Preparation method of nanometer emulsion of oregano essential oil and application of nanometer emulsion of oregano essential oil in fresh-keeping of fragrant pears

Abstract

The application discloses a preparation method of a nanometer oil emulsion of oregano essential oil and application of the nanometer oil emulsion in fresh-keeping of fragrant pears and belongs to the technical field of fresh-keeping of agricultural products. The preparation method comprises the following steps: 1) a certain mass of deacetylated chitosan is weighed, and a certain volume of deionized water and glacial acetic acid are added to perform an acetylation reaction of the chitosan; 2) a magnetic stirrer is used to heat and stir until the solution is clear, and a certain volume of a mixed solution of oregano essential oil and Tween 80 is added; 3) the solution is homogenized by ultrasonic waves to obtain the nanometer oil emulsion of the oregano essential oil; and 4) the fragrant pears after harvesting are immersed in the nanometer oil emulsion of the oregano essential oil, so that the surface of the fragrant pears is uniformly coated with a layer of fresh-keeping emulsion, the fragrant pears are dried at room temperature until the emulsion on the surface of the fragrant pears is dried, and the fragrant pears after coating are stored at room temperature. The nanometer oil emulsion of the oregano essential oil prepared by the preparation method solves the problem that the oregano essential oil as an oily substance is not easy to be dissolved in water, the oregano essential oil can be wrapped in water to form a nanometer oil emulsion, the nanometer oil emulsion can be applied to fruits such as fragrant pears in a way of immersion or smearing, the oregano essential oil can play a role in killing microorganisms and adjusting physiological metabolism of the fragrant pears, and the oregano essential oil is favorable for maintaining the storage quality of the fragrant pears.

Description

Technical Field

[0001] This invention belongs to the field of agricultural product preservation technology, specifically relating to a preparation process based on oregano essential oil nanoemulsion and its application in the storage and preservation of fragrant pears, particularly targeting Penicillium expansum (…). Penicillium expansum This method helps control postharvest diseases and regulate fruit quality by inhibiting pathogen infection and regulating fruit physiological metabolism through multiple barrier effects, thereby improving the storage quality of fragrant pears. Background Technology

[0002] Fragrant pears are rich in nutrients, but they are susceptible to Penicillium expansion mold during post-harvest storage and transportation. Penicillium expansum Pollution leads to decay and spoilage. While traditional chemical fungicides (such as pyrimethanil) can inhibit decay-causing fungi like Penicillium expansum, long-term use can easily result in pesticide residues and induce microbial resistance, threatening environmental safety and human health. Therefore, developing alternative technologies for chemical fungicides is of great significance for sustainable environmental development and protecting human health.

[0003] Plant essential oils are a class of natural products extracted from plants with broad-spectrum antibacterial activity. Due to their green and safe characteristics, they are widely used in preservation research. However, plant essential oils have limitations such as high volatility, poor water solubility, and the potential for damage to fruits at high concentrations, which restricts their application in preservation. Our previous research showed that oregano essential oil has a highly effective antibacterial effect against Penicillium expansum, demonstrating significant application potential in agricultural product preservation. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing oregano essential oil nanoemulsion and its application in the preservation of fragrant pears. This method solves the problems of oregano essential oil's poor solubility and high volatility in aqueous phase, thus improving its film-forming and sustained-release properties. The oregano essential oil nanoemulsion prepared by this invention can significantly inhibit the rotting of fragrant pears caused by Penicillium expansum, reduce the water loss rate of fragrant pears during storage, improve the antioxidant capacity of fragrant pears, and enhance the quality of fragrant pears during storage.

[0005] To achieve the objective of this invention, in a first aspect, this invention provides a method for preparing oregano essential oil nanoemulsion, characterized by comprising the following steps: Step 1: Weigh a certain mass of deacetylated chitosan, add a certain volume of deionized water and glacial acetic acid to carry out the chitosan acetylation reaction; Step 2: Heat and stir the solution with a magnetic stirrer until it becomes clear, then add a certain volume of oregano oil-Tween 80 mixed solution; Step 3: Homogenize the solution using ultrasound to obtain oregano essential oil nanoemulsion.

[0006] The magnetic stirrer has a stirring time of 30 minutes, a rotation speed of 1200 rpm, and a heating temperature of 60℃.

[0007] The ultrasonic treatment consisted of intermittent ultrasonic treatment at 400 W power for 40 min, with 3 s of ultrasonic treatment followed by a 4 s pause.

