Methyl ferulate-grapefruit essential oil nanoemulsion, and preparation method and application thereof

Abstract

The application discloses methyl ferulate-grapefruit essential oil nanoemulsion as well as a preparation method and application thereof, the preparation method comprises the following steps: dissolving methyl ferulate in grapefruit essential oil to obtain an oil phase, adding Tween 80 and water into the oil phase in sequence, stirring to obtain a coarse emulsion, and then treating the coarse emulsion through ultrasonic treatment to obtain the methyl ferulate-grapefruit essential oil nanoemulsion. The preparation process is simple, convenient, green and safe, methyl ferulate and grapefruit essential oil can be co-stabilized in a nano system, the water solubility and dispersity of methyl ferulate are improved, and the obtained methyl ferulate-grapefruit essential oil nanoemulsion can be used for inhibiting alternaria alternata and alternaria alternata toxin and preserving fruits such as pears, cherry tomatoes and jujube fruits.

Description

Technical Field

[0001] This invention belongs to the field of food packaging technology, and relates to a methyl ferulic acid-grapefruit essential oil nanoemulsion, its preparation method and application, specifically to a methyl ferulic acid-grapefruit essential oil nanoemulsion based on ultrasonic nanoemulsification technology, its preparation method and application. Background Technology

[0002] Alternaria is one of the most typical pathogens causing postharvest diseases. During the transplanting process, it produces Alternaria toxins, which accelerate the infection process, ultimately causing black spot disease in fruits such as jujubes, pears, citrus, and tomatoes. This severely reduces the quality of agricultural products and causes huge economic losses to my country's agricultural sector. Furthermore, these Alternaria toxins can accumulate in the body and contaminate agricultural products and their processed forms, eventually posing adverse health risks when ingested.

[0003] Ferulic acid is a phenolic acid compound widely found in the epidermis of fruits and vegetables. It is synthesized via the phenylpropane route and cross-linked with cell wall polysaccharides through ester bonds. Esterified ferulic acid, due to its closer resemblance to the form in which it exists in plant epidermis, exhibits stronger antibacterial activity against Alternaria alternata compared to ferulic acid, more effectively inhibiting mycelial growth and spore germination. Furthermore, in terms of biotoxicity, the median lethal dose (LD50) of methyl ferulic acid is 1189 mg / kg, significantly higher than that of ferulic acid (LD50 = 194 mg / kg). Therefore, methyl ferulic acid is more environmentally friendly and safer than ferulic acid, making it a promising fruit and vegetable preservative. However, no studies have yet reported on the application of methyl ferulic acid in fruit preservation, primarily because its water insolubility limits its application in fruit and vegetable preservation. Currently, the common method for dissolving methyl ferulic acid is to dissolve it in ethanol and other organic reagents. These organic reagents are highly volatile and tend to cause methyl ferulic acid to crystallize and aggregate, hindering its uniform distribution on the fruit surface. Furthermore, the residue of these organic reagents may cause fruit quality deterioration and food safety issues. In summary, traditional organic reagent dissolution methods are unfavorable for the application of methyl ferulic acid in fruit preservation. Therefore, there is an urgent need to improve the water solubility and dispersibility of methyl ferulic acid to expand its application range. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a methyl ferulic acid-grapefruit essential oil nanoemulsion with excellent stability, antibacterial properties against Alternaria alternata, and fruit preservation properties, as well as its preparation method and application.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0006] A method for preparing a methyl ferulic acid-grapefruit essential oil nanoemulsion includes the following steps:

[0007] (1) Add methyl ferulic acid to grapefruit essential oil and stir to dissolve it in a hot water bath at 60℃~70℃ to obtain the oil phase;

[0008] (2) Add Tween 80 to the oil phase and stir to obtain an oil phase containing emulsifier;

[0009] (3) Add water to the oil phase containing emulsifier, and after stirring, obtain a crude emulsion;

[0010] (4) The crude emulsion was subjected to ultrasonic treatment in an ice bath to obtain methyl ferulic acid-grapefruit essential oil nanoemulsion.

[0011] In the above-mentioned method for preparing methyl ferulic acid-grapefruit essential oil nanoemulsion, preferably, in step (1), the mass of methyl ferulic acid is 10% to 80% of the mass of grapefruit essential oil.

[0012] In the above-mentioned method for preparing methyl ferulic acid-grapefruit essential oil nanoemulsion, preferably, the mass ratio of grapefruit essential oil in step (1) to Tween 80 in step (2) is 1:0.5-3.

[0013] In the above-mentioned method for preparing methyl ferulic acid-grapefruit essential oil nanoemulsion, preferably, the mass of water in step (3) is 10 to 20 times the mass of grapefruit essential oil in step (1).

[0014] In the above-mentioned method for preparing methyl ferulic acid-grapefruit essential oil nanoemulsion, preferably, in step (2), the stirring speed is 500 rpm to 1000 rpm and the stirring time is 5 min to 20 min; in step (3), the stirring speed is 500 rpm to 1000 rpm and the stirring time is 15 min to 45 min.

[0015] In the above-mentioned method for preparing methyl ferulic acid-grapefruit essential oil nanoemulsion, preferably, in step (4), the power of the ultrasound is 200W to 400W, and the duration of the ultrasound is 5min to 20min.

[0016] In the above-mentioned method for preparing methyl ferulic acid-grapefruit essential oil nanoemulsion, preferably, in step (4), the particle size of the obtained methyl ferulic acid-grapefruit essential oil nanoemulsion is 15nm to 120nm.

[0017] As a general technical concept, the present invention also provides a method for preparing the above-mentioned methyl ferulic acid-grapefruit essential oil nanoemulsion, which yields a methyl ferulic acid-grapefruit essential oil nanoemulsion.

[0018] As a general technical concept, the present invention also provides an application of the above-mentioned methyl ferulic acid-grapefruit essential oil nanoemulsion in fruit preservation.

[0019] In the above-described applications, preferably, the fruit includes pears, cherry tomatoes, or dates.

