A pickering emulsion containing menthol and a method of preparing the same

Abstract

The present application belongs to the field of food technology, and particularly relates to a Pickering emulsion containing menthol and a preparation method thereof. The Pickering emulsion containing menthol is prepared by taking menthol and cyclodextrin composite particles as a solid particle emulsifier and soybean oil as an oil phase, and the mass ratio of the menthol to the cyclodextrin is 1:2-5. The Pickering emulsion containing menthol prepared by the present application exhibits good storage stability and rheological properties within 30 days, and in addition, the emulsion also exhibits excellent antioxidant and antibacterial properties. In an in vitro simulated digestion experiment, the release rate of free fatty acids in the emulsion in the small intestine is 76.64+ / -1.08%, and the emulsion has good sustained-release property and improves the bioavailability of menthol.

Description

Technical Field

[0001] This invention belongs to the field of food technology, specifically relating to a menthol-containing Pickering emulsion and its preparation method. Background Technology

[0002] Menthol (C 10 H 20 O,(1R,2S,5R)-2-isopropyl-5-methylcyclohexanol is a naturally occurring chiral volatile cyclic terpene alcohol, primarily extracted from the peppermint plant *Mentha x piperita* (Laminoideae family), and possesses the characteristic peppermint aroma. However, menthol is unstable, sensitive to light, heat, and oxygen, and easily lost through degradation and volatilization. Furthermore, it has a certain degree of irritation, thus severely limiting many of its potential applications.

[0003] To improve the stability of menthol and slow down its degradation and volatilization, existing studies often employ micro / nano-carrier encapsulation techniques for its protection and utilization. For example, Zhu et al. successfully prepared an inclusion complex using hydroxypropyl cyclodextrin as the host and menthol as the guest. However, the release rate of the inclusion complex was temperature-limited, with a higher release rate only observed at 279.1℃. Additionally, patent CN112705131A discloses a eutectic solvent / hydroxypropyl-β-cyclodextrin-menthol microcapsule, which improves microcapsule stability by adding hexadecyltrimethylammonium chloride. Furthermore, in other studies, inclusion complexes were prepared between starch and menthol with varying amylose contents; even with adsorption times exceeding one day, the highest menthol mass fraction in the inclusion complex was only 5.5%.

[0004] Pickering emulsions, as a green alternative to traditional surfactant-stabilized emulsions, achieve effective emulsion stabilization with a lower colloidal particle loading. Solid particles, rather than surfactants, stabilize Pickering emulsions. Due to the irreversible adsorption of these solid particles at the oil-water interface, a dense barrier is formed, resulting in superior stability compared to traditional emulsions.

[0005] Cyclodextrin (CD) is a collective term for a series of cyclic oligosaccharides produced by the action of cyclodextrin glucosyltransferase on glucose polymers such as starch, maltose, and glycogen. Its unique molecular structure, which is hydrophilic on the outside and hydrophobic on the inside, gives it excellent encapsulation ability. Through inclusion, it can protect some easily oxidized and decomposed active substances. For example, Sha et al. prepared catechin / β-cyclodextrin inclusion complexes by saturated aqueous solution method and investigated the effect of the inclusion complexes on oral instant films.

[0006] Furthermore, using cyclodextrin as an encapsulating agent to prepare Pickering emulsions of active ingredients yields products that combine the advantages of both methods. For example, patent CN113519822A discloses a temperature-responsive cyclodextrin nanoparticle Pickering emulsion, which uses cyclodextrin to encapsulate sensitive ingredients such as quercetin, retinol, and lycopene, ultimately obtaining a temperature-responsive cyclodextrin nanoparticle Pickering emulsion. However, the Pickering emulsion prepared using the method in this patent is significantly affected by temperature changes.

[0007] Currently, research on menthol both domestically and internationally mainly focuses on the development of peppermint flavorings and peppermint essential oils. There is a lack of effective and widely applicable loading methods to address the unstable and volatile nature of menthol. Using Pickering emulsions can serve as an excellent carrier for loading menthol, transforming it from a crystalline structure into an emulsion form, which has broad application value. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a menthol-containing Pickering emulsion and its preparation method.

[0009] The Pickering emulsion containing menthol provided by this invention is specifically prepared using menthol and cyclodextrin composite particles as solid particle emulsifiers and soybean oil as the oil phase; the mass ratio of menthol to cyclodextrin is 1:2 to 5.

[0010] Preferably, the cyclodextrin is selected from any one of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

[0011] More preferably, the cyclodextrin is β-cyclodextrin.

[0012] In fact, cyclodextrin molecular inclusion technology has already been applied and can indeed effectively improve the solubility and stability of functional active ingredients. However, the existing encapsulation process for menthol mainly focuses on using complexes or encapsulating peppermint oil. Firstly, this has limitations in application scope, and secondly, peppermint oil contains many other types of compounds. Therefore, compared with other technologies, directly encapsulating menthol crystals in the form of emulsions can reduce interference from other factors in application and has good stability.

[0013] The β-cyclodextrin used in this invention has a hollow, cyclic molecular structure resembling an oil cake, formed by seven pyranose molecules linked by α-1-4 glycosidic bonds. It possesses a hydrophilic outer layer and a hydrophobic center. Due to its size-matching effect and central hydrophobicity, molecules of appropriate size, shape, and hydrophobicity can interact with β-cyclodextrin through non-covalent bonds to form stable inclusion complexes. Among various cyclodextrins, β-cyclodextrin exhibits superior emulsifying ability, not only providing excellent protection for guest molecules but also effectively improving their stability, solubility, and bioavailability.

