Litsea cubeba essential oil preparation and application thereof

Abstract

The invention discloses a litsea cubeba essential oil preparation. The preparation is prepared by uniformly mixing a chitosan quaternary ammonium salt solution with the mass percent concentration of 1% and a litsea cubeba essential oil ethanol mixed solution according to the volume ratio of 1: 4, the volume ratio of the litsea cubeba essential oil to the absolute ethyl alcohol in the litsea cubeba essential oil and ethyl alcohol mixed solution is 2: 3. The prepared litsea cubeba essential oil preparation is good in dispersity and stability, the short-term and long-term bacteriostatic effects on aspergillus flavus both reach 100%, and the litsea cubeba essential oil preparation is obviously better than litsea cubeba essential oil or chitosan quaternary ammonium salt independently used, is also better than chitosan quaternary ammonium salt-litsea cubeba essential oil preparations of other formulas, is better than common mildew preventives in the market, and is suitable for industrial production. The preparation method is simple, non-toxic and harmless, does not pollute the environment, is low in production cost, is used for preventing or inhibiting the growth of the aspergillus flavus, and can reduce the damage of the aspergillus flavus to grains, nuts or wooden living goods and the like.

Description

Technical Field

[0001] The present application relates to the technical field of plant product preservation, and in particular to a litsea cubeba essential oil preparation and application thereof. Background Art

[0002] Litsea cubeba is a perennial shrub or small tree of the genus Litsea Lam in the family Lauraceae. It 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, and the essential oil content of the peel is 3% to 5%. Litsea cubeba essential oil is an active secondary metabolite of Litsea cubeba. Litsea cubeba essential oil produced from fresh Litsea cubeba fruit is a unique natural fragrance product in my country and is a food additive allowed for use in the national standard.

[0003] A large number of studies have shown that Litsea cubeba essential oil has a broad-spectrum antibacterial property and is an indispensable natural antibacterial material. It is known as the natural nemesis of Aspergillus flavus and has great application potential and promotion value. However, Litsea cubeba oil has the characteristic of being volatile. At present, there are methods for stabilizing and protecting essential oils by encapsulating them in shells or forming films, such as microencapsulation technology, gelation, etc., but their processes are complicated and costly. Quaternary ammonium chitosan (QAC), as a typical derivative of chitosan, has biodegradability, biocompatibility and good film-forming properties. The introduction of quaternary ammonium salt groups can weaken the hydrogen bonds between chitosan molecular chains, improve the water solubility and antibacterial activity of chitosan, and can effectively lock the essential oil components and release them slowly. Based on this, it is necessary to provide a Litsea cubeba essential oil product with a sustained-release effect, good antibacterial effect and high stability. Summary of the invention

[0004] The invention provides a litsea cubeba essential oil preparation. The litsea cubeba essential oil preparation is prepared by uniformly mixing a chitosan quaternary ammonium salt solution with a mass percentage concentration of 1% and a litsea cubeba essential oil ethanol mixed liquid in a volume ratio of 1:4; the volume ratio of the litsea cubeba essential oil to anhydrous ethanol in the litsea cubeba essential oil ethanol mixed liquid is 2:3.

[0005] 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 particles are evenly dispersed, thereby obtaining the litsea cubeba essential oil preparation.

[0006] The dispersion was performed using a high-speed shearing disperser, specifically, the dispersion was performed for 3 minutes at a speed of 10000 r / min.

[0007] 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 ultrasonicated at 70°C to make it uniform.

[0008] The present invention also provides an application of the aforementioned Litsea cubeba essential oil preparation in preventing or inhibiting the growth of Aspergillus flavus.

[0009] Specifically, the application includes inhibiting the growth of Aspergillus flavus on grains, nuts or wooden daily necessities.

[0010] More specifically, the aforementioned Litsea cubeba essential oil preparation is covered on the surface of the cereals, nuts or wooden daily necessities to achieve the purpose of inhibiting the growth of Aspergillus flavus on the cereals, nuts or wooden daily necessities.

[0011] The beneficial effects of the present invention include:

[0012] (1) The Litsea cubeba essential oil preparation prepared by the present invention has good dispersibility and stability, an average particle size of 955.5 nm, a Zeta potential range of +4.16 to +37.4 mv, and no precipitation, discoloration, or stratification occurs after being placed at room temperature for 2 months.