[0020] The main innovation of this invention is as follows:

[0021] To improve the water solubility and dispersibility of methyl ferulate, ultrasonic nanoemulsification was employed to load methyl ferulate into a nanoemulsion. Since methyl ferulate is a solid powder at room temperature, it cannot be directly converted into a nanoemulsion using ultrasonic nanoemulsification. Therefore, a suitable oil phase is needed to provide a lipid-soluble environment for methyl ferulate, followed by ultrasonic nanoemulsification to uniformly disperse it as oil-in-water droplets. However, different oil phases exhibit significantly different properties due to their varying compositions. Medium-chain triglycerides and corn oil are commonly chosen as oil phases for nanoemulsion preparation due to their relatively stable properties; however, nanoemulsions prepared from these two oil phases typically have large particle sizes and lack antibacterial and antioxidant properties, failing to meet application requirements. Essential oils are volatile oils extracted from plants, possessing antibacterial and antioxidant properties. They can not only provide a lipid-soluble environment for methyl ferulic acid but also enhance its antibacterial and antioxidant properties. However, compared to nanoemulsions prepared from medium-chain triglycerides and corn oil, nanoemulsions prepared from volatile essential oils are unstable. In particular, some highly water-soluble essential oils, such as citrus essential oils, are prone to Ostwald ripening, ultimately leading to nanoemulsion stratification. The applicant conducted extensive experiments and selected several essential oils (such as oregano, thyme, clove, and grapefruit oil) as the lipid-soluble oil phase for methyl ferulic acid. The applicant found that grapefruit oil can stably support methyl ferulic acid without crystallization or excessively large nanoemulsion particle size. Research and experiments have shown that methyl ferulate can be successfully and stably loaded into the oil phase of grapefruit essential oil using nanoemulsion technology. Furthermore, it was discovered that methyl ferulate can act as an Ostwald ripening inhibitor, suppressing the Ostwald ripening process of the nanoemulsion, thus improving its stability and reducing its particle size. The methyl ferulate-grapefruit essential oil nanoemulsion obtained in this invention exhibits stronger antibacterial activity and the ability to inhibit Alternaria alternata toxin production compared to methyl ferulate alone.

[0022] Compared with the prior art, the advantages of the present invention are as follows:

[0023] 1. This invention prepares a methyl ferulic acid-grapefruit essential oil nanoemulsion using ultrasonic emulsification technology. This nanoemulsion can co-stabilize methyl ferulic acid and grapefruit essential oil in a nanoscale system. On the one hand, the hydrophobic methyl ferulic acid can act as an Ostwald ripening inhibitor, increasing the hydrophobicity of grapefruit essential oil and thus inhibiting the Ostwald ripening of the grapefruit essential oil nanoemulsion. On the other hand, grapefruit essential oil provides a lipophilic environment for methyl ferulic acid, allowing methyl ferulic acid to dissolve in the essential oil. The methyl ferulic acid-grapefruit essential oil nanoemulsion prepared by this invention effectively improves the water solubility and dispersibility of methyl ferulic acid. Furthermore, this nanoemulsion has a very small particle size (15.66 nm) and a polymer dispersibility index (0.09), and exhibits excellent stability, remaining stable for more than one month at 4°C and 25°C.

[0024] 2. Compared to methyl ferulic acid dissolved in 1% (v / v) ethanol, the methyl ferulic acid-grapefruit oil nanoemulsion prepared in this invention exhibits stronger inhibitory activity against Alternaria alternata, with both the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) reduced by four times. It also more effectively controls the development of pear black spot disease and inhibits the accumulation of Alternaria alternata toxins. Adding methyl ferulic acid-grapefruit oil nanoemulsion to carboxymethyl cellulose imparts antibacterial properties to the cellulose. The resulting methyl ferulic acid-grapefruit oil nanoemulsion coating forms a dense protective film on the fruit surface, reducing the weight loss rate of pears, cherry tomatoes, and dates, maintaining good quality, and extending shelf life. Attached Figure Description

[0025] Figure 1 The process flow diagram shows the preparation method of methyl ferulic acid-grapefruit essential oil nanoemulsion in Examples 1-5 of this invention.

[0026] Figure 2 The images show the appearance and turbidity of the methyl ferulic acid-grapefruit essential oil nanoemulsions of Examples 1-5 and the grapefruit essential oil nanoemulsion of Comparative Example 1.

[0027] Figure 3 The particle size, PDI, and ζ-potential diagrams are shown for the methyl ferulic acid-grapefruit essential oil nanoemulsions of Examples 1-5 and the grapefruit essential oil nanoemulsion of Comparative Example 1.

[0028] Figure 4 Transmission electron microscopy images of the methyl ferulic acid-grapefruit essential oil nanoemulsion of Example 3 and the grapefruit essential oil nanoemulsion of Comparative Example 1.

[0029] Figure 5 The images show the oil-water interfacial tension of the methyl ferulic acid-grapefruit essential oil nanoemulsions of Examples 1-5 and the grapefruit essential oil nanoemulsion of Comparative Example 1.

[0030] Figure 6Viscosity diagrams of the methyl ferulic acid-grapefruit essential oil nanoemulsions of Examples 1-5 and the grapefruit essential oil nanoemulsion of Comparative Example 1.

[0031] Figure 7 Fourier transform infrared spectra of methyl ferulic acid, grapefruit essential oil, Tween 80, methyl ferulic acid-grapefruit essential oil nanoemulsions of Examples 2, 3, and 5 of this invention, and grapefruit essential oil nanoemulsion of Comparative Example 1.

[0032] Figure 8 The particle size variation diagrams of the methyl ferulic acid-grapefruit essential oil nanoemulsions of Examples 1-5 and Comparative Example 1 at different pH values ​​are shown.

[0033] Figure 9 The particle size variation diagrams of the methyl ferulic acid-grapefruit essential oil nanoemulsions of Examples 1-5 and the grapefruit essential oil nanoemulsion of Comparative Example 1 under different concentrations of salt ions are shown.

[0034] Figure 10 The graphs show the changes in particle size and PDI of the methyl ferulic acid-grapefruit essential oil nanoemulsion of Example 3 and the grapefruit essential oil nanoemulsion of Comparative Example 1 during storage at 4°C and 25°C.