[0014] Furthermore, the present invention also provides a method for preparing the above-mentioned menthol-containing Pickering emulsion, specifically comprising the following steps:

[0015] Preparation of S1, Menthol / Cyclodextrin Composite Particles

[0016] Cyclodextrin was dispersed in water to form a suspension, and then menthol was slowly added to it while stirring. After the menthol was added, stirring was continued to allow the two to react fully. After the reaction was completed, the reaction solution was refrigerated and allowed to settle. The precipitate was filtered, washed, and freeze-dried to obtain menthol / cyclodextrin composite granular powder.

[0017] S2, Preparation of Pickering emulsion containing menthol

[0018] The menthol / cyclodextrin composite particle powder obtained in S1 is dissolved in water to obtain an aqueous solution of menthol / cyclodextrin composite particles. Then, it is mixed with soybean oil and homogenized to obtain a Pickering emulsion containing menthol.

[0019] In the above method for preparing the menthol-containing Pickering emulsion, preferably, the mass ratio of cyclodextrin to water in S1 is 1:5-10, the mass ratio of menthol to cyclodextrin is 1:2-5, the temperature at which cyclodextrin is dispersed in water is 40-50°C, the temperature at which the reaction is stirred is 40-50°C, and the reaction time is 2-5 hours.

[0020] In this invention, when preparing the Pickering emulsion, a mass ratio of cyclodextrin to water that is too low or too high will affect the formation of the final Pickering emulsion complex, resulting in a decrease in the loading rate of menthol obtained in the end.

[0021] In addition, excessively high temperatures during the dispersion of cyclodextrin and menthol can also cause menthol to volatilize, while excessively low temperatures can easily cause menthol to precipitate. Both of these results will lead to a decrease in the loading rate of menthol.

[0022] More preferably, in S1, the mass ratio of cyclodextrin to water is 1:8-10, the mass ratio of menthol to cyclodextrin is 1:3-4, the temperature at which cyclodextrin is dispersed in water is 45-50℃, the temperature at which the reaction is stirred is 45-50℃, and the reaction time is 3-4 hours.

[0023] Preferably, in S2, the menthol / cyclodextrin composite particle aqueous solution has a mass volume fraction of 1-5%, and soybean oil accounts for 40-80% of the Pickering emulsion volume.

[0024] More preferably, in S2, the menthol / cyclodextrin composite particle aqueous solution has a mass-volume fraction of 3-5%, and soybean oil accounts for 40-60% of the Pickering emulsion volume.

[0025] Preferably, in S2, the homogenization conditions are: homogenization rate of 15000-25000 rpm and homogenization time of 1-5 min.

[0026] Preferably, in S2, the homogenization conditions are: homogenization rate of 15,000 to 20,000 rpm and homogenization time of 3 to 5 min.

[0027] Furthermore, the present invention provides a method for preparing a menthol-containing Pickering emulsion, as detailed below:

[0028] Preparation of S1, Menthol / β-Cyclodextrin Composite Particles

[0029] β-Cyclodextrin was dispersed in water at 40–45°C to form a suspension. Menthol was then slowly added to the suspension while stirring. After the menthol was added, the mixture was stirred and reacted at 40–45°C for 2–5 hours. After the reaction was completed, the reaction solution was refrigerated at 0–10°C for 12–36 hours to allow it to settle. The precipitate was then filtered, washed, and freeze-dried to obtain menthol / β-cyclodextrin composite granular powder.

[0030] S2, Preparation of Pickering emulsion containing menthol

[0031] The menthol / β-cyclodextrin composite particle powder obtained in S1 was dissolved in water to obtain an aqueous solution of menthol / β-cyclodextrin composite particles. The mass volume fraction of the aqueous solution of menthol / β-cyclodextrin composite particles was 1-5%. Then, it was mixed with soybean oil and homogenized at a homogenization rate of 15,000-20,000 rpm for 1-5 min to obtain a Pickering emulsion containing menthol. Soybean oil accounted for 40-80% of the volume of the Pickering emulsion.

[0032] The beneficial effects of this invention are as follows:

[0033] (1) This invention provides a Pickering emulsion stabilized by menthol / β-cyclodextrin composite particles. The obtained menthol-containing Pickering emulsion exhibits good rheological properties and storage stability within 30 days. In addition, in an in vitro simulated digestion experiment, the menthol-containing Pickering emulsion exhibits good sustained-release effect, with a free fatty acid release rate of 76.64±1.08% in the small intestine.

[0034] (2) The method for preparing menthol-containing Pickering emulsion provided in this invention is simple to operate and the menthol encapsulation rate in the obtained Pickering emulsion is high, reaching 89.7%. Attached Figure Description

[0035] Figure 1 The infrared spectrum of the menthol / β-cyclodextrin composite particles provided in Example 1 of this invention;

[0036] Figure 2 The XRD pattern of the menthol / β-cyclodextrin composite particles provided in Example 1 of this invention;

[0037] Figure 3 The particle size distribution diagram of the menthol / β-cyclodextrin composite particle-stabilized Pickering emulsion provided in Example 2 of this invention;

[0038] Figure 4 This is the standard curve of menthol encapsulation rate provided in Example 2 of the present invention;

[0039] Figure 5 Storage stability diagram of the menthol / β-cyclodextrin composite particle-stabilized Pickering emulsion provided in Example 2 of this invention;