[0013] (2) The litsea cubeba essential oil preparation of the present invention overcomes the characteristic of litsea cubeba essential oil being volatile, effectively prolongs the duration of action of litsea cubeba, and further improves the long-term antibacterial effect on Aspergillus flavus. Both the short-term and long-term antibacterial effects reach 100%, which is significantly better than using litsea cubeba essential oil or chitosan quaternary ammonium salt alone, and is also better than chitosan quaternary ammonium salt-litsea cubeba essential oil preparations of other formulations.

[0014] (3) The antibacterial effect of the litsea cubeba oil preparation prepared by the present invention on Aspergillus flavus is significantly better than that of the common antifungal agents on the market, such as natamycin, sorbic acid, and sodium benzoate. Compared with natamycin, sorbic acid, and sodium benzoate, the inhibition rates are increased by 93.11%, 96.85%, and 89.13%, respectively.

[0015] (4) The essential oil preparation of Litsea cubeba of the present invention can effectively and long-term reduce the content of aflatoxin in plant products such as peanuts that are easily contaminated by Aspergillus flavus. The aflatoxin content in peanuts after treatment for 72 hours was reduced by 98.78% to 2.51 μg / kg, which is far lower than the national food safety standard of 20 μg / kg (GB2761-2017).

[0016] (5) The preparation method of the Litsea cubeba essential oil preparation of the present invention is simple, has few components, is non-toxic and harmless, does not pollute the environment, and has low production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the inhibitory activity of the Litsea cubeba essential oil preparations prepared from the Litsea cubeba essential oil ethanol mixtures with different ratios in Example 3 against Aspergillus flavus;

[0018] Figure 2 The particle size distribution diagram of the Litsea cubeba essential oil preparation prepared by formula 4 in Example 3;

[0019] Figure 3 The Zeta potential diagram of the Litsea cubeba essential oil preparation prepared by Formula 4 in Example 3;

[0020] Figure 4 This is a comparison of the inhibitory activity of the Fructus Litseae Litseae essential oil preparation and natamycin, sorbic acid and sodium benzoate on Aspergillus flavus in Example 4;

[0021] Figure 5 This is a comparison chart of the contamination of peanuts with Aspergillus flavus 24, 48, and 72 hours after treatment with the Litsea cubeba essential oil preparation and Litsea cubeba essential oil in Example 5. DETAILED DESCRIPTION

[0022] The present invention is further illustrated and described below in conjunction with embodiments, but the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the present invention and embodiments, all other inventions and embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0025] In the process of dropping the ethanol mixture of Litsea cubeba essential oil into the chitosan quaternary ammonium salt solution, since the chitosan quaternary ammonium salt solution accounts for a large proportion, a preliminary test to explore the concentration of chitosan quaternary ammonium salt was first carried out in this application to obtain a preliminary concentration of chitosan quaternary ammonium salt. During the dropping process, with the addition of the ethanol mixture of Litsea cubeba essential oil, the dispersibility of the preparation will change. Too much ethanol mixture of Litsea cubeba essential oil will cause the dispersibility to deteriorate. Therefore, this application subsequently further optimizes the ratio of chitosan quaternary ammonium salt to ethanol mixture of Litsea cubeba essential oil. Finally, considering the best antibacterial effect in application, further research is conducted to determine the optimal concentration of Litsea cubeba essential oil.

[0026] Example 1, determination of chitosan quaternary ammonium salt concentration

[0027] (1) Preparation of chitosan quaternary ammonium salt solution

[0028] Weigh 0.5g, 1g, 1.5g, and 2g of chitosan quaternary ammonium salt (hydroxypropyltrimethylammonium chloride chitosan is used in this embodiment) powder and pour them into 100mL of pure water respectively, and ultrasonicate them at 70°C to make them uniform to obtain chitosan quaternary ammonium salt solutions with mass volume percentage concentrations of 0.5%, 1%, 1.5%, and 2%, respectively.

[0029] (2) Preparation of Litsea cubeba essential oil preparation

[0030] Take 1 mL of ethanol mixture (500 μL of ethanol + 500 μL of anhydrous ethanol) of Litsea cubeba essential oil (extracted from fresh Litsea cubeba fruit by steam distillation), slowly drip it into the above 9 mL of chitosan quaternary ammonium salt solution of different concentrations, and use a high-speed shear disperser at 10000 r / min for 3 min to achieve uniform dispersion of the particles.