[0035] Figure 11 The images show the appearance of the methyl ferulic acid-grapefruit essential oil nanoemulsion of Example 3 and the grapefruit essential oil nanoemulsion of Comparative Example 1 at different concentrations inhibiting the growth of Alternaria mycelia.

[0036] Figure 12 The graphs show the inhibition of Alternaria mycelial growth by different concentrations of the methyl ferulic acid-grapefruit essential oil nanoemulsion of Example 3 and the grapefruit essential oil nanoemulsion of Comparative Example 1.

[0037] Figure 13 The images show the MIC and MFC diagrams of the methyl ferulic acid-grapefruit essential oil nanoemulsion of Example 3 and the grapefruit essential oil nanoemulsion of Comparative Example 1, as well as the methyl ferulic acid.

[0038] Figure 14 Images show pear black spot disease during storage of the methyl ferulic acid-grapefruit essential oil nanoemulsion treatment in Example 3 of the present invention, the grapefruit essential oil nanoemulsion treatment in Comparative Example 1, and the control group without any treatment.

[0039] Figure 15 The graphs show the changes in the diameter of pear black spot disease lesions in Example 3 of the present invention treated with methyl ferulic acid-grapefruit essential oil nanoemulsion, Comparative Example 1 treated with grapefruit essential oil nanoemulsion, and the control group without any treatment.

[0040] Figure 16The graph shows the TeA, AME, and AOH toxin content of the methyl ferulic acid-grapefruit essential oil nanoemulsion treatment in Example 3 of the present invention, the grapefruit essential oil nanoemulsion treatment in Comparative Example 1, and the control group without any treatment.

[0041] Figure 17 The images show the appearance, weight loss, hardness, soluble solids, and total acidity of pears treated with carboxymethyl cellulose coating containing the methyl ferulic acid-grapefruit essential oil nanoemulsion of Example 3 of this invention, as well as control group 1 without any treatment and control group 2 treated only with carboxymethyl cellulose coating during storage.

[0042] Figure 18 The images show the appearance, weight loss, hardness, soluble solids, and total acidity of cherry tomatoes during storage, including those treated with carboxymethyl cellulose coating containing the methyl ferulic acid-grapefruit essential oil nanoemulsion of Example 3 of this invention, control group 1 without any treatment, and control group 2 treated only with carboxymethyl cellulose coating.

[0043] Figure 19 The images show the appearance, weight loss, hardness, soluble solids, and total acidity of jujubes treated with carboxymethyl cellulose coating containing the methyl ferulic acid-grapefruit essential oil nanoemulsion of Example 3 of this invention, as well as control group 1 without any treatment and control group 2 treated only with carboxymethyl cellulose coating during storage. Detailed Implementation

[0044] 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. All materials and instruments used in the following embodiments are commercially available. Methyl ferulic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and grapefruit essential oil was purchased from Ji'an Huatianbao Traditional Chinese Medicine Biological Products Factory.

[0045] In the following embodiments, the relevant detection and characterization are as follows:

[0046] (1) Particle size, polymer dispersibility index (PDI), and zeta potential: The nanoemulsion was diluted 10 times with deionized water to avoid multiple scattering effects. The particle size, PDI, and zeta potential of the nanoemulsion were measured using a nanoparticle size analyzer. The absorbance of the nanoemulsion was measured at a wavelength of 600 nm, which is the turbidity.

[0047] (2) Transmission electron microscopy: 10 μL of nanoemulsion was dropped onto a copper grating, and after standing for 10 min, the excess liquid was aspirated. Then, 2% phosphotungstic acid was dropped onto the copper grating and stained for 1–3 min. After removing the excess staining solution and allowing it to air dry for 10 min, images were taken using a transmission electron microscope at 100 kV.

[0048] (3) Interfacial tension: The interfacial tension between oil and water was determined by the pendant drop method using a contact angle measuring instrument.

[0049] (4) Viscosity: The viscosity of the nanoemulsion was measured using a rotational rheometer at 25°C, with a shear rate range of 1–100 s⁻¹. -1 .

[0050] (5) Fourier transform infrared spectroscopy: The concentrations of methyl ferulic acid, Tween 80, grapefruit essential oil, and nanoemulsion were analyzed using a Fourier transform infrared spectroscopy instrument in the range of 4000–4000 cm⁻¹. -1 Infrared spectrum in the wavenumber range.

[0051] (6) pH stability and salt ion stability: The pH of the nanoemulsion was adjusted to 1, 3, 5, 7, 9 and 11 using 1M NaOH or HCl. Different masses of NaCl were added to achieve sodium chloride concentrations of 100 mM, 200 mM, 300 mM, 400 mM and 500 mM in the NEs. After standing at room temperature for 12 h, the particle size of the nanoemulsion was measured.

[0052] (7) Storage stability: The nanoemulsions were stored at 4℃ and 25℃ respectively, and the particle size and PDI were measured on days 0, 5, 10, 15, 20, 25 and 30.

[0053] (8) Inhibition of Alternaria mycelial growth by nanoemulsion: A certain volume of nanoemulsion was added to sterilized PDA medium to make the volume fraction of nanoemulsion in PDA medium reach 0%, 0.25%, 0.5%, 1%, 2% and 4%. Then, a 7 mm mycelial cake was taken with a punch and placed in the center of a cooled and solidified PDA plate (90 mm). The plate was placed in a constant temperature and humidity incubator at 28℃ and 90% relative humidity for 7 days and the mycelial diameter was measured.

[0054] (9) Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MFC): The MICs of methyl ferulic acid, grapefruit oil nanoemulsion, and methyl ferulic acid-grapefruit oil nanoemulsion were determined using the 96-well plate microdilution method. The lowest concentration in the wells where no mycelial growth was observed after culturing the 96-well plates at 28°C and 90% RH for 3 days was the MIC. 100 μL of PDB medium with no obvious mycelial growth was transferred from the wells to PDA plates, spread, and then incubated at 28°C and 90% RH for 3 days. The lowest concentration in the plates where no mycelial growth was observed was the MFC.

