fragrance product in my country and a food additive permitted under national standards. Numerous studies have shown that Litsea cubeba essential oil has broad-spectrum antibacterial properties, making it an indispensable natural antibacterial material. It is known as a natural nemesis of aflatoxin and has enormous application potential and promotional value. However, Litsea cubeba oil is volatile. Currently, methods exist to stabilize and protect the essential oil by encapsulating it in the shell or forming a film, such as microencapsulation technology and gelation, but these processes are complex and costly. Quaternary ammonium chitosan (QAC), a typical derivative of chitosan, possesses biodegradability, biocompatibility, and excellent film-forming properties. The introduction of quaternary ammonium groups weakens the hydrogen bonds between chitosan molecular chains, improving its water solubility and antibacterial activity, effectively locking in essential oil components and providing a slow-release effect. Therefore, there is a need to provide a Litsea cubeba essential oil product with a sustained-release effect, good antibacterial effect, and high stability. Summary of the Invention

[0003] This invention provides a Litsea cubeba essential oil preparation, which is prepared by mixing a 1% (w / w) chitosan quaternary ammonium salt solution with a Litsea cubeba essential oil ethanol mixture at a volume ratio of 1:4; the volume ratio of Litsea cubeba essential oil to anhydrous ethanol in the Litsea cubeba essential oil ethanol mixture is 2:3.

[0004] Preferably, the Litsea cubeba essential oil ethanol mixture is slowly dripped into the chitosan quaternary ammonium salt solution and dispersed to obtain a dispersion in which the microparticles are evenly dispersed, thus obtaining the Litsea cubeba essential oil preparation.

[0005] Dispersion was performed using a high-speed shear disperser. Specifically, dispersion was carried out for 3 minutes at a speed of 10,000 r / min using a high-speed shear disperser.

[0006] The chitosan quaternary ammonium salt solution is prepared as follows: 1g of chitosan quaternary ammonium salt powder is poured into 100mL of purified water and ultrasonicated at 70℃ to make it uniform.

[0007] The present invention also provides the application of the aforementioned Litsea cubeba essential oil preparation in preventing or inhibiting the growth of Aspergillus flavus.

[0008] Specifically, the application includes inhibiting the growth of Aspergillus flavus on wooden household products.

[0009] More specifically, the aforementioned Litsea cubeba essential oil preparation is applied to the surface of the wooden household items to inhibit the growth of Aspergillus flavus on the wooden household items.

[0010] The beneficial effects of this invention include:

[0011] (1) The Litsea cubeba essential oil preparation prepared by the present invention has good dispersibility and stability, with an average particle size of 955.5 nm and a Zeta potential range of +4.16 ~ +37.4 mV. After being placed at room temperature for 2 months, no precipitation, discoloration or stratification occurred.

[0012] (2) The Litsea cubeba essential oil preparation of the present invention overcomes the volatile characteristics of Litsea cubeba essential oil, effectively prolongs the duration of action of Litsea cubeba, and further improves the long-term antibacterial effect against Aspergillus flavus. Its short-term and long-term antibacterial effects both reach 100%, which is significantly better than using Litsea cubeba essential oil or chitosan quaternary ammonium salt alone, and also better than other chitosan quaternary ammonium salt-Litsea cubeba essential oil preparations.

[0013] (3) The Litsea cubeba oil preparation prepared in this invention has a significantly better antibacterial effect against Aspergillus flavus than the commonly used antifungal agents natamycin, sorbic acid and sodium benzoate on the market. The inhibition rate is increased by 93.11%, 96.85% and 89.13% respectively compared with natamycin, sorbic acid and sodium benzoate.

[0014] (4) The Litsea cubeba essential oil preparation of the present invention can reduce the content of aflatoxin in plant products such as peanuts that are easily contaminated by Aspergillus flavus in a long-term and efficient manner. After 72 hours of treatment, the content of aflatoxin in peanuts was reduced by 98.78% to 2.51 μg / kg.