[0040] Figure 6 This is a microstructure diagram of the menthol / β-cyclodextrin composite particle-stabilized Pickering emulsion provided in Example 2 of the present invention;

[0041] Figure 7 Rheological characterization of the menthol / β-cyclodextrin composite particle-stabilized Pickering emulsion provided in Example 2 of this invention;

[0042] Figure 8 Antioxidant characterization of the menthol / β-cyclodextrin composite particle-stabilized Pickering emulsion provided in Example 3 of this invention;

[0043] Figure 9 In vitro digestion characterization of the menthol / β-cyclodextrin composite particle-stabilized Pickering emulsion provided in Example 3 of this invention;

[0044] Figure 10 Characterization of the antibacterial properties of the menthol / β-cyclodextrin composite particle-stabilized Pickering emulsion provided in Example 3 of the present invention;

[0045] Figure 11 The stability of Pickering emulsions obtained from different oil phases in Example 4 of this invention after 30 days of storage;

[0046] Figure 12The stability of Pickering emulsion prepared at different concentrations of menthol / β-cyclodextrin composite particles in Example 5 of this invention after 30 days of storage.

[0047] Figure 13 The stability of Pickering emulsions prepared under different oil phase ratios in Example 6 of this invention after 30 days of storage;

[0048] Figure 14 The stability of Pickering emulsions obtained at different homogenization rates in Example 7 of this invention after 30 days of storage;

[0049] Figure 15 This describes the stability of Pickering emulsions obtained under different homogenization times in Example 8 of the present invention after 30 days of storage. Detailed Implementation

[0050] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0051] Example 1

[0052] Prepare menthol / β-cyclodextrin composite particles.

[0053] First, take 10g of β-cyclodextrin and disperse it in 80g of distilled water at 45℃ to make a suspension. Then, slowly add 3g of menthol while stirring. After the menthol is added, continue stirring and react at 45℃ for 3h. The resulting suspension is stored in a refrigerator at 5℃ for 24h to allow it to stand and precipitate. Filter the precipitate, wash it with anhydrous ethanol, and finally freeze-dry it to obtain a menthol / β-cyclodextrin composite granular powder.

[0054] The various indicators of the menthol / β-cyclodextrin composite particles prepared in this embodiment were tested, and the specific test results are as follows:

[0055] Appendix Figure 1 Fourier transform infrared (FT-IR) scans of menthol, β-cyclodextrin (β-CD), menthol / β-cyclodextrin composite particles, and menthol / β-cyclodextrin mixtures.

[0056] Appendix Figure 1 In the image, the black line indicates the characteristic absorption peak of menthol at 3240 cm⁻¹. -1 The peak at 2871 cm⁻¹ represents the vibrational absorption peak of the hydroxyl group (-OH). -1 The absorption peak of the rocking vibration of the CH bond is at 1445 cm⁻¹. -1 The vibrational absorption peak of the CH bond.

[0057] The red line indicates the characteristic absorption peak of β-cyclodextrin at 3281 cm⁻¹. -1 The stretching vibration peak of the hydroxyl group (-OH) at 2923 cm⁻¹ -1 The stretching vibration peak of the CH bond, 1022 cm⁻¹ -1 The absorption peak of the stretching vibration of the C=O bond (COC) at that location.

[0058] The infrared spectrum of the simple mixture of menthol and β-cyclodextrin, represented by the green line, is a simple superposition of the individual peaks of the infrared spectra of β-cyclodextrin and menthol, without any shift of the characteristic peaks.

[0059] The blue line represents the -OH vibrational stretching absorption peak from wavenumber 3240 cm⁻¹ in the infrared spectrum of the menthol / β-cyclodextrin complex particles. -1 The absorption peak of the CH bond rocking vibration shifted from 2871 cm⁻¹ to 3303 cm⁻¹. -1 It moved to 2867cm -1 At this point, the vibrational absorption peak of alkyl groups is from 1445 cm⁻¹. -1 The location has moved to 1440cm. -1 Place.

[0060] The above results indicate that the hydrophobic volatile compounds in menthol are subjected to intermolecular forces, which cause menthol to be encapsulated into the cavity of β-cyclodextrin, forming menthol / β-cyclodextrin composite particles.

[0061] Appendix Figure 2 The figures show the X-ray diffraction (XRD) patterns of menthol, β-cyclodextrin (β-CD), menthol / β-cyclodextrin composite particles, and menthol / β-cyclodextrin mixtures. The figures show that the XRD patterns of menthol and β-cyclodextrin have many strong crystal absorption peaks, which are typical crystal structures.

[0062] In the XRD pattern of the menthol / β-cyclodextrin composite particles, the characteristic peak of menthol shifted from 8.22° to 6.7°, from 17.08° to 17.68°, and from 19.34° to 20.24°, and the characteristic peaks at 12.57°, 20.4°, and 38.3° disappeared.

[0063] The XRD spectrum of the menthol / β-cyclodextrin mixture is a simple superposition of the XRD spectra of menthol and β-cyclodextrin, and the elution positions of the characteristic peaks are almost identical to those of menthol and β-cyclodextrin.

[0064] The above results confirm that the menthol / β-cyclodextrin composite particles in Example 1 were successfully prepared, which is of great significance for the subsequent study of their application in Pickering emulsion.

[0065] Example 2

[0066] Preparation of a Pickering emulsion containing menthol.