[0055] (10) Inoculation and drug treatment of pear with Alternaria alternata: Fruits of similar maturity, uniform size, without mechanical damage, and free from pathogen infection were selected for in vivo experiments. All fruits were rinsed with clean water to remove surface dust, then disinfected by soaking in 2% (v / v) sodium hypochlorite for 3 minutes, and finally rinsed 3 times with distilled water. After the pear surface dried, a 5 mm deep and 3 mm wide wound was made at the equator of the pear using a sterile needle. Then, 10 μL of 1×10⁻⁶ sodium hypochlorite solution was added to the wound site. 6 The spore suspension was prepared using 10 μL of either 46% grapefruit oil nanoemulsion (Comparative Example 1) or 10 μL of 46% methyl ferulic acid-grapefruit oil nanoemulsion (Example 3). Finally, all fruits were placed in polyethylene resealable bags and stored in a constant temperature and humidity incubator at 28°C and 90% RH. The vertical diameter of the black spot lesions was measured on days 4, 6, and 8, and the average value was calculated.

[0056] (11) Detection of the content of three Alternaria toxins in pears: TeA, AME, and AOH. First, add 5g of pear black spot homogenate sample to a centrifuge tube, then add 2mL of deionized water and 5mL of acetonitrile containing 1.5% formic acid (v / v). Then, immediately vortex for 30s and ultrasonically extract at 400W for 30min. Next, add 0.5g of sodium chloride and 4g of anhydrous magnesium sulfate to the centrifuge tube, vortex for 30s, and then place the sample in a shaker and shake for 30min. Centrifuge the sample at 2420×g for 5min to separate the organic and aqueous phases. Use a syringe to aspirate the organic phase and pass it through a 0.22μm polytetrafluoroethylene filter. Finally, inject it into a brown sample bottle and detect the content using ultra-high performance liquid chromatography-mass spectrometry. Chromatographic conditions: EclipsePlus C18 HPLC column (1.8 μm, 2.1 × 150 mm); column temperature 30℃; mobile phase A: 0.1% formic acid solution, mobile phase B: acetonitrile; flow rate 0.5 mL / min; injection volume 10 μL. Gradient elution program: 0.0–0.5 min, 70% A; 0.5–1.0 min, 70%–10% A; 1.0–3.0 min, 10% A; 3.0–3.1 min, 10%–70% A; 3.1–5.5 min, 70% A. Mass spectrometry conditions: electrospray ionization source in negative ion mode; capillary interface voltage 3.2 kV; source temperature 230℃; quadrupole temperature 150℃. Multiple reaction monitoring (MRM) mode was used for qualitative and quantitative analysis of Alternaria alternata toxins.

[0057] (12) Fruit Preservation Nanoemulsion Coating: Fruits of similar maturity, uniform size, without mechanical damage, and free from pathogen infection were selected for preservation experiments. All fruits were rinsed with clean water to remove surface dust and air-dried naturally. A 2% (w / v) carboxymethyl cellulose solution was prepared, with glycerol added as a plasticizer and stirred thoroughly. The mass of glycerol was 25% of the mass of carboxymethyl cellulose. Subsequently, methyl ferulate-grapefruit essential oil nanoemulsion was added, with the volume of methyl ferulate-grapefruit essential oil nanoemulsion being 4% of the volume of the carboxymethyl cellulose solution (without glycerol). After stirring for 12 hours, a coating solution containing 4% methyl ferulate-grapefruit essential oil nanoemulsion was obtained. The fruits were immersed in the coating solution and, after being fully coated, were placed at room temperature to air-dry naturally. Subsequently, the fruits were packed into PE plastic boxes in groups of 6 pears, 20 cherry tomatoes, and 10 jujubes, and stored in a constant temperature and humidity incubator at 28°C and 80% relative humidity.

[0058] (13) Weight loss rate: The weight loss of fruit during storage was determined by gravimetric method.

[0059] Calculate the weight loss rate using the following formula:

[0060] Weight loss rate (%) = [(W0-W1) / W0] × 100% (2)

[0061] Where W0 and W1 are the initial weight of the fruit and the weight of the fruit at different storage times, respectively.

[0062] (14) Firmness: The firmness of the fruit was determined using a texture analyzer. A TA-39 probe was used to puncture the equatorial region of the fruit. The puncture depths were 5 mm and 8 mm, and the testing speed was 1 mm / s. The results are expressed in N / mm². 2 Expressed in units.

[0063] (15) Determination of soluble solids and total acid content: The soluble solids and total acid content of the fruit were determined using a handheld refractometer. The fruit was juiced and centrifuged at 2420×g for 10 min. The supernatant was collected and dropped into the refractometer probe for soluble solids determination. The supernatant was then diluted 50 times for total acid content determination.

[0064] Example 1:

[0065] A method for preparing the methyl ferulic acid-grapefruit essential oil nanoemulsion of the present invention, as follows: Figure 1 As shown, it includes the following steps:

[0066] (1) Add methyl ferulic acid to grapefruit essential oil and stir to dissolve it in a hot water bath at 60°C to obtain the oil phase. The mass of methyl ferulic acid is 10% of the mass of grapefruit essential oil.

[0067] (2) Add Tween 80 to the oil phase, stir at 1000 rpm for 10 min, and after stirring evenly, an oil phase containing emulsifier is obtained. The mass ratio of grapefruit essential oil to Tween 80 (i.e. polysorbate-80) is 1:2.

[0068] (3) Add water to the oil phase containing emulsifier, stir at 1000 rpm for 30 min to obtain crude emulsion, the mass of water is 20 times the mass of grapefruit essential oil;

[0069] (4) The crude emulsion was placed in an ultrasonic homogenizer and ultrasonicated in an ice bath (0℃) with an ultrasonic power of 300W and an ultrasonic time of 15min to obtain methyl ferulic acid-grapefruit essential oil nanoemulsion.

[0070] The methyl ferulic acid-grapefruit essential oil nanoemulsion prepared in this embodiment can be used for the preservation of pears, cherry tomatoes, and jujubes.