[0008] The oregano essential oil nanoemulsion is composed of an aqueous phase and an organic phase containing oregano essential oil. The aqueous phase is deionized water, and the organic phase consists of oregano essential oil, Tween 80, chitosan, and acetic acid. The oregano essential oil mass ratio is 0.04%, the final concentration of Tween 80 is 0.1%, the chitosan content is 0.1%, and the acetic acid content is 1%.

[0009] An oregano essential oil nanoemulsion and its application in the preservation of fragrant pears.

[0010] The oregano essential oil nanoemulsion is composed of an aqueous phase and an organic phase containing oregano essential oil. The aqueous phase is deionized water, and the organic phase consists of oregano essential oil, Tween 80, chitosan, and acetic acid. The oregano essential oil mass ratio is 0.04%, the final concentration of Tween 80 is 0.1%, the chitosan content is 0.1%, and the acetic acid content is 1%.

[0011] The preservation steps are as follows: After harvesting, the fragrant pears are immersed in oregano essential oil nano-emulsion to coat their surface evenly with a layer of preservative emulsion. They are then air-dried at room temperature for 10 minutes until the emulsion on the surface of the fragrant pears is dry. The coated fragrant pears are then stored at room temperature.

[0012] The pear was immersed in the oregano essential oil nanoemulsion for 1 minute.

[0013] Compared with the prior art, the present invention has at least the following technical effects or beneficial effects. The oregano essential oil nanoemulsion prepared by the preparation method of the present invention solves the problem that oregano essential oil, as an oily substance, is not easily soluble in the aqueous phase. After the oregano essential oil is encapsulated in the aqueous phase to form a nanoemulsion, it can be applied to fruits such as pears by immersion or application. The oregano essential oil can kill microorganisms and regulate the physiological metabolism of pears, which is beneficial to maintaining the storage quality of pears. Attached Figure Description

[0014] Figure 1 Graph showing the inhibitory effect of oregano essential oil on the germination of Penicillium spores. Figure 2 : Effect of oregano essential oil nanoemulsion coating on the inhibition of lesion development in pear under simulated inoculation conditions (Figure) Figure 3 Image showing the effect of oregano essential oil nano-emulsion coating on improving the firmness of pears. Figure 4 Graph showing the effect of oregano essential oil nanoemulsion coating on reducing water loss rate (A) and inhibiting respiration rate (B) in pears. Figure 5Effects of oregano essential oil nanoemulsion coating on soluble sugars (A) and titratable acid (B) of pear Figure 6 : Effect of oregano essential oil nanoemulsion coating on enhancing the antioxidant enzyme system of pear Detailed Implementation

[0015] To make the technical solution of the present invention clearer, the following description is provided in conjunction with the appendix. Figure 1-6 The present invention will be described in detail below. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the embodiments are conventional technical means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0016] Example 1: A method for preparing an oregano essential oil nanoemulsion The oregano oil nanoemulsion is composed of an aqueous phase and an organic phase containing oregano oil. The aqueous phase is deionized water, and the organic phase consists of oregano oil, Tween 80, chitosan, and acetic acid. The mass percentages of oregano oil are 0.04%, Tween 80 is 0.1%, chitosan is 1%, and acetic acid is 1%. The preparation steps are as follows: Step 1: Weigh a certain mass of chitosan and dissolve it in deionized water. Stir until the chitosan is completely dissolved, then add a certain volume of glacial acetic acid. Stir well and then add a certain volume of oregano essential oil-Tween 80 solution. Step 2: Stir the above mixture magnetically for 30 minutes at a speed of 800 rpm; Step 3: Homogenize the solution using ultrasound. The ultrasound treatment is performed intermittently at 400W power for 20 minutes, with a 5-second ultrasound cycle followed by a 3-second pause, to obtain the oregano essential oil nanoemulsion.

[0017] The oregano essential oil nanoemulsion had a particle size of (332.9 ± 7.5) nm and a polydispersity index (PDI) of 0.209 ± 0.003, indicating that the prepared oregano essential oil nanoemulsion had a relatively uniform particle size.

[0018] The oregano essential oil nanoemulsion prepared using the above method and its application in the preservation of fragrant pears are as follows: Step 1: After harvesting, soak the fragrant pears in oregano essential oil nano-emulsion to coat their surface evenly with a layer of preservative emulsion. Let them air dry at room temperature for 10 minutes until the emulsion on the surface of the fragrant pears is dry. Store the coated fragrant pears at room temperature.

[0019] The pear was immersed in the oregano essential oil nanoemulsion for 1 minute.