[0015] (5) The preparation method of the Litsea cubeba essential oil preparation of the present invention is simple, has few components and is non-toxic and harmless, does not pollute the environment, and has low production cost. Attached Figure Description

[0016] Figure 1 The graph shows the inhibitory activity of Litsea cubeba essential oil preparations prepared from different ratios of Litsea cubeba essential oil and ethanol mixtures against Aspergillus flavus in Example 3.

[0017] Figure 2 This is a particle size distribution diagram of the Litsea cubeba essential oil preparation prepared according to formula four in Example 3;

[0018] Figure 3The Zeta potential diagram is shown for the Litsea cubeba essential oil preparation prepared according to formula four in Example 3.

[0019] Figure 4 This is a comparison chart of the inhibitory activity of Litsea cubeba essential oil preparation in Example 4 against natamycin, sorbic acid, and sodium benzoate against aflatoxin. Figure 5 This is a comparison chart of the contamination of peanuts with Aspergillus flavus after treatment with Litsea cubeba essential oil and Litsea cubeba essential oil for 24, 48, and 72 hours in Example 5. Detailed Implementation

[0020] The present invention will be further described and illustrated below with reference to embodiments. However, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the present invention and the embodiments, all other inventions and embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0023] In the process of adding the Litsea cubeba essential oil-ethanol mixture dropwise to the chitosan quaternary ammonium salt solution, due to the large proportion of chitosan quaternary ammonium salt in the solution, a preliminary experiment was conducted to explore the concentration of chitosan quaternary ammonium salt and obtain an initial concentration. During the dropwise addition, the dispersibility of the formulation changed with the addition of the Litsea cubeba essential oil-ethanol mixture; excessive Litsea cubeba essential oil-ethanol mixture led to poor dispersibility. Therefore, this application further optimized the ratio of chitosan quaternary ammonium salt to the Litsea cubeba essential oil-ethanol mixture. Finally, considering the optimal antibacterial effect in application, further research was conducted to determine the optimal concentration of Litsea cubeba essential oil.

[0024] Example 1: Determination of chitosan quaternary ammonium salt concentration

[0025] (1) Preparation of chitosan quaternary ammonium salt solution

[0026] Weigh 0.5g, 1g, 1.5g, and 2g of chitosan quaternary ammonium salt (hydroxypropyltrimethylammonium chloride chitosan was used in this example) powder and pour them into 100mL of purified water. Sonicate at 70℃ to make them uniform, and obtain chitosan quaternary ammonium salt solutions with mass-volume percentage concentrations of 0.5%, 1%, 1.5%, and 2%, respectively.

[0027] (2) Preparation of Litsea cubeba essential oil preparations

[0028] Take 1 mL of Litsea cubeba essential oil (extracted from fresh Litsea cubeba fruit by steam distillation) and ethanol mixture (500 μL Litsea cubeba essential oil + 500 μL anhydrous ethanol), and slowly drop it into the above 9 mL chitosan quaternary ammonium salt solution of different concentrations. Use a high-speed shear disperser at 10000 r / min for 3 min to achieve uniform particle dispersion.

[0029] The dispersibility of the Litsea cubeba essential oil preparation prepared in step (2) was studied. The particle size and potential of Litsea cubeba essential oil preparations prepared with chitosan quaternary ammonium salt solutions of different concentrations were tested. The results are shown in Table 1.

[0030] Table 1. Particle size and potential of Litsea cubeba essential oil preparations prepared from chitosan quaternary ammonium salt solutions of different concentrations.

[0031]

[0032] As can be seen from Table 1, the Litsea cubeba essential oil preparation containing a 1% chitosan quaternary ammonium salt solution has a smaller particle size and better dispersibility. Therefore, a 1% chitosan quaternary ammonium salt solution was ultimately selected.

