Seville orange flower Pickering emulsion as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of food industry, and particularly relates to seville orange flower Pickering emulsion as well as a preparation method and application thereof. The seville orange flower Pickering emulsion which has good stability under the conditions of extreme pH, high ionic strength, temperature fluctuation and the like is prepared by taking nano esterified starch as solid colloidal particles and taking a mixed oil phase of seville orange flower essential oil and carrier oil as a dispersion phase. Performance test results show that when the emulsion is heated at 80 DEG C for 1 h, the particle size of the emulsion is basically kept unchanged, the polydispersity index (PDI) is slightly reduced or kept stable, and the zeta potential is slightly reduced, which indicates that the emulsion has good thermal stability; under the strongly alkaline condition that the pH is 11, the particle size and zeta potential of the emulsion are slightly changed, and only the polydispersity index is slightly increased; in a high-concentration NaCl environment, various parameters of the emulsion are not obviously changed, and the emulsion shows relatively high ion interference resistance.

Description

Technical Field

[0001] The present application belongs to the technical field of food industry, and specifically relates to a Daisy Pickering emulsion with water as a continuous phase, Daisy essential oil as a dispersed phase, and esterified modified starch as solid particles. Background Art

[0002] As an important medicinal and edible plant resource, the essential oil extracted from daisy flower is rich in a variety of compounds with significant antibacterial and antioxidant activities. These natural active ingredients give it broad application prospects in the development of functional foods, natural preservatives, and cosmetics. However, the inherent high volatility, low water solubility, and instability of daisy flower essential oil during processing and storage seriously affect the maintenance and effective utilization of its antibacterial and other biological activities. Therefore, the development of a stable emulsion delivery system is of vital importance for protecting the active ingredients of daisy flower essential oil and improving its dispersibility and bioavailability in the application system.

[0003] Currently, the most widely used emulsification technologies in the food industry include microemulsions, Pickering emulsification, high-speed shear dispersion emulsification, and membrane emulsification. Pickering emulsification is widely used in food emulsification due to its excellent compatibility with food matrices, nutrient protection, and controlled release. The key difference between Pickering emulsions and traditional emulsions is that the former uses solid colloidal particles as emulsion stabilizers, while the latter uses surfactants. Therefore, the stability of Pickering emulsions is highly dependent on the type and properties of the solid colloidal particles. During use, the stabilization mechanism of solid colloidal particles lies in the fact that the dispersed solid particles adsorb at the oil / water interface, forming a physical barrier that prevents aggregation of emulsion droplets and slows emulsification. To obtain Pickering emulsions of varying types, properties, and requirements, it is essential to select the appropriate solid colloidal particles. Food-grade solid particles used to stabilize Pickering emulsions fall into six main categories: polysaccharide particles, protein-based particles, flavonoid particles, composite particles, food-grade waxes, and fatty acids.

[0004] To address the poor water solubility of daikon radish and the volatility of its active ingredients, patent application number 202410208301.7 uses Pickering emulsification technology to encapsulate daikon radish essential oil by constructing a Pickering emulsion system using wheat alcohol-soluble protein powder as protein nanoparticles, effectively solving the technical problem of the volatility of daikon radish essential oil. However, the applicant discovered that this Pickering emulsion had poor physical stability and low stability in complex food systems such as those with extreme pH, high ionic strength, and temperature fluctuations. It was prone to aggregation, stratification, or leakage of active ingredients, limiting the application of daikon radish as a highly effective antimicrobial delivery system. Summary of the Invention

[0005] The present application provides a daikon radish Pickering emulsion that has good stability under extreme pH, high ionic strength, and temperature fluctuations, which is specifically achieved through the following technical solutions: A method for preparing a daisy flower Pickering emulsion comprises the following steps: using nano-esterified starch as solid colloidal particles, using a mixed oil phase of daisy flower essential oil and a carrier oil as a dispersed phase, and using water as a continuous phase; and subjecting the mixture to homogenized ultrasonic treatment, so that the solid colloidal particles are coated on the surface of oil droplets of the mixed oil phase, thereby obtaining the daisy flower Pickering emulsion; in the daisy flower Pickering emulsion, the mass volume concentration of the solid colloidal particles is 3-8%, the mass volume concentration of the daisy flower essential oil is 1-3%, and the mass volume concentration of the carrier oil is 4-6%.