[0071] Example 2

[0072] A method for preparing methyl ferulic acid-grapefruit essential oil nanoemulsion of the present invention is basically the same as that in Example 1, except that in step (1), the mass of methyl ferulic acid is 20% of the mass of grapefruit essential oil.

[0073] Example 3

[0074] A method for preparing methyl ferulic acid-grapefruit essential oil nanoemulsion of the present invention is basically the same as that in Example 1, except that in step (1), the mass of methyl ferulic acid is 40% of the mass of grapefruit essential oil.

[0075] Example 4

[0076] A method for preparing methyl ferulic acid-grapefruit essential oil nanoemulsion of the present invention is basically the same as that in Example 1, except that in step (1), the mass of methyl ferulic acid is 60% of the mass of grapefruit essential oil.

[0077] Example 5

[0078] A method for preparing methyl ferulic acid-grapefruit essential oil nanoemulsion of the present invention is basically the same as that in Example 1, except that in step (1), the mass of methyl ferulic acid is 80% of the mass of grapefruit essential oil.

[0079] Comparative Example 1

[0080] A method for preparing grapefruit essential oil nanoemulsion, the preparation process is basically the same as that in Example 1, the only difference is that methyl ferulic acid is not added in step (1).

[0081] Comparative Example 2

[0082] A method for preparing a ferulic acid-grapefruit essential oil nanoemulsion was described. The preparation process was basically the same as in Example 3, except that in step (1), ferulic acid was added to grapefruit essential oil. The resulting nanoemulsion exhibited poor stability and was prone to aging and separation.

[0083] Comparative Example 3

[0084] A method for preparing p-coumaric acid-grapefruit essential oil nanoemulsion is provided. The preparation process is basically the same as that in Example 3, except that in step (1), p-coumaric acid is added to grapefruit essential oil. As a result, p-coumaric acid cannot be dispersed in the emulsion system and crystallizes out.

[0085] Comparative Example 4

[0086] A method for preparing methyl ferulic acid nanoemulsion was described. The preparation process was basically the same as in Example 3, except that: in step (1), methyl ferulic acid was not added to any essential oil; and in step (2), the mass ratio of methyl ferulic acid to Tween 80 was 1:2. As a result, methyl ferulic acid nanoemulsion could not be prepared, and methyl ferulic acid crystals precipitated.

[0087] Comparative Example 5

[0088] A method for preparing a methyl ferulic acid-oregano essential oil nanoemulsion is described. The preparation process is basically the same as in Example 3, except that in step (1), methyl ferulic acid is added to oregano essential oil. As a result, a nanoemulsion (particle size > 200 nm) could not be prepared, and the emulsion stability was poor, with methyl ferulic acid crystallizing out.

[0089] Comparative Example 6

[0090] A method for preparing a methyl ferulic acid-thyme essential oil nanoemulsion was disclosed. The preparation process was basically the same as in Example 3, except that in step (1), methyl ferulic acid was added to the thyme essential oil. The resulting nanoemulsion had a larger particle size (>200 nm) and poor performance.

[0091] Comparative Example 7

[0092] A method for preparing a methyl ferulic acid-clove oil nanoemulsion was disclosed. The preparation process was basically the same as in Example 3, except that in step (1), methyl ferulic acid was added to clove oil. The resulting nanoemulsion had a larger particle size (>200 nm) and poor performance.

[0093] Data Analysis:

[0094] (1) Turbidity, particle size, PDI and ζ potential of nanoemulsions

[0095] Figure 2The images show the appearance and turbidity of the methyl ferulic acid-grapefruit essential oil nanoemulsions of Examples 1-5 and the grapefruit essential oil nanoemulsion of Comparative Example 1. Figure 3 The particle size, PDI, and zeta potential diagrams are shown for the methyl ferulic acid-grapefruit oil nanoemulsions of Examples 1-5 and the grapefruit oil nanoemulsion of Comparative Example 1. Figure 2 As shown in (1) and (2), the turbidity of Examples 1-5 first decreased and then increased with the increase of methyl ferulic acid (MF) content, ranging from 0.06 to 1.18. Example 3 had the lowest turbidity at 0.06, while Comparative Example 1 had the highest at 2.68. Figure 3 It can be seen that in Examples 1-5, with the increase of methyl ferulic acid (MF) content, the particle size (15.66 nm to 71.80 nm) and PDI (0.09 to 0.42) both showed a trend of first decreasing and then increasing. When the mass of methyl ferulic acid was 40% of the mass of grapefruit essential oil, the particle size and PDI were the smallest, at 15.66 nm and 0.09, respectively. Figure 2 The nanoemulsion in Example 3 was the clearest. However, when the MF content exceeded 40%, the particle size of the nanoemulsion increased. Figure 2 The nanoemulsions of Examples 4 and 5 gradually became turbid, with particle sizes reaching 33.71 nm and 71.80 nm, respectively, but still significantly lower than that of Comparative Example 1. These results indicate that when the methyl ferulic acid content is 40%, the entire system gradually approaches saturation, and the interfacial stability reaches its optimal level. Furthermore, the addition of methyl ferulic acid improves the overall hydrophobicity of the oil phase. The main component of grapefruit essential oil is D-limonene, which has high solubility in water (13.5 mg / L). Compared to other essential oils, it is more prone to Ostwald ripening, meaning it easily diffuses, migrates, and aggregates into large droplets in water. Methyl ferulic acid can be used as a ripening inhibitor to increase the hydrophobicity of the oil phase, thereby mitigating Ostwald ripening and reducing droplet size. It is worth noting that the zeta potential does not completely affect the particle size and PDI of NEs (nanoemulsions). Since Tween 80 is a nonionic surfactant, it mainly relies on steric repulsion rather than electrostatic repulsion to stabilize the system. Therefore, even with a zeta potential above -30, it still has a very small particle size and PDI.