[0020] Example 2: Minimum inhibitory concentration and minimum bactericidal concentration of oregano essential oil against Penicillium expansum. In this embodiment, the concentrations of oregano essential oil were 0%, 0.01%, 0.02%, 0.04%, and 0.1%. The culture medium containing the above essential oil was prepared as a solid culture medium. 100 μL of Penicillium spore suspension (7.5 × 10⁻⁶) was taken. 3 Oregano essential oil (CFU / mL) was evenly spread on the surface of the culture medium, with the medium without added essential oil serving as a blank control. The plates were incubated at 28 ℃ for 48 h, and the number of colonies was recorded. The lowest essential oil concentration at which no colonies grew on the plate after 48 h was defined as the minimum inhibitory concentration (MIC) of oregano essential oil against *Penicillium expansum*. The plates without colonies were incubated for another 48 h, and the lowest essential oil concentration at which no colonies grew after 96 h was defined as the minimum fungicidal concentration (MFC) of oregano essential oil against *Penicillium expansum*.

[0021] Tests showed that the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MCC) of oregano essential oil against Penicillium expansum were 0.02% and 0.04%, respectively, indicating that oregano essential oil exhibited a good inhibitory effect on Penicillium expansum.

[0022] Example 3: Inhibitory effect of oregano essential oil on the germination of Penicillium spores In this embodiment, the concentrations of oregano essential oil are 0, 0.01%, 0.02%, and 0.04%. 450 μL of spore suspension (1×10⁻⁶) is added to liquid PDB culture medium containing the above concentrations of oregano essential oil. 7 The culture medium was prepared at 28 ℃ and 220 r / min using a medium without added essential oil as a control group. At regular intervals, 100 μL of the culture medium was aspirated and centrifuged at 13000 r / min for 5 min to obtain the precipitated spores. After staining with lactic acid phenol blue, the spores were observed under a microscope to assess germination.

[0023] Figure 1 The image shows the inhibitory effect of oregano essential oil on the germination of Penicillium spores. Figure 1 It was found that oregano essential oil had a significant inhibitory effect on the germination of Penicillium spores, and the inhibitory effect increased with the increase of oregano essential oil concentration. At 12 h, the spore germination rate of the control group was 34.18%, while the spores in the essential oil treatment group did not germinate at all; at 48 h, the spore germination rate of the control group was 100%, while the spore germination rate of the 0.01% oregano essential oil treatment group was only 3.33%, and the spores in the 0.02% and 0.04% oregano essential oil treatment groups did not germinate at all.

[0024] Example 4: Inhibitory effect of oregano essential oil nanoemulsion on the development of lesions in pears under simulated pollution conditions The oregano essential oil nanoemulsion described in this embodiment is the oregano essential oil nanoemulsion prepared in Example 1. After harvesting, the fragrant pears were soaked in 0.01% sodium hypochlorite for 10 min, rinsed thoroughly with sterile water, and air-dried. Sterile inoculation needles were used to drill holes (2 mm × 2 mm) at the equator on the surface of the fragrant pears to simulate surface damage. 5 μL of Penicillium spore suspension (1 × 10⁻⁶) was inoculated at the damaged sites. 7 (CFU / mL) The above-mentioned fragrant pears were immersed in oregano essential oil nanoemulsion and left to stand for 1 minute to allow the surface to be evenly coated with a layer of preservative emulsion. They were then air-dried at room temperature for 10 minutes until the emulsion on the surface of the fragrant pears was dry. The disease status of the fragrant pears was recorded at a fixed time every day, and the diameter of the lesions was measured and the area of ​​the lesions was calculated. The control group and the essential oil treatment group each contained 5 fragrant pears. The control group was immersed in water after inoculation.

[0025] Figure 2 This is a diagram illustrating the inhibitory effect of oregano essential oil nanoemulsion on the development of pear lesions under simulated pollution conditions, as described in this embodiment of the invention. Figure 2 It can be seen that the use of oregano essential oil nanoemulsion has a significant inhibitory effect on the development of lesions in fragrant pears caused by Penicillium expansion contamination. After 7 days of storage, the lesion area of ​​fragrant pears in the essential oil treatment group was reduced by 64% compared with that in the control group.

[0026] Example 5: Effect of Oregano Essential Oil Nanoemulsion on Improving the Firmness of Pears The oregano essential oil nanoemulsion mentioned in this embodiment is the oregano essential oil nanoemulsion prepared in Example 1. The fragrant pear is either an untreated fragrant pear or a fragrant pear obtained by soaking in the oregano essential oil nanoemulsion according to Example 1.

[0027] The firmness of the fragrant pear was measured using a texture analyzer. Three points were selected on the pear surface at the equator. The pressure gauge probe was placed vertically at the equatorial surface of the fruit and pierced the flesh until the probe penetrated the pulp. The maximum force during the piercing process was recorded, and the unit was Newtons (N). The probe diameter was 0.5 cm, the force sensor range was 250 N, the trigger force was 0.05 N, the speed was 60 mm / min, and the penetration distance was 8 mm.