[0031] The dispersibility of the Litsea cubeba essential oil preparation prepared in step (2) was studied, and the particle size and potential of the Litsea cubeba essential oil preparation prepared with chitosan quaternary ammonium salt solutions of different concentrations were tested. The results are shown in Table 1.

[0032] Table 1 Particle size and potential results of Litsea cubeba essential oil preparations prepared with chitosan quaternary ammonium salt solutions at different concentrations

[0033]

[0034]

[0035] As can be seen from Table 1, the particle size of the Litsea cubeba essential oil preparation containing 1% chitosan quaternary ammonium salt solution is smaller and its dispersibility is better, so the chitosan quaternary ammonium salt with a concentration of 1% is finally selected.

[0036] Example 2: Study on the ratio of ethanol mixture of Litsea cubeba essential oil and 1% chitosan quaternary ammonium salt

[0037] In order to further optimize the ratio of chitosan quaternary ammonium salt to Litsea cubeba essential oil, the following experiment was set up:

[0038] Take 1mL of ethanol mixture of Litsea cubeba essential oil (500μL of Litsea cubeba essential oil + 500μL of anhydrous ethanol), slowly drip it into 1mL, 2mL, 3mL, 4mL, 5mL of chitosan quaternary ammonium salt solution with a mass volume percentage concentration of 1%, and use a high-speed shear disperser at 10000r / min for 3min to achieve the purpose of uniform dispersion of particles. The volume ratios of ethanol mixture of Litsea cubeba essential oil and chitosan quaternary ammonium salt solution are 1:1, 1:2, 1:3, 1:4, 1:5 respectively.

[0039] The dispersibility of the prepared Litsea cubeba essential oil preparation was studied, and the particle size and potential of the Litsea cubeba essential oil preparation prepared by mixing Litsea cubeba essential oil ethanol with 1% chitosan quaternary ammonium salt in different volume ratios were tested. The results are shown in Table 2.

[0040] Table 2 Particle size and potential results of Litsea cubeba essential oil preparations prepared by mixing different ratios of Litsea cubeba essential oil ethanol solution with 1% chitosan quaternary ammonium salt

[0041]

[0042] As can be seen from Table 2, when the volume ratio of the ethanol mixture of Litsea cubeba essential oil and the chitosan quaternary ammonium salt with a mass volume percentage concentration of 1% is 1:4, the particle size of the preparation is smaller and its dispersibility is better. Therefore, the ethanol mixture of Litsea cubeba essential oil and the chitosan quaternary ammonium salt with a mass volume percentage concentration of 1% are finally selected to be mixed in a volume ratio of 1:4 to prepare the Litsea cubeba essential oil preparation.

[0043] Example 3: Screening of Litsea cubeba essential oil preparations prepared from different ratios of Litsea cubeba essential oil ethanol mixtures

[0044] Different concentrations of ethanol mixtures of Litsea cubeba essential oil were prepared according to Table 3. 1 mL of the above ethanol mixture of Litsea cubeba essential oil was slowly dripped into 4 mL of chitosan quaternary ammonium salt solution with a mass volume percentage concentration of 1%, and a high-speed shear disperser was used at 10000 r / min for 3 minutes to achieve uniform dispersion of the particles.

[0045] Table 3 Litsea cubeba essential oil preparations with different formulations

[0046]

[0047] (1) The antibacterial activity of Litsea cubeba essential oil preparations prepared from ethanol mixtures of Litsea cubeba essential oils in different proportions against Aspergillus flavus was determined by the growth rate method.