[0096] (2) Morphology of nanoemulsions

[0097] Figure 4These are transmission electron microscopy (TEM) images of the methyl ferulic acid-grapefruit essential oil nanoemulsion of Example 3 and the grapefruit essential oil nanoemulsion of Comparative Example 1. To more intuitively observe the morphology of the NEs, the grapefruit essential oil nanoemulsion and the methyl ferulic acid-grapefruit essential oil nanoemulsion were observed using TEM. As shown in the figure, the nanoemulsions all exhibit monodisperse spherical morphology, indicating the formation of an oil-in-water nanosystem. Notably, compared to the grapefruit essential oil nanoemulsion, the methyl ferulic acid-grapefruit essential oil nanoemulsion has a significantly smaller particle size and more uniform morphology.

[0098] (3) Oil-water interfacial tension of nanoemulsions

[0099] Figure 5 The images show the oil-water interfacial tension of the methyl ferulic acid-grapefruit essential oil nanoemulsions of Examples 1-5 and the grapefruit essential oil nanoemulsion of Comparative Example 1. Figure 5 It is observed that the interfacial tension decreases with the addition of methyl ferulate. This phenomenon can be attributed to methyl ferulate, as an Ostwald ripening inhibitor, promoting the formation of smaller particles, thereby reducing the interfacial tension between oil and water. However, interfacial tension is not the decisive factor affecting particle size. As the methyl ferulate content gradually increases, the total oil phase content also gradually increases, which can lead to an overload of the oil phase in a system with a fixed emulsifier content, resulting in an increase in particle size. On the one hand, the addition of methyl ferulate can significantly reduce the particle size of the nanosystem; on the other hand, the nanosystem also stabilizes methyl ferulate, and the two complement each other to form a stable emulsion system.

[0100] (4) Viscosity of nanoemulsion

[0101] Figure 6 The figures show the viscosity of the methyl ferulic acid-grapefruit oil nanoemulsions of Examples 1-5 and the grapefruit oil nanoemulsion of Comparative Example 1. Emulsion viscosity is an indicator of its physical stability. Nanofluids typically exhibit low viscosity and Newtonian fluid characteristics; lower viscosity generally indicates better stability. As shown, the viscosity of all nanoemulsions is less than 0.01, indicating that methyl ferulic acid has good stability within the nanoemulsion. Furthermore, the addition of methyl ferulic acid transforms the system from a non-Newtonian fluid to a Newtonian fluid, demonstrating that methyl ferulic acid improves the stability of the nanoemulsion. Additionally, Example 3 exhibits the lowest viscosity, suggesting it may be the most stable system, consistent with the particle size results.

[0102] (5) Fourier transform infrared spectrum of nanoemulsion

[0103] Figure 7Fourier transform infrared (FTIR) spectra of methyl ferulic acid, grapefruit essential oil, Tween 80, the methyl ferulic acid-grapefruit essential oil nanoemulsions of Examples 2, 3, and 5 of this invention, and the grapefruit essential oil nanoemulsion of Comparative Example 1 are shown. The interactions between the components in the nanoemulsions were analyzed using Fourier transform infrared spectroscopy. As shown in the figure, no new characteristic peaks appeared in Examples 2, 3, and 5, indicating that there were no significant chemical interactions between the nanoemulsion components. Methyl ferulic acid showed OH stretching (3399 cm⁻¹). -1 CH stretching (2947cm) -1 C=O stretching (1718cm) -1 ), aliphatic C=C stretch (1634cm) -1 Aromatic C=C stretching (1604~1436cm) -1 ) and COC asymmetric stretch (1265cm) -1 Characteristic peaks such as 3399 cm⁻¹ were observed. In Examples 2, 3, and 5, the intensity of the characteristic peak of methyl ferulic acid increased with increasing methyl ferulic acid content, indicating that methyl ferulic acid is stably present in the nanoemulsion. Furthermore, with increasing methyl ferulic acid content, the intensity of the characteristic peak of methyl ferulic acid increased. -1 The increased intensity of the OH stretching vibration at the molecule and its shift to lower wavenumbers indicate the presence of hydrogen bonding interactions between molecules. In summary, methyl ferulate, when incorporated into the nanoemulsion, retains the absorption peaks of its original active groups, and there is no evidence of new chemical reactions occurring between the nanoemulsion components. This retention of the antibacterial activity of methyl ferulate further confirms its stability within the nanoemulsion.

[0104] (6) pH stability and salt ion stability of nanoemulsions

[0105] Figure 8 The particle size variation diagrams of the methyl ferulic acid-grapefruit essential oil nanoemulsions of Examples 1-5 and Comparative Example 1 at different pH values ​​are shown. Figure 9 The figures show the particle size variations of the methyl ferulic acid-grapefruit oil nanoemulsions of Examples 1-5 and the grapefruit oil nanoemulsion of Comparative Example 1 under different salt ion concentrations. To evaluate the versatility and stability of NEs, the particle size of the nanoemulsions was tested at pH values ​​from 1 to 11 and salt ion concentrations from 0 mM to 500 mM. Figure 8 It can be seen that the particle size of all nanoemulsions remained basically unchanged at different pH levels, indicating good pH stability. Notably, the particle size of Example 3 remained consistently within 20 nm, showing minimal influence from pH, indicating that Example 3 exhibited the best pH stability. Furthermore, from... Figure 9 It can be seen that the particle size of each group fluctuates only slightly with changes in salt ion concentration, with Example 3 having the smallest particle size. The results indicate that Example 3 exhibits excellent pH and salt ion stability.

[0106] (7) Storage stability of nanoemulsions

[0107] Figure 10 (1) and Figure 10 (2) The graphs show the changes in particle size and PDI of the methyl ferulic acid-grapefruit oil nanoemulsion of Example 3 and the grapefruit oil nanoemulsion of Comparative Example 1 during storage at 4°C and 25°C. The storage stability of the nanoemulsions was evaluated by measuring their particle size and PDI during 30 days of storage at 4°C and 25°C. Figure 10 (1) As shown, the particle size of Example 3 hardly changed at 4°C. Figure 10 (2) The PDI shown fluctuates slightly but remains less than 0.2. This indicates that Example 3 has excellent storage stability at 4°C. At 25°C, the particle size and PDI of Example 3 gradually increase, while those of Comparative Example 1 gradually decrease. This is because Ostwald ripening occurred in Comparative Example 1, causing the emulsion droplets to gradually aggregate. Grapefruit essential oil is gradually released and volatilized at 25°C over time, resulting in a reduction in total oil content, leaving only emulsion droplets with smaller particle sizes. However, in Example 3, due to the presence of methyl ferulate, Ostwald ripening is delayed. Therefore, with prolonged storage time, the nanoemulsion particle size and PDI increase slowly, while the amount of grapefruit essential oil volatilized is small and methyl ferulate did not precipitate. In summary, Example 3 has good storage stability. Methyl ferulate can be stably present in the nanoemulsion and, in turn, acts as an Ostwald ripening inhibitor to stabilize the nanoemulsion.