[0067] Pickering emulsion was prepared using the menthol / β-cyclodextrin composite particle powder obtained in Example 1; the specific method is as follows:

[0068] First, the menthol / β-cyclodextrin composite particle powder obtained in Example 1 was dissolved in water to prepare aqueous solutions of menthol / β-cyclodextrin composite particles with mass volume fractions of 1%, 2%, 3%, 4%, and 5% (w / v). Then, the solution was mixed with soybean oil and homogenized at 20,000 rpm for 3 min to finally obtain a Pickering emulsion containing menthol, wherein soybean oil accounted for 60% of the volume of the Pickering emulsion.

[0069] Meanwhile, a control was prepared using only β-cyclodextrin without the addition of menthol, with the experimental conditions being exactly the same as those for preparing the Pickering emulsion using menthol / β-cyclodextrin composite granules.

[0070] The relevant parameters of Pickering emulsions containing menthol and Pickering emulsions without menthol were detected using aqueous solutions of menthol / β-cyclodextrin composite particles with different mass volume fractions.

[0071] 2.1 Determination of particle size, zeta potential and inclusion fraction of Pickering emulsion.

[0072] Methods for measuring the particle size and zeta potential of Pickering emulsions: The particle size distribution and zeta potential of the prepared Pickering emulsion samples were measured using a Malvern particle size analyzer. Specific results are shown in Table 1 and appendix below. Figure 3 .

[0073] Table 1. Particle size and zeta potential of Pickering emulsion

[0074]

[0075] Appendix Figure 3 In the figure, (a) shows the particle size distribution of Pickering emulsion (Menthol / β-CD composite particles) with different concentrations (or mass-volume fractions) and Pickering emulsion (β-CD) without menthol, and (b) shows the zeta potential distribution of Pickering emulsion with different concentrations and Pickering emulsion (β-CD) without menthol.

[0076] Appendix Figure 3The results show that changes in the crystal structure of the menthol / β-cyclodextrin composite particles affect the interfacial behavior during Pickering emulsion formation. This is because when the concentration of β-cyclodextrin increases to above 3 wt%, the particles adsorbed at the interface are already saturated. However, due to the hydrogen bonding interactions between β-cyclodextrin molecules, excess β-cyclodextrin particles in the system will crystallize and aggregate on the solid shell. When menthol combines with β-cyclodextrin, the hydrogen bonding forces between β-cyclodextrin molecules decrease, preventing this process.

[0077] Figure 3 The particle size of the Pickering emulsion without menthol was significantly smaller than that of the Pickering emulsion prepared from menthol / β-cyclodextrin composite particles. Furthermore, the particle size of the obtained emulsion gradually decreased with increasing concentration of the menthol / β-cyclodextrin composite particle aqueous solution. The absolute value of the zeta potential showed a trend of first increasing and then decreasing. When the concentration of the menthol / β-cyclodextrin composite particle aqueous solution was between 2% and 5%, the absolute value of the zeta potential was greater than 30 mV, indicating good emulsion stability.

[0078] 2.2 Determination of the encapsulation efficiency of Pickering emulsion inclusion complex, the specific method is as follows:

[0079] Referring to and improving upon the "Tobacco Industry Standards of the People's Republic of China," gas chromatography was employed, using a DB-WAX (30m × 250μm × 0.25μm) polyethylene glycol gas capillary column. Column temperature was programmed: initial temperature 70℃, held for 2 min; then increased to 120℃ at a rate of 15℃ / min, held for 3 min; then increased to 160℃ at a rate of 5℃ / min, held for 1 min; finally increased to 240℃ at a rate of 20℃ / min, held for 5 min. A flame ionization detector (FID) was used, with both the injection port and detector temperatures at 250℃. The carrier gas was nitrogen; the flow rate was 1.0 mL / min; the split ratio was 10:1; and the injection volume was 1 μL.

[0080] Preparation of reference solution: Accurately weigh 19.89 mg of menthol reference standard, place it in a 25 mL volumetric flask, dissolve it in ethyl acetate and dilute to the mark, shake well to obtain a reference standard stock solution of 795.60 μg / mL.

[0081] The menthol encapsulation rate was calculated by injection under chromatographic conditions, and the obtained standard curve is shown in the appendix. Figure 4 .

[0082] Accurately weigh 19.89 mg of menthol standard, dissolve and dilute to volume with ethyl acetate to prepare a standard stock solution of 795.60 μg / mL. Pipette 0.5, 1.0, 2.0, 4.0, 5.0, and 8.0 mL of the stock solution into 10 mL volumetric flasks, dilute to the mark with ethyl acetate, and mix well. Perform the determination under the above chromatographic conditions, establish a linear regression equation, and the results are shown in the attached figure. Figure 4 As shown. The regression equation is: y = 0.02253x + 0.47914, R0 2 =0.99972.

[0083] The results showed that the menthol concentration and peak area had a good linear fit in the range of 39.78–795.6 μg / mL.

[0084] The encapsulation efficiency of menthol in the β-cyclodextrin solution was 89.7±2.32%. Excellent menthol encapsulation efficiency is a necessary condition for the menthol / β-CD composite particles to stabilize Pickering emulsion.

[0085] 2.3 Investigation into the stability of Pickering emulsion.

[0086] To further verify the effect of the concentration of the menthol / β-cyclodextrin composite particle aqueous solution prepared in Example 2 of this invention on the stability of the final Pickering emulsion, a stability experiment was conducted on the obtained Pickering emulsion.