[0048] Dispense the prepared YPG culture medium into tissue culture bottles, pour 100 mL into each bottle, and when it cools to about 55°C, add 1 mL of the elm lily essential oil preparation prepared in Formula 1 to Formula 4, and shake well immediately. After mixing, pour evenly into 90 mm culture dishes. Similarly, add 1 ml of 1% chitosan quaternary ammonium salt solution and 1 ml of elm lily essential oil ethanol solution of different concentrations (the concentration of elm lily essential oil is the same as that in Formula 1 to Formula 4) to every 100 ml of YPG as a control. Use a sterilized 5 mm puncher to punch out a bacterial cake on the cultured Aspergillus flavus plate, use tweezers to stick the bacterial cake upside down on the above plate, culture at 28°C, and measure the colony diameter by the cross method at 48, 72, 96, 120, and 144 hours. The antibacterial results are as follows. Figure 1 As shown in Table 4, the inhibition rate = (the diameter of the colony in the control group - the diameter of the colony in the treatment group) / (the diameter of the colony in the control group - the diameter of the bacterial cake) × 100%.

[0049] Table 4 Inhibition rate of Litsea cubeba essential oil preparations against Aspergillus flavus prepared from different ratios of Litsea cubeba essential oil ethanol mixtures

[0050]

[0051]

[0052] pass Figure 1As can be seen from Table 4, the antibacterial effect of the Litsea cubeba essential oil preparation of Formula 4 is unexpectedly good, significantly better than the chitosan quaternary ammonium salt-Litsea cubeba essential oil preparations of the other three groups of formulas, and also better than the effect of using chitosan quaternary ammonium salt and Litsea cubeba essential oil alone, and its antibacterial rate can still reach 100% until 144h. The ratios of Litsea cubeba essential oil and chitosan quaternary ammonium salt in Formulas 1 to 4 are different, which will lead to different release amounts of Litsea cubeba essential oil, so compared with using Litsea cubeba essential oil of the same concentration alone, there will be different differences in the antibacterial effect. However, the short-term and long-term antibacterial rates of Formula 4 both reach 100%, indicating that the release effect of Litsea cubeba essential oil in Formula 4 is the best, and the antibacterial effect is not affected by the different release amounts in different periods, and the concentration of this formula preparation is close to the minimum inhibitory concentration of Litsea cubeba essential oil, so this formula has an advantage in inhibiting Aspergillus flavus compared to Litsea cubeba essential oil.

[0053] (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.

[0054] like Figure 2 and 3 As shown, the Litsea cubeba essential oil preparation prepared by formula 4 has good uniformity and an average particle size of 955.5nm ( Figure 2 ), zeta potential range is +4.16~+37.4mv( Figure 3 ).

[0055] In addition, the Litsea cubeba essential oil preparation with ratio 4 was placed at room temperature for 2 months, and no precipitation, discoloration or stratification occurred.

[0056] In summary, Formula 4 has the best antibacterial effect and good dispersibility and stability, and is suitable for product promotion.

[0057] Example 4: Comparison of the antibacterial effect of Litsea cubeba essential oil preparation and conventional mildew inhibitors on Aspergillus flavus

[0058] The common fungicides on the market, natamycin (MIC: 5 mg / kg), sorbic acid (MIC: 200 mg / kg), and benzoic acid (MIC: 1000 mg / kg), were selected for comparison of their inhibitory activity against Aspergillus flavus. The concentration used was the minimum inhibitory concentration for Aspergillus flavus in the literature.

[0059] The toxicity equation was obtained based on the correlation between the logarithm of concentration and the inhibition rate. According to the toxicity equation, when the y value is 1, the concentration corresponding to x is the minimum inhibitory concentration (MIC); when the y value is 0.5, the concentration corresponding to x is the half-maximal effect concentration (EC 50). As shown in Table 5, the minimum inhibitory concentration (MIC) of the Litsea cubeba oil preparation against Aspergillus flavus was 74.74 μL / mL, and the half-maximal effect concentration (EC50) was 23.97 μL / mL. The essential oil concentration of formula 4 was 80 μL / mL, which was close to the MIC, so the Litsea cubeba essential oil preparation of formula 4 was selected for the antibacterial effect comparison experiment.

[0060] Table 5 Analysis of the inhibitory activity of Aspergillus flavus from Litsea cubeba essential oil preparation

[0061]

[0062]

[0063] Antibacterial effect Figure 4 Compared with natamycin, sorbic acid and sodium benzoate, the Litsea cubeba essential oil preparation prepared by formula 4 has a significantly better antibacterial effect on Aspergillus flavus, with the inhibition rates increased by 93.11%, 96.85% and 89.13% respectively.