[0108] (8) Antibacterial properties of nanoemulsions against Alternaria alterniflora

[0109] Figure 11 The images show the appearance of the methyl ferulic acid-grapefruit essential oil nanoemulsion of Example 3 and the grapefruit essential oil nanoemulsion of Comparative Example 1 at different concentrations, inhibiting the mycelial growth of Alternaria alterniflora. Figure 12 This image shows the inhibition of Alternaria mycelial growth by different concentrations of the methyl ferulic acid-grapefruit essential oil nanoemulsion of Example 3 and the grapefruit essential oil nanoemulsion of Comparative Example 1. Figure 11 As shown, the inhibitory effects of Comparative Example 1 and Example 3 on Alternaria were evaluated on PDA plates. Figure 11 and Figure 12 It was found that grapefruit essential oil nanoemulsion had a certain inhibitory effect on the mycelial growth of Alternaria alternata, but the inhibitory effect was not significant with increasing concentration. In contrast, methyl ferulate-grapefruit essential oil nanoemulsion showed a significant inhibitory effect on the mycelial growth of Alternaria alternata in a dose-dependent manner. The mycelial diameter treated with 4% methyl ferulate-grapefruit essential oil nanoemulsion was only 20.90±0.64 mm, a reduction of 68.09% compared to 4% grapefruit essential oil nanoemulsion.

[0110] (9) MIC and MFC of nanoemulsions

[0111] Figure 13 The images show the MIC and MFC diagrams of the methyl ferulic acid-grapefruit essential oil nanoemulsion of Example 3 and the grapefruit essential oil nanoemulsion of Comparative Example 1, as well as that of methyl ferulic acid. The comparison of the MIC and MFC of the methyl ferulic acid-grapefruit essential oil nanoemulsion, the grapefruit essential oil nanoemulsion, and methyl ferulic acid was used to evaluate whether the nanoemulsion could enhance the antibacterial properties of methyl ferulic acid. Figure 13 The results show that the MIC and MFC values ​​of the methyl ferulic acid-grapefruit oil nanoemulsion are only 0.8 mg / mL and 1.6 mg / mL, respectively, which are 4 times lower than those of methyl ferulic acid, while the MIC and MFC values ​​of the grapefruit oil nanoemulsion are much higher. These results indicate that methyl ferulic acid can be more effectively loaded into the nanoemulsion to exert its antibacterial activity.

[0112] (10) Inhibitory effect of nanoemulsion on pear black spot disease and Alternaria toxin production

[0113] Figure 14 and Figure 15 The images show the pear black spot disease and the change in the diameter of the black spot disease lesions, respectively, for the pear treated with methyl ferulic acid-grapefruit essential oil nanoemulsion in Example 3 of the present invention, the pear treated with grapefruit essential oil nanoemulsion in Comparative Example 1, and the control group without any treatment. Figure 16 (1), (2), and (3) are graphs showing the TeA, AME, and AOH toxin contents of the methyl ferulic acid-grapefruit essential oil nanoemulsion treatment in Example 3 of the present invention, the grapefruit essential oil nanoemulsion treatment in Comparative Example 1, and the control group without any treatment. Korla fragrant pears are a typical fruit infected by Alternaria alternata, such as... Figure 15 As shown, pears infected with Alternaria alternata develop black spot disease. In the control group, the lesions gradually enlarged during storage, reaching 31.93±1.90 mm by day 8. Grapefruit essential oil nanoemulsion showed a certain inhibitory effect on black spot disease, with the lesion diameter at day 8 being 28.86±1.05 mm. In Example 3, the diameter at day 8 was 10.29±0.45 mm, a reduction of 67.77% compared to the control group. This indicates that the methyl ferulic acid-grapefruit essential oil nanoemulsion has a stronger effect in inhibiting Alternaria alternata infection and alleviating black spot symptoms. Alternaria toxins are fungal toxins produced by Alternaria alternata. They not only harm plants but also accumulate in agricultural products and flow into processed products, ultimately harming human health. Therefore, three Alternaria toxins—TeA, AME, and AOH—produced by Alternaria alternata during pear storage were tested. Figure 16As shown, AME was not detected in Example 3, while in other groups, the three toxins gradually accumulated. After 8 days of storage, the control group had the highest levels of the three toxins, while the levels of TeA and AOH in Example 3 were reduced by 23.81 and 20.37 times, respectively, compared to the control group. This indicates that methyl ferulic acid-grapefruit essential oil nanoemulsion can significantly inhibit the production of Alternaria toxins.

[0114] (11) Preservation effect of nanoemulsion coating on pears, cherry tomatoes and jujubes

[0115] Figure 17 (1)-(5) Figure 18 (1)-(5) and Figure 19 (1)-(5) show the appearance, weight loss, hardness, soluble solids, and total acid changes of pears, cherry tomatoes, and jujubes during storage, respectively, for pears, cherry tomatoes, and jujubes treated with carboxymethyl cellulose coating containing the methyl ferulic acid-grapefruit oil nanoemulsion of Example 3 of this invention, and for control group 1 (without any treatment) and control group 2 (treated only with carboxymethyl cellulose coating). Korla pears, cherry tomatoes, and jujubes are typical fruits infected by Alternaria alternata, resulting in severe post-harvest diseases that lead to a decline in their quality. Carboxymethyl cellulose is a safe, non-toxic, and biodegradable linear polysaccharide that can form a solution with a specific viscosity, thus effectively adhering to the surface of fruits. However, it lacks antibacterial properties, which limits its widespread application. Therefore, the methyl ferulic acid-grapefruit oil nanoemulsion prepared above was added to a carboxymethyl cellulose solution to coat Korla pears, cherry tomatoes, and jujubes, and its preservation effect was evaluated by measuring the weight loss, hardness, soluble solids, and total acid during storage.