[0087] Pickering emulsions stabilized with different concentrations of menthol / β-cyclodextrin composite particles were monitored for 30 days. The changes in the Pickering emulsions over time are shown in the appendix. Figure 5 (a) See Appendix for the variation in particle size. Figure 5 (b) Specific data are shown in Table 2 below.

[0088] Table 2. Variation of particle size (μm) in Pickering emulsion over time (d).

[0089] 0 10 20 30 1％ 13.02±0.35a 19.14±0.52a 28.27±0.44a 37.04±1.28a 2％ 11.59±0.43b 16.35±0.97b 25.72±0.84b 32.85±1.05b 3％ 10.63±0.49c 14.24±0.65c 19.22±0.63c 24.17±0.72c 4％ 9.65±0.53c 13.41±0.67c 17.74±0.94c 22.23±0.96c 5％ 8.47±0.66c 12.27±0.73c 15.94±0.75c 20.34±0.94c

[0090] Figure 5 Figure (a) shows the stability of Pickering emulsion during storage. It was observed that when the concentration of the menthol / β-cyclodextrin composite particle aqueous solution was below 2%, the emulsion exhibited demulsification. This is because when the amount of menthol / β-cyclodextrin composite particles added was too small, the emulsifier particles adsorbed at the oil-water interface were insufficient to form a complete protective shell for the emulsion. Therefore, the particle size of the obtained Pickering emulsion showed a relatively obvious change over time.

[0091] When the concentration of the menthol / β-cyclodextrin composite particle aqueous solution is between 3% and 5%, the emulsification value of the resulting Pickering emulsion is 100%, and the emulsion remains stable during long-term storage. The particle size of the emulsion remains basically unchanged within 30 days.

[0092] Figure 5 (b) The stability of these emulsions over 30 days was also effectively demonstrated, indicating that the Pickering emulsion prepared in this invention when the concentration of the menthol / β-cyclodextrin composite particle aqueous solution is 3-5% has good storage stability.

[0093] 2.4 Microstructural characterization of Pickering emulsion.

[0094] The prepared Pickering emulsion was characterized by optical microscopy, fluorescence microscopy, and scanning electron microscopy, respectively.

[0095] Figure 6 The images show the microstructures of Pickering emulsions obtained from aqueous solutions of menthol / β-cyclodextrin composite particles at different concentrations. (a) to (e) are optical micrographs of Pickering emulsions obtained from aqueous solutions of menthol / β-cyclodextrin composite particles at concentrations of 1–5% (w / v). The microscope magnification is 100x and the scale bar is 10 μm.

[0096] As shown in the figure, when the concentration of the menthol / β-cyclodextrin composite particle aqueous solution is 1%, that is, when the concentration of the menthol / β-cyclodextrin composite particle aqueous solution is 1%, the menthol / β-cyclodextrin composite particle aqueous solution is 1%. Figure 6 (a) The emulsion droplets in the field of view are relatively large in size and few in number. When the concentration of the menthol / β-cyclodextrin complex particle aqueous solution increases from 1% to 3%, the size of the emulsion droplets gradually decreases. When the concentration of the menthol / β-cyclodextrin complex particle aqueous solution increases from 3% to 5%, the droplet size of the emulsion does not change significantly, indicating that the number of complex particles in the Pickering emulsion system has reached saturation. That is, when the concentration of the menthol / β-cyclodextrin complex particle aqueous solution is 3wt%, saturation is reached, and further increasing the concentration of the complex particles will not have a significant impact on the microstructure and stability of the Pickering emulsion.

[0097] In addition, before observing the Pickering emulsion under a fluorescence microscope, the oil phase of the Pickering emulsion and the menthol / β-cyclodextrin complex particles were stained with Nile blue and Nile red, respectively. The oil droplets were stained green and the menthol / β-cyclodextrin complex particles were stained red.

[0098] Figures (a1) to (e1) and (a2) to (e2) are fluorescence micrographs of the Pickering emulsion. Figures (a1) to (e1) show that the internal oil phase of the Pickering emulsion is green, and the size of the oil droplets gradually decreases with increasing concentration of the menthol / β-cyclodextrin composite particle aqueous solution. Figures (a2) to (e2) show that the outer shell of the emulsion droplets composed of menthol / β-cyclodextrin composite particles is stained red. With increasing concentration of the menthol / β-cyclodextrin composite particle aqueous solution, the droplet size of the Pickering emulsion gradually decreases, but the thickness of the droplet shell does not change significantly. This is because the excess menthol / β-cyclodextrin composite particles are mostly uniformly distributed in the aqueous phase of the emulsion, preventing the droplets from approaching each other, enhancing the stability of the emulsion, and avoiding the crystallization and aggregation of molecules at the oil-water interface.

[0099] Further analysis of the particle microstructure was performed using scanning electron microscopy. Figure 6 In the figure, (f), (g), and (h) are scanning electron images of β-cyclodextrin, menthol, and menthol / β-cyclodextrin composite particles, respectively. As can be seen from the figure, the structures of β-cyclodextrin and menthol are lamellar, while the structure of the composite particles changes from an irregular state to a regular state, resulting in individually dispersed, relatively uniform spherical particles with a particle size of about 10 μm. They have good water solubility and strong stability.

[0100] 2.5 Rheological properties characterization of Pickering emulsion.

[0101] The experiment was conducted using an MCR302 rheometer and a PP50 flat plate measurement system with a gap size of 1 mm.