[0033] Example 2: Study on the ratio of Litsea cubeba essential oil ethanol mixture to 1% chitosan quaternary ammonium salt

[0034] To further optimize the ratio of chitosan quaternary ammonium salt to Litsea cubeba essential oil, the following experiment was conducted:

[0035] Take 1 mL of a Litsea cubeba essential oil-ethanol mixture (500 μL Litsea cubeba essential oil + 500 μL anhydrous ethanol) and slowly add it dropwise to 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of a 1% (w / v) chitosan quaternary ammonium salt solution. Use a high-speed shear disperser at 10000 rpm for 3 minutes to achieve uniform particle dispersion. The volume ratios of the Litsea cubeba essential oil-ethanol mixture to the chitosan quaternary ammonium salt solution are 1:1, 1:2, 1:3, 1:4, and 1:5, respectively.

[0036] The dispersibility of the prepared Litsea cubeba essential oil formulation was studied. The particle size and potential of the Litsea cubeba essential oil formulation prepared by mixing Litsea cubeba essential oil with ethanol and 1% chitosan quaternary ammonium salt at different volume ratios were tested. The results are shown in Table 2.

[0037] Table 2. Particle size and potential results of Litsea cubeba essential oil formulations prepared by different ratios of Litsea cubeba essential oil ethanol mixtures with 1% chitosan quaternary ammonium salt.

[0038] As can be seen from Table 2, when the volume ratio of the Litsea cubeba essential oil ethanol mixture to chitosan quaternary ammonium salt with a mass-volume percentage concentration of 1% is 1:4, the particle size of the formulation is smaller and its dispersibility is better. Therefore, the Litsea cubeba essential oil formulation was finally prepared by mixing the Litsea cubeba essential oil ethanol mixture with chitosan quaternary ammonium salt with a mass-volume percentage concentration of 1% at a volume ratio of 1:4.

[0039] Example 3: Screening of Litsea cubeba essential oil preparations prepared from Litsea cubeba essential oil-ethanol mixtures with different ratios.

[0040] Prepare different concentrations of Litsea cubeba essential oil-ethanol mixtures according to Table 3. Take 1 mL of the above Litsea cubeba essential oil-ethanol mixture and slowly add it dropwise to 4 mL of chitosan quaternary ammonium salt solution with a mass-volume percentage concentration of 1%. Use a high-speed shear disperser at 10000 r / min for 3 min to achieve uniform particle dispersion.

[0041] Table 3. Litsea cubeba essential oil preparations with different formulations

[0042] (1) The growth rate method was used to determine the antibacterial activity of Litsea cubeba essential oil preparations prepared by different ratios of Litsea cubeba essential oil and ethanol mixture against Aspergillus flavus.

[0043] Dispense the prepared YPG medium into tissue culture flasks, adding 100 mL to each flask. After cooling to approximately 55 °C, add 1 mL of the *Litsea cubeba* essential oil preparations from formulations one through four to each flask, and immediately shake well. Pour the mixture evenly into 90 mm petri dishes. Similarly, add 1 mL of 1% chitosan quaternary ammonium salt solution and 1 mL of *Litsea cubeba* essential oil ethanol solution of different concentrations (the *Litsea cubeba* essential oil concentration is consistent with that in formulations one through four) to each 100 mL of YPG as controls. Use a sterile 5 mm punch to collect mycelial cakes from the cultured *Aspergillus flavus* agar plates. Use tweezers to invert the mycelial cakes onto the plates and incubate at 28 °C. Measure the colony diameter using the cross-sectional method at 48, 72, 96, 120, and 144 h. The antibacterial results are as follows: Figure 1 As shown in Table 4, the inhibition rate is calculated as follows: (Coronavirus diameter of control group - Coronavirus diameter of treatment group) / (Coronavirus diameter of control group - Coronavirus cake diameter) × 100%.

[0044] Table 4. Antibacterial rate of Litsea cubeba essential oil preparations prepared with different ratios of Litsea cubeba essential oil and ethanol mixtures against Aspergillus flavus.