[0116] Depend on Figure 17 (1) It can be seen that on the 15th day of storage, black spots appeared on the pears of control group 1 and control group 2, indicating that they were infected with mold. In contrast, the pears of Example 3 did not show any lesions or mold infection during storage. Similarly, as Figure 18 As shown in (1), the cherry tomatoes treated in control group 1 and control group 2 showed obvious wrinkling and fungal infection on the epidermis on day 4, and severe shrinkage and rotting on day 8, while the cherry tomatoes in Example 3 only showed slight wrinkling. Figure 19 As shown in (1), on the 9th day of storage, the skin of the jujubes in control group 1 and control group 2 showed severe wrinkling and black spots, while some of the jujubes in Example 3 showed slight wrinkling.

[0117] Figure 17 (2) Figure 18 (2) and Figure 19(2) indicates that the weight loss rate of Example 3 is less than that of Control Group 1 and Control Group 2, which shows that the methyl ferulic acid-grapefruit essential oil nanoemulsion coating can inhibit fruit respiration, thereby slowing down water loss and maintaining the weight of the fruit.

[0118] Figure 17 (3) Figure 18 (3) and Figure 19 (3) indicates that at the end of storage, the hardness of Example 3 was greater than that of Control Group 1 and Control Group 2, indicating that the methyl ferulic acid-grapefruit essential oil nanoemulsion coating maintained the integrity of the cell wall, delayed the senescence of the fruit, and thus maintained the hardness of the fruit.

[0119] Figure 17 (4), 18(4), and 19(4) indicate that the methyl ferulic acid-grapefruit essential oil nanoemulsion coating effectively maintains the soluble solids content of the fruit. Figure 17 In (4), after 15 days of storage, the soluble solids content of pears in control group 1 was higher than that in example 3. This was because severe fungal infection led to the decomposition of organic compounds in the cell wall. Similarly, as Figure 18 (4) The same results were also observed in cherry tomatoes on day 8 of storage, while the soluble solids in jujubes in Example 3 remained at a high level throughout.

[0120] During storage, total acid, as a substrate for cellular respiration, is gradually consumed, and its levels decrease with prolonged storage time. Figure 17 As shown in (5), 18(5), and 19(5), compared with control groups 1 and 2, the total acid content of Example 3 decreased more slowly, indicating that the methyl ferulic acid-grapefruit oil nanoemulsion coating delayed the degradation of total acid, thereby maintaining the fruit flavor. In conclusion, the methyl ferulic acid-grapefruit oil nanoemulsion coating can maintain the quality of pears, cherry tomatoes, and jujubes and effectively extend their shelf life.

[0121] This invention successfully stabilizes methyl ferulate in a grapefruit oil-based nanoemulsion using ultrasonic emulsification technology, improving its water solubility and dispersibility and broadening its application range. Simultaneously, methyl ferulate acts as an Ostwald ripening inhibitor to stabilize the nanoemulsion. The methyl ferulate-grapefruit oil nanoemulsion exhibits good stability and antibacterial properties, and can inhibit the production of Alternaria toxin. Its addition to carboxymethyl cellulose for fruit coating maintains the quality of pears, cherry tomatoes, and dates, and effectively extends their shelf life.

[0122] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A method for preparing a methyl ferulic acid-grapefruit essential oil nanoemulsion, characterized in that, Includes the following steps: (1) Add methyl ferulic acid to grapefruit essential oil and stir to dissolve it in a hot water bath at 60℃~70℃ to obtain the oil phase; (2) Add Tween 80 to the oil phase and stir to obtain an oil phase containing emulsifier; (3) Add water to the oil phase containing emulsifier, and after stirring, obtain a crude emulsion; (4) The crude emulsion was subjected to ultrasonic treatment in an ice bath to obtain methyl ferulic acid-grapefruit essential oil nanoemulsion.

2. The method for preparing the methyl ferulic acid-grapefruit essential oil nanoemulsion according to claim 1, characterized in that, In step (1), the mass of the methyl ferulic acid is 10% to 80% of the mass of grapefruit essential oil.

3. The method for preparing the methyl ferulic acid-grapefruit essential oil nanoemulsion according to claim 1, characterized in that, The mass ratio of grapefruit essential oil in step (1) to Tween 80 in step (2) is 1:0.5 to 3.

4. The method for preparing the methyl ferulic acid-grapefruit essential oil nanoemulsion according to claim 1, characterized in that, The mass of water in step (3) is 10 to 20 times the mass of grapefruit essential oil in step (1).

5. The method for preparing the methyl ferulic acid-grapefruit essential oil nanoemulsion according to any one of claims 1 to 4, characterized in that, In step (2), the stirring speed is 500 rpm to 1000 rpm and the stirring time is 5 min to 20 min; in step (3), the stirring speed is 500 rpm to 1000 rpm and the stirring time is 15 min to 45 min.

6. The method for preparing the methyl ferulic acid-grapefruit essential oil nanoemulsion according to any one of claims 1 to 4, characterized in that, In step (4), the power of the ultrasound is 200W to 400W, and the duration of the ultrasound is 5min to 20min.

7. The method for preparing the methyl ferulic acid-grapefruit essential oil nanoemulsion according to any one of claims 1 to 4, characterized in that, In step (4), the particle size of the obtained methyl ferulic acid-grapefruit essential oil nanoemulsion is 15nm to 120nm.

8. A methyl ferulic acid-grapefruit essential oil nanoemulsion prepared by the method described in any one of claims 1 to 7.

9. The application of the methyl ferulic acid-grapefruit essential oil nanoemulsion as described in claim 8 in fruit preservation.

10. The application according to claim 9, characterized in that, The fruits mentioned include pears, cherry tomatoes, or dates.

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