[0028] Figure 3 The figure shows the effect of oregano essential oil nano-emulsion coating on improving the hardness of pears in an embodiment of the present invention. As can be seen from the figure, compared with the control group, the use of oregano essential oil nano-emulsion is beneficial to maintaining the hardness of pears during storage and improving the storage quality of pears.

[0029] Example 6: Effect of Oregano Essential Oil Nanoemulsion on Reducing Water Loss and Inhibiting Respiration Rate in Pears The oregano essential oil nanoemulsion mentioned in this embodiment is the oregano essential oil nanoemulsion prepared in Example 1. The fragrant pear is either an untreated fragrant pear or a fragrant pear obtained by soaking in the oregano essential oil nanoemulsion according to Example 1.

[0030] The control group consisted of untreated pears, while the essential oil treatment group consisted of pears soaked in oregano essential oil nanoemulsion as described in Example 1. Each group contained 7 pears. Respiration rate and water loss were measured every 3 days for a period of 15 days. For respiration rate measurement, the pears in both groups were weighed and placed in a fumigation tank. Carbon dioxide concentration was detected using gas chromatography at an injection port temperature of 120°C, a Propark Q column (80-100 mesh) at 70°C, a TCD detector temperature of 200°C, H2 as the carrier gas, and a column flow rate of 25 mL / min. Respiration rate was measured in mLCO2 / (kg·h). Weight loss was determined by weighing and reducing the weight of the pears every three days for a 15-day storage period. The average value of the results was taken.

[0031] Figure 4 This is a graph showing the effect of oregano essential oil nanoemulsion coating on reducing the water loss rate and inhibiting the respiration rate of pears in an embodiment of the present invention. As can be seen from the graph, compared with the control group, the use of oregano essential oil nanoemulsion can inhibit the water loss of pears during storage and reduce the respiration of pears, which is beneficial to maintaining the moisture content of pears and reducing the respiration consumption of organic matter.

[0032] Example 7: Effect of Oregano Essential Oil Nanoemulsion on Soluble Sugar and Titratable Acid Content in Pear The oregano essential oil nanoemulsion mentioned in this embodiment is the oregano essential oil nanoemulsion prepared in Example 1. The fragrant pear is either an untreated fragrant pear or a fragrant pear obtained by soaking in the oregano essential oil nanoemulsion according to Example 1.

[0033] The control group consisted of untreated pears, while the essential oil treatment group consisted of pears soaked in oregano essential oil nanoemulsion as described in Example 1. Each group contained 24 pears, and measurements were taken every 3 days for a total of 15 days. Titratable acid and soluble sugar contents were determined using an acidity meter and a saccharimeter, respectively. Specifically, the pears were peeled, chopped, and juiced using a juicer. The juice was then centrifuged to obtain clear juice. The soluble reducing sugar and titratable acid contents in the clear juice were measured using a handheld saccharimeter and an acidity meter, respectively. The measurements were repeated three times, and the average value was taken. Both soluble sugar and titratable acid contents are expressed as a percentage (%).

[0034] Figure 5 The figure shows the effect of oregano essential oil nanoemulsion on the soluble sugar and titratable acid content of pears in this embodiment of the invention. As can be seen from the figure, compared with the control group, the use of oregano essential oil nanoemulsion can delay the decrease in the soluble sugar content of pears during storage and reduce the titratable acid content of pears, which is beneficial to maintaining the flavor quality of pears.

[0035] Example 8: Effect of Oregano Essential Oil Nanoemulsion on Enhancing the Antioxidant Enzyme System of Pear The oregano essential oil nanoemulsion mentioned in this embodiment is the oregano essential oil nanoemulsion prepared in Example 1. The fragrant pear is either an untreated fragrant pear or a fragrant pear obtained by soaking in the oregano essential oil nanoemulsion according to Example 1.

[0036] The control group consisted of untreated fragrant pears, while the essential oil treatment group consisted of fragrant pears soaked in oregano essential oil nanoemulsion as described in Example 1. Each group contained 24 fragrant pears, and measurements were taken every 3 days for a period of 15 days. The pear samples were cut into small pieces, and 10 g of each piece was ground into powder in liquid nitrogen. 90 mL of pre-cooled potassium phosphate buffer (100 mM, pH 7.4) was added, and the homogenized mixture was centrifuged at 13000 rpm for 10 min. The supernatant was the crude enzyme extract.