[0102] Strain scanning experiment: frequency 1 Hz, strain range 0.01–100%.

[0103] Frequency scanning experiment: In the linear viscoelastic region, the angular frequency range is 1-10 rad / s, and the frequency range is 0.1-100 rad / s.

[0104] Steady-state shear test: shear rate range 0.1–100 s. -1 .

[0105] Figure 7 Rheological results of Pickering emulsions prepared from aqueous solutions (1–5%, w / v) of menthol / β-cyclodextrin composite particles at different concentrations.

[0106] The rheological properties of Pickering emulsions can provide insights into their stability and microstructure, which is crucial for emulsion applications. Here, G′ represents the energy recovered or stored in the material during each deformation cycle, and G” represents the energy lost through viscous dissipation during each deformation cycle.

[0107] The results show that when the concentration of the menthol / β-cyclodextrin composite particle aqueous solution is 1%, the G' and G” values ​​of the emulsion are both low. When the concentration of the menthol / β-cyclodextrin composite particle aqueous solution increases from 1% to 3%, the G' and G” values ​​of the Pickerling emulsion are much higher than those of 1%, and the storage modulus of the Pickerling emulsion increases rapidly. When the concentration of the menthol / β-cyclodextrin composite particle aqueous solution increases from 3% to 5%, the G' and G” values ​​of the Pickerling emulsions prepared from different concentrations of menthol / β-cyclodextrin composite particles are similar. This is because the adsorption of the composite particles at the interface reaches saturation, and more composite particles are free in the continuous phase.

[0108] The above rheological results indicate that the storage modulus of the Pickering emulsion prepared from menthol / β-cyclodextrin composite particles is higher than that of the loss modulus, and the elastic properties of the Pickering emulsion are dominant, exhibiting viscoelasticity similar to that of a gel.

[0109] Furthermore, as shown in Figure (c), the apparent viscosity (η) of all Pickering emulsions decreases with increasing shear rate, exhibiting typical shear thinning and pseudoplastic non-Newtonian fluid characteristics.

[0110] Example 3

[0111] Performance determination of Pickering emulsion containing menthol.

[0112] 3.1 Determination of the antioxidant capacity of Pickering emulsion containing menthol.

[0113] The antioxidant capacity of menthol-containing Pickering emulsion was evaluated using malondialdehyde (MDA) content. The content of secondary oxide MDA was determined by the thiobarbituric acid method. The absorbance was measured at 600 nm, 532 nm, and 450 nm according to the MDA kit instructions, and the results were calculated.

[0114] The preparation method of Pickering emulsion containing menthol is as follows:

[0115] The menthol / β-cyclodextrin composite particle powder obtained in Example 1 was dissolved in water to prepare an aqueous solution of menthol / β-cyclodextrin composite particles with a mass volume fraction of 3%. Then, it was mixed with soybean oil with a volume fraction of 60% and homogenized at 20,000 rpm for 3 min to finally obtain a Pickering emulsion containing menthol (Composite particles).

[0116] Meanwhile, a menthol-free Pickering emulsion (β-CD) obtained using the exact same method without the addition of menthol was used as a control; soybean oil was used as a blank control.

[0117] Figure 8 Table 3 shows the trends of MDA content (mmol / L) in soybean oil, Pickering emulsion without menthol, and Pickering emulsion with menthol as a function of storage time.

[0118] Table 3. Changes in MDA content with storage time

[0119]

[0120] The results showed that the MDA content of soybean oil increased most significantly over time, followed by Pickering emulsion without menthol. The MDA content of Pickering emulsion stabilized with menthol / β-cyclodextrin complex particles was the lowest. This may be because in the menthol-containing Pickering emulsion, the menthol / β-cyclodextrin complex solid particles adsorbed at the oil-water interface to form a dense protective shell, effectively isolating external oxygen and helping to slow down the oxidation rate of oils in the emulsion.

[0121] 3.2 In vitro digestion characterization of Pickering emulsion containing menthol.

[0122] Simulated oral digestion: Mix the emulsion and simulated oral fluid at a ratio of 10:7 (v / v), dilute with water, adjust the pH to 7.0, and incubate at 100 rpm and 37°C for 5 min in a constant temperature shaker.

[0123] Simulated gastric digestion: The oral digestion sample and simulated gastric juice were mixed at a ratio of 10:7.5 (v / v), and the pH was adjusted to 2.0. The mixture was then incubated at 37°C and 100 rpm for 2 hours in a constant temperature shaker.

[0124] Simulated intestinal digestion: The gastric digested sample and SIF were mixed at a ratio of 2:1 (v / v), the pH was adjusted to 7.0, and the mixture was incubated in a constant-temperature shaker at 100 rpm and 37°C for 2 hours. During the experiment, 0.1M NaOH solution was continuously added to maintain the pH of the mixed solution at 7. The release rate of FFA in the sample was calculated based on the volume of NaOH solution added. The calculation formula is as follows:

[0125]

[0126] In the formula, M trigliycerides V represents the average molecular weight of fat (g / mol); NaOH C represents the volume (mL) of NaOH solution consumed during simulated small intestinal digestion at time t; NaOH The concentration (mol / L) of the standard NaOH solution used in the titration. Wherein, SSF—simulated oral fluid; SGF—simulated gastric fluid; SIF—simulated intestinal fluid.

[0127] Figure 9Table 4 shows the release rates of free fatty acids in a simulated small intestinal solution for soybean oil, Pickering emulsion without menthol, and Pickering emulsion with menthol. The original data are shown in Table 4.