[0045] pass Figure 1As shown in Table 4, the antibacterial effect of the Litsea cubeba essential oil preparation in Formula 4 was unexpectedly good, significantly better than the chitosan quaternary ammonium salt-Litsea cubeba essential oil preparations in the other three formulas, and also better than the effect of using chitosan quaternary ammonium salt and Litsea cubeba essential oil alone. Its antibacterial rate remained at 100% even after 144 hours. The different ratios of Litsea cubeba essential oil and chitosan quaternary ammonium salt in Formulas 1 to 4 resulted in different release amounts of Litsea cubeba essential oil, leading to varying antibacterial effects compared to using the same concentration of Litsea cubeba essential oil alone. However, Formula 4 achieved 100% antibacterial rate in both short and long term, indicating that the release effect of Litsea cubeba essential oil in Formula 4 was the best, and the antibacterial effect was not affected by the different release amounts at different times. Furthermore, the concentration of this formulation was close to the minimum inhibitory concentration (MIC) of Litsea cubeba essential oil, thus this formulation had an advantage over Litsea cubeba essential oil in inhibiting Aspergillus flavus.

[0046] (2) The dispersibility of the Litsea cubeba essential oil preparation prepared by Formula 4 was studied, and the particle size and potential of the Litsea cubeba essential oil preparation were tested.

[0047] like Figure 2 and 3 As shown, the Litsea cubeba essential oil preparation prepared by Formula 4 exhibits good homogeneity, with an average particle size of 955.5 nm. Figure 2 The zeta potential ranges from +4.16 to +37.4 mV. Figure 3 ).

[0048] In addition, when the Litsea cubeba essential oil preparation with a ratio of 4 was left at room temperature for 2 months, no precipitation, discoloration, or stratification was observed.

[0049] In summary, Formula 4 exhibits the best antibacterial effect and good dispersibility and stability, making it suitable for promotion as a product.

[0050] Example 4: Comparison of the antifungal effects of Litsea cubeba essential oil preparation and conventional antifungal agents against Aspergillus flavus.

[0051] Natamycin (MIC: 5 mg / kg), sorbic acid (MIC: 200 mg / kg), and benzoic acid (MIC: 1000 mg / kg), commonly available antifungal agents, were selected for comparison of their inhibitory activity against Aspergillus flavus. The concentrations used were the minimum inhibitory concentrations against Aspergillus flavus found in the literature.

[0052] The virulence equation was derived based on the correlation between the logarithm of concentration and the inhibition rate. According to the virulence equation, when y = 1, the concentration corresponding to x is the minimum inhibitory concentration (MIC); when y = 0.5, the concentration corresponding to x is the half-maximal effective concentration (EC5). 50As shown in Table 5, the minimum inhibitory concentration (MIC) of Litsea cubeba oil preparation against Aspergillus flavus was 74.74 μL / mL, and the half-maximal effective concentration (EC50) was 23.97 μL / mL. The concentration of essential oil in Formula 4 was 80 μL / mL, which is close to the MIC. Therefore, Litsea cubeba essential oil preparation of Formula 4 was selected for the comparative experiment on antibacterial effect.

[0053] Table 5. Analysis of the inhibitory activity of Litsea cubeba essential oil preparations against Aspergillus flavus.

[0054] Antibacterial effect such as Figure 4 As shown. Compared with natamycin, sorbic acid, and sodium benzoate, the Litsea cubeba essential oil preparation prepared by formula four showed significantly better antibacterial effect against Aspergillus flavus, with inhibition rates increased by 93.11%, 96.85%, and 89.13%, respectively.