[0037] For the peroxidase activity assay, 200 µL of crude extract was taken and 3.6 mL of reaction buffer was added. The buffer contained micritoxyphenol (0.1%), hydrogen peroxide (0.05%), and 100 mM potassium phosphate buffer (pH 7.4). Peroxidase activity was determined by monitoring the increase in absorbance at 470 nm at room temperature.

[0038] For the determination of superoxide dismutase activity, the crude extract was reacted with tetrazolium blue under sunlight for 20 minutes, and the absorbance at 560 nm was recorded. One unit of superoxide dismutase activity was defined as the amount of enzyme required to inhibit 50% of the tetrazolium blue photoreduction reaction.

[0039] Catalase activity was determined by detecting changes in absorbance at 240 nm in the presence of hydrogen peroxide. One unit of catalase activity was defined as the amount of enzyme required to catalyze 1 µmol of hydrogen peroxide per minute.

[0040] For the determination of polyphenol oxidase activity, 100 µL of crude extract was taken and 1 mL of reaction buffer was added. The buffer contained 100 mM phosphate (pH 6.8) and 50 mM catechol. Enzyme activity was determined by monitoring the change in absorbance at 420 nm. One unit of polyphenol oxidase activity was defined as the amount of enzyme required to catalyze the oxidation of 1 nmol of catechol per minute.

[0041] Figure 6This is a diagram illustrating the effect of oregano essential oil nanoemulsion on improving the antioxidant enzyme system of pear in an embodiment of the present invention. As can be seen from the diagram, compared with the control group, the use of oregano essential oil nanoemulsion can increase the activity of superoxide dismutase in pear during storage, inhibit the decline of catalase activity, increase peroxidase activity, and reduce polyphenol oxidase activity. This is beneficial for eliminating the adverse effects of reactive oxygen species on pear, maintaining the redox balance of pear during storage, and delaying the deterioration of pear quality.

Claims

1. A method for preparing an oregano essential oil nanoemulsion for preserving fragrant pears, characterized in that, Includes the following steps: 1) Weigh a certain mass of deacetylated chitosan, add a certain volume of deionized water and glacial acetic acid to carry out the chitosan acetylation reaction; 2) Heat and stir the solution with a magnetic stirrer until it becomes clear, then add a certain volume of oregano oil-Tween 80 mixed solution; 3) The solution was homogenized by ultrasound to obtain oregano essential oil nanoemulsion.

2. The method for preparing the oregano essential oil nanoemulsion for preserving pears as described in claim 1, characterized in that, In the oregano essential oil nanoemulsion, the volume fraction of oregano essential oil is 0.04%, the volume fraction of chitosan is 0.1%, the volume fraction of Tween 80 is 0.1%, and the volume fraction of acetic acid is 1%.

3. The method for preparing the oregano essential oil nanoemulsion for preserving pears as described in claim 1, characterized in that, In step 2), the magnetic stirrer is used for 30 minutes, at a speed of 1200 rpm, and at a heating temperature of 60°C.

4. The method for preparing the oregano essential oil nanoemulsion for preserving pears as described in claim 1, characterized in that, In step 3), the ultrasonic treatment is intermittent ultrasonic treatment at 400 W power for 40 min, with 3 seconds of ultrasonic treatment followed by a 4-second pause.

5. An oregano essential oil nanoemulsion for preserving fragrant pears, characterized in that, It was obtained by the preparation method of oregano essential oil nanoemulsion as described in steps 1 to 4.

6. An oregano essential oil nanoemulsion as described in claim 5 and its application in the preservation of fragrant pears.

7. The oregano essential oil nanoemulsion according to claim 6 and its application in the preservation of fragrant pears, characterized in that: 1) The oregano essential oil nanoemulsion is composed of an aqueous phase and an organic phase containing oregano essential oil. The aqueous phase is deionized water, and the organic phase is composed of oregano essential oil, Tween 80, chitosan, and acetic acid. The mass volume fraction of oregano essential oil is 0.04%, the final concentration of Tween 80 is 0.1%, the proportion of chitosan is 0.1%, and the proportion of acetic acid is 1%.

8. The oregano essential oil nanoemulsion according to claim 7 and its application in the preservation of fragrant pears, characterized in that, The preservation steps are as follows: After harvesting, the fragrant pears are immersed in oregano essential oil nano-emulsion to coat their surface evenly with a layer of preservation emulsion. They are then air-dried at room temperature for 10 minutes until the emulsion on the surface of the fragrant pears is dry. The coated fragrant pears are then stored at room temperature.

9. The oregano essential oil nanoemulsion according to claim 8 and its application in the preservation of fragrant pears, characterized in that: The pear was immersed in the oregano essential oil nanoemulsion for 1 minute.

Images