[0128] Table 4. Release rate (%) of free fatty acids from endocritine emulsion in small intestinal solution at different times (min).

[0129]

[0130] The results show that within the first 30 minutes of digestion, the free fatty acid content of soybean oil, pickering emulsion without menthol, and pickering emulsion with menthol all increased rapidly, indicating that the oils could be hydrolyzed more quickly. This is because fats are broken down in the initial stage of digestion, generating a large amount of glycerol and free fatty acids. After 2 hours of digestion, the release rates of free fatty acids among the three showed significant differences, at 96.67±0.69%, 68.06±1.23%, and 76.64±1.08%, respectively.

[0131] The reason for the above phenomenon is that soybean oil cannot achieve a good sustained-release effect in the intestinal fluid environment, and the release rate of free fatty acids is high throughout. The interfacial layer formed by menthol / β-cyclodextrin composite particles has good barrier properties, which can effectively resist and slow down the hydrolysis of digestive enzymes, thereby delaying the decomposition of oil and reducing the release rate of menthol to a certain extent. In contrast, the droplet protective shell of Pickering emulsion obtained without menthol and only protected by β-cyclodextrin is thicker, and the protective shell of some droplets is not completely destroyed. Therefore, the release rate of free fatty acids in Pickering emulsion without menthol is lower.

[0132] The above results indicate that the Pickering emulsion stabilized by menthol / β-cyclodextrin composite particles provides a good sustained-release effect for menthol, which helps reduce the irritation of menthol.

[0133] 3.3 Characterization of the antibacterial properties of Pickering emulsion containing menthol.

[0134] Drug-resistant Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were inoculated into beef extract peptone liquid medium and cultured in shake flasks at 37°C and 200 rpm overnight. The next day, the bacterial suspension was re-inoculated into fresh beef extract peptone liquid medium at a ratio of 1:40 and cultured under the same conditions for about 3 hours. At this point, the bacteria were in the logarithmic phase. The bacterial suspension in the logarithmic phase was then inoculated into fresh beef extract peptone liquid medium at a ratio of 1:40 and cultured on a shaker for 12 hours at 37°C and 200 rpm.

[0135] Among them, the menthol / β-cyclodextrin composite particle-stabilized Pickering emulsion group and the menthol solution group of the same concentration were used as two drug-treated groups. Meanwhile, the menthol-free Pickering emulsion was used as the control group, and the bacterial suspension without any added drugs was used as the blank group.

[0136] The bacterial suspensions of each blank group, control group, and drug-treated group were serially diluted, and 150 μL of each was spread on solid beef extract peptone medium. After incubation in an incubator for 12 h, the number of bacterial colonies on solid plates in different groups was counted by CFU method. The experiment was repeated three times and the average value was taken.

[0137] Figure 10 Table 5 shows plate colony diagrams for antibacterial tests of Escherichia coli and Staphylococcus aureus, respectively. The original data are shown in Table 5.

[0138] Table 5. Plate colony count of Pickering emulsion for antibacterial test

[0139]

[0140] The results showed that the total number of colonies in the Staphylococcus aureus group, the blank group, and the control group was approximately 2.5 × 10⁻⁶. 9 CFU / mL, the total bacterial count in the L-menthol group was 2×10⁻⁶. 8 The total bacterial count in the Pickering emulsion group stabilized with menthol / β-cyclodextrin complex particles was 1×10⁻⁶ CFU / mL. 8 CFU / mL.

[0141] For the Escherichia coli group, the total bacterial count in both the blank group and the control group was 6 × 10⁻⁶. 7 The total bacterial count in the L-menthol group was 1.2 × 10⁻⁶ CFU / mL. 7 The total bacterial count of the Pickering emulsion group stabilized with menthol / β-cyclodextrin complex particles was 6 × 10⁻⁶ CFU / mL. 6 CFU / mL.

[0142] It is evident that the addition of Pickering emulsion without menthol did not affect the total bacterial count. However, the total bacterial count of the L-menthol group was greater than that of the Pickering emulsion group with menthol / β-cyclodextrin complex particles stabilized. This indicates that Pickering emulsion has a protective effect on L-menthol, allowing the antibacterial effect of L-menthol to be fully exerted.

[0143] Example 4

[0144] This embodiment focuses on investigating the effects of different sources and types of oil phases (soybean oil, flaxseed oil, and fish oil) on the storage stability of the prepared Pickering emulsion. The stability of the prepared Pickering emulsion after 30 days of storage is shown in the appendix. Figure 11 As shown.

[0145] The figure shows that the menthol-containing Pickering emulsions prepared using different types of oil phases all exhibit good emulsifying properties and good storage stability. Therefore, different types of oil phase formulations can be selected to prepare the corresponding Pickering emulsions according to the actual application.

[0146] Example 5

[0147] This embodiment focuses on investigating the effect of the concentration of the aqueous solution of menthol / β-cyclodextrin composite particles on the storage stability of the emulsion.

[0148] Figure 12 The stability of Pickering emulsions obtained with different concentrations (1%–5%) of menthol / β-cyclodextrin composite particles in aqueous solution was shown after 30 days of storage.

[0149] The figure shows that the Pickering emulsion prepared when the concentration of the menthol / β-cyclodextrin composite particle aqueous solution is set to 3% exhibits the best stability.

[0150] Example 6

[0151] This embodiment focuses on investigating the effect of the volume fraction of the oil phase (i.e., the oil phase percentage) in the Pickering emulsion on the storage stability of the prepared Pickering emulsion.