[0055] Example 5: Inhibitory effect of Litsea cubeba essential oil preparation on peanut aflatoxin

[0056] Select 20 peanuts of the same size and add 1 ml of Aspergillus flavus spores (10 6 (CFU / mL) was mixed with 1 ml of Litsea cubeba essential oil preparation from Formula 4 and 1 ml of Litsea cubeba essential oil-ethanol mixture (Litsea cubeba essential oil concentration 80 μL / mL), respectively, and evenly applied to the peanut surface. The mixtures were then placed in 90 mm agar plates and incubated at 28°C. The level of Aspergillus contamination in peanuts was recorded every 24 hours, and the aflatoxin content in peanuts was measured after 72 hours. The peanuts treated with 1 ml of anhydrous ethanol served as the control group (CK). The Aspergillus contamination levels in peanuts after 24, 48, and 72 hours after treatment with Litsea cubeba essential oil preparation and Litsea cubeba essential oil are shown below. Figure 5 As shown in Table 6, the contamination rate (contamination rate = (number of peanuts contaminated with Aspergillus flavus / total number of peanuts) × 100%) and the aflatoxin content after 72 hours are shown in Table 7.

[0057] Table 6. Contamination rate of peanuts by Aspergillus flavus after treatment with chitosan quaternary ammonium salt-Litsea cubeba essential oil preparation and Litsea cubeba essential oil.

[0058] Table 7. Aflatoxin content after 72 hours

[0059] Combination Figure 5 As shown in Tables 6-7, the short-term and long-term antibacterial effects of the Litsea cubeba essential oil preparation of Formula 4 on Aspergillus flavus are significantly better than those of Litsea cubeba essential oil. After 72 hours, the aflatoxin content of peanuts treated with the Litsea cubeba essential oil preparation of Formula 4 was 2.51±0.54ug / kg, which was 98.78% and 95.68% lower than that of untreated peanuts and peanuts treated with Litsea cubeba essential oil, respectively.

[0060] In summary, the *Litsea cubeba* essential oil preparation prepared by Formula 4 of this invention exhibits good dispersibility and demonstrates significantly superior antibacterial effects against *Aspergillus flavus* compared to other chitosan quaternary ammonium salt-*Litsea cubeba* essential oil preparations. It is also superior to using *Litsea cubeba* essential oil or chitosan quaternary ammonium salt alone, and even more superior to commonly available antifungal agents such as natamycin, sorbic acid, and sodium benzoate. Furthermore, the *Litsea cubeba* essential oil preparation of this invention overcomes the volatility of *Litsea cubeba* essential oil, effectively prolonging its duration of action and further enhancing its long-term antibacterial effect against *Aspergillus flavus*. Its preparation method is simple, involves few components, is non-toxic and harmless, does not pollute the environment, and has low production costs. When used to prevent or inhibit the growth of *Aspergillus flavus*, it can reduce its damage to wooden household products.

Claims

1. A formulation of Litsea cubeba essential oil, characterized in that, The preparation of the Litsea cubeba essential oil is prepared by mixing 1% chitosan quaternary ammonium salt solution with Litsea cubeba essential oil ethanol mixture in a volume ratio of 4:1; the volume ratio of Litsea cubeba essential oil to anhydrous ethanol in the Litsea cubeba essential oil ethanol mixture is 2:3; the chitosan quaternary ammonium salt is hydroxypropyl trimethyl ammonium chloride chitosan.

2. The Litsea cubeba essential oil preparation according to claim 1, characterized in that, The Litsea cubeba essential oil ethanol mixture is slowly dropped into the chitosan quaternary ammonium salt solution and dispersed to obtain a dispersion liquid with uniformly dispersed microparticles, thereby obtaining the preparation of the Litsea cubeba essential oil.

3. The Litsea cubeba essential oil preparation according to claim 2, characterized in that, A high-speed shearing disperser is used for dispersion.

4. The Litsea cubeba essential oil preparation according to claim 3, characterized in that, The high-speed shearing disperser is used for dispersion at a speed of 10000r / min for 3min.

5. The Litsea cubeba essential oil preparation according to claim 1, characterized in that, The preparation method of the chitosan quaternary ammonium salt solution is as follows: 1g of chitosan quaternary ammonium salt powder is poured into 100mL of pure water, and ultrasonic is used at 70℃ to make it uniform.

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