[0064] Example 5: Inhibitory effect of Litsea cubeba essential oil preparation on aflatoxin in peanuts

[0065] Select 20 peanuts of the same size and add 1 ml of Aspergillus flavus spores (10 6 cfu / mL) were mixed evenly with 1ml of the Litsea cubeba essential oil preparation of formula 4 and 1ml of Litsea cubeba essential oil ethanol mixture (the concentration of Litsea cubeba essential oil was 80μL / mL), evenly spread on the surface of peanuts, placed in a 90mm plate, and cultured at 28℃. The aflatoxin contamination of peanuts by Aspergillus flavus was counted every 24h, and the aflatoxin content of peanuts was measured after 72h. The peanuts treated with 1ml of anhydrous ethanol were used as the control group (CK). The contamination of peanuts with aflatoxin by Aspergillus flavus after 24, 48, and 72h after being treated with Litsea cubeba essential oil preparation and Litsea cubeba essential oil is shown in the figure. Figure 5 The contamination rate (contamination rate = (number of peanuts contaminated by Aspergillus flavus / total number of peanuts) × 100%) is shown in Table 6, and the aflatoxin content after 72 hours is shown in Table 7.

[0066] Table 6 Contamination rate of peanuts with Aspergillus flavus after treatment with chitosan quaternary ammonium salt-Litsea cubeba essential oil preparation and Litsea cubeba essential oil

[0067]

[0068] Table 7 Aflatoxin content after 72 hours

[0069]

[0070] Combination Figure 5As can be seen from Tables 6-7, the short-term and long-term antibacterial effects of the lilacinus scoparia essential oil preparation of Formula 4 on Aspergillus flavus are significantly better than those of the lilacinus scoparia essential oil. After 72 hours, the aflatoxin content of peanuts treated with the lilacinus scoparia essential oil preparation of Formula 4 was 2.51±0.54ug / kg, which was 98.78% and 95.68% lower than those of the untreated and lilacinus scoparia essential oil-treated peanuts, respectively, and far lower than the national food safety standard of 20μg / kg (GB2761-2017).

[0071] From the above, it can be seen that the Litsea cubeba essential oil preparation prepared by the fourth formula of the present invention has good dispersibility, and its antibacterial effect on Aspergillus flavus is significantly better than the chitosan quaternary ammonium salt-Litsea cubeba essential oil preparation of other formulas, and is also better than using Litsea cubeba essential oil or chitosan quaternary ammonium salt alone, and is better than common mildew inhibitors natamycin, sorbic acid, and sodium benzoate on the market. And the Litsea cubeba essential oil preparation of the present invention overcomes the volatile characteristics of Litsea cubeba essential oil, effectively prolongs the effect time of Litsea cubeba, and further improves the long-term antibacterial effect on Aspergillus flavus. Its preparation method is simple, the components are few and nontoxic, it will not pollute the environment, and the production cost is low. It is used to prevent or inhibit the growth of Aspergillus flavus, and its damage to cereals, nuts or wooden daily necessities can be reduced.

Claims

1. A Litsea cubeba essential oil preparation, characterized in that The litsea cubeba essential oil preparation is prepared by mixing a chitosan quaternary ammonium salt solution with a mass percentage concentration of 1% and a litsea cubeba essential oil ethanol mixture in a volume ratio of 1:4; the volume ratio of the litsea cubeba essential oil to anhydrous ethanol in the litsea cubeba essential oil ethanol mixture is 2:

3.

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

3. The Litsea cubeba essential oil preparation according to claim 2, characterized in that Use a high-speed shear disperser for dispersion.

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

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

6. The Litsea cubeba essential oil preparation according to claim 1, characterized in that The chitosan quaternary ammonium salt is hydroxypropyltrimethylammonium chloride chitosan.

7. Use of the Litsea cubeba essential oil preparation according to any one of claims 1 to 6 in preventing or inhibiting the growth of Aspergillus flavus.

8. The use according to claim 7, characterized in that: The application includes inhibiting the growth of Aspergillus flavus on grains, nuts or wooden daily necessities.

9. The use according to claim 8, characterized in that: The Litsea cubeba essential oil preparation described in any one of claims 1 to 6 is covered on the surface of the cereals, nuts or wooden daily necessities to achieve the purpose of inhibiting the growth of Aspergillus flavus on the cereals, nuts or wooden daily necessities.

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