[0152] This embodiment measured the stability of Pickering emulsions prepared from soybean oil with different oil phase ratios (40%, 50%, 60%, 70%, and 80%). The stability of Pickering emulsions obtained with different oil-water ratios after 30 days of storage is shown in the attached figure. Figure 13 As shown.

[0153] The figure shows that when the oil phase content is 40% and 50%, the aqueous phase of the emulsion precipitates during storage, and stratification can be observed. When the oil phase content of the emulsion increases to 60%, the emulsification value of the emulsion is 100%, exhibiting good storage stability. This is because as the oil phase volume increases, water molecules gradually participate in the emulsification process, and no excess water molecules precipitate. Therefore, the emulsification value of the emulsion gradually increases, and the stability remains good with no further stratification. When the oil phase volume increases from 60% to 80%, a large amount of oil phase precipitates in the upper layer of the Pickering emulsion, resulting in a phase inversion from O / W type to W / O type emulsion. This is because when the oil phase volume is too high, due to the low density of oil, the particles in the emulsion system cannot prevent excessive oil molecules from floating to the surface.

[0154] Example 7

[0155] This embodiment focuses on investigating the effect of homogenization rate on the stability of Pickering emulsion.

[0156] Pickering emulsions were obtained by homogenization at different homogenization rates (15000 r / min, 18000 r / min, 20000 r / min, 22000 r / min, and 25000 r / min). The stability of the Pickering emulsions obtained at different homogenization rates is shown in the attached figure. Figure 14 As shown.

[0157] The results show that as the homogenization rate increases, the emulsification value of the Pickering emulsion increases from 81.74% to 84.94%. The emulsified phase increases and the precipitated aqueous phase decreases. This is because homogenization can evenly disperse oil droplets in the aqueous phase. When the homogenization rate is too low, solid particles cannot effectively reach the oil-water interface to form a solid shell. Finally, when the homogenization rate reaches 20,000 rpm / min, the emulsification value of the emulsion is 100%, exhibiting good stability during long-term storage. Furthermore, as the homogenization rate increases, the emulsion no longer undergoes significant changes.

[0158] Example 8

[0159] This embodiment focuses on investigating the effect of homogenization time on the stability of Pickering emulsion.

[0160] Pickering emulsions were obtained by homogenization at different times (1 min, 2 min, 3 min, 4 min, and 5 min). The stability of the Pickering emulsions obtained at different homogenization times is shown in the attached figure. Figure 15 As shown.

[0161] The figure shows that when the homogenization time is less than 3 minutes, the emulsification value of the Pickering emulsion does not reach 100%, and the aqueous phase precipitates and separates during storage, resulting in poor storage stability. This is because the homogenization time is insufficient to ensure that the solid particles of the emulsion are stable at the oil-water interface, and the aqueous phase gradually precipitates and separates. As the homogenization time increases, when the homogenization time is between 3 and 5 minutes, the emulsification value of the emulsion is 100%. This indicates that when the homogenization time is 3 minutes, the composite particles adsorbed at the oil-water interface are relatively saturated, resulting in a higher emulsification value and stronger stability of the emulsion.

[0162] An orthogonal experiment was conducted on the factors affecting the stability of Pickering emulsion. The results and analysis of the orthogonal experiment are shown in Tables 6 and 7 below.

[0163] Table 6 Orthogonal Experiment Parameters

[0164]

[0165] Table 7 Results of the orthogonal experiment

[0166]

[0167]

[0168] The results above show that the priority order of factors affecting the emulsification value of Pickering emulsions is C>B>A>D, i.e., homogenization rate > oil phase ratio > particle concentration > homogenization time. The optimal combination of factors obtained from the orthogonal experiment was an oil phase ratio of 60%, a composite particle aqueous solution concentration of 3%, a homogenization rate of 20,000 rpm / min, and a homogenization time of 3 min. Pickering emulsions prepared under these conditions, repeated three times, remained stable during long-term storage.

Claims

1. A method for preparing a menthol-containing Pickering emulsion, characterized in that, The Pickering emulsion is prepared using menthol and β-cyclodextrin composite particles as solid particle emulsifiers and soybean oil as the oil phase. The method for preparing the menthol-containing Pickering emulsion is as follows: S1, Preparation of menthol and β-cyclodextrin composite particles Take 10g of β-cyclodextrin and disperse it in 80g of distilled water at 45℃ to make a suspension. Then, while stirring, slowly add 3g of menthol. After the menthol is added, continue stirring and react at 45℃ for 3h. The obtained suspension is stored in a refrigerator at 5℃ for 24h and allowed to stand to precipitate. Filter, wash the precipitate with anhydrous ethanol, and finally freeze-dry to obtain a composite granular powder of menthol and β-cyclodextrin. S2, Preparation of Pickering Emulsion The menthol and β-cyclodextrin composite particle powder obtained in S1 was dissolved in water to prepare an aqueous solution of menthol and β-cyclodextrin composite particles with a mass volume fraction of 3%. Then, it was mixed with soybean oil with a volume fraction of 60% and homogenized at 20,000 rpm for 3 min to finally obtain a Pickering emulsion containing menthol. The Pickering emulsion allows the antibacterial effect of menthol to be fully exerted, and also has a good sustained-release effect on menthol. After 2 hours of simulated in vitro digestion, the free fatty acid release rate is 76.64%.

Images