[0006] Preferably, the preparation step of the nano-esterified starch comprises: esterifying the refined starch with 2-octenylsuccinic anhydride under alkaline conditions to introduce amphiphilic groups on the surface of the refined starch to obtain esterified starch.

[0007] Preferably, the preparation step of the nano-esterified starch further comprises: gelatinizing the suspension of the esterified starch, cooling the suspension to make the system temperature lower than the gelatinization temperature to promote recrystallization of the esterified starch, and performing ultrasonic crushing to obtain the nano-esterified starch.

[0008] Preferably, the refined starch is obtained by removing protein from crude starch through alkali washing, and the content of amylopectin in the crude starch is higher than that of amylose.

[0009] A daisy flower Pickering emulsion is prepared by using any one of the preparation methods.

[0010] Preferably, when the pH value of the Daidaihua Pickering emulsion is 11, the particle size is 280-310 nm, the polydispersity index is 0.25-0.35, and the potential is -30--40.

[0011] Preferably, when the Daidaihua Pickering emulsion is heated at 80° C. for 1 hour, its particle size is less than 300 nm, the polydispersity index is ≤0.2, and the potential is -28 to -33.

[0012] Preferably, when the NaCl concentration of the Daidaihua Pickering emulsion is 200 mM, the particle size is 350-400 nm, the polydispersity index is ≤0.2, and the potential is -10--5.

[0013] Preferably, the particle size of the Daidaihua Pickering emulsion is 200-230 nm, the polydispersity index is 0.1-0.2, and the potential is -30--40.

[0014] Application of the above-mentioned Daidaihua Pickering emulsion in the preparation of food and medicine.

[0015] Compared with the prior art, this application has the following beneficial effects: This application uses nano-esterified starch as solid colloidal particles and a mixture of daisy essential oil and carrier oil as the dispersed phase to prepare a daisy Pickering emulsion that exhibits excellent stability under extreme pH, high ionic strength, and temperature fluctuations. Performance testing results show that after heating at 80°C for 1 hour, the emulsion's particle size remains essentially unchanged, the polydispersity index (PDI) decreases slightly or remains stable, and the potential decreases only slightly, demonstrating excellent thermal stability. Under strongly alkaline conditions of pH 11, the emulsion's particle size and potential change minimally, with only a slight increase in the polydispersity index. Under strongly acidic conditions of pH 3, despite a slight increase in particle size, potential, and polydispersity index, the oil phase remains uniformly dispersed throughout the emulsion. In a high NaCl concentration, all emulsion parameters remain unchanged, demonstrating strong resistance to ionic interference. In contrast, a control emulsion using standard esterified starch as a stabilizer exhibits significant instability under the same conditions. For example, after heating at 80°C for 1 hour, its particle size and polydispersity index are nearly twice that before heating, and the potential drops significantly, indicating that the emulsion structure has obviously aggregated and the thermal stability is poor. Under the strong acidic condition of pH 3, the emulsion in the control group quickly showed particle aggregation and droplet merging, accompanied by obvious stratification, and the emulsion stability decreased significantly. This further shows that the nano-esterified starch used in this application has better interfacial stabilization ability and environmental tolerance, and can effectively improve the overall stability of the Pickering emulsion. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] To clearly introduce the embodiments, the following briefly introduces the drawings: Figure 1 Particle size (a), polydispersity index (b), and ζ-potential (c) of the Pickering emulsion of daidaihua; Figure 2 This is a laser confocal scanning microscope image of the Pickering emulsion of Dai Dai Hua; Figure 3 This is a transmission electron micrograph of Pickering emulsion of Daidaihua; Figure 4 Type test chart for Daidaihua Pickering Lotion; Figure 5 Storage stability graphs of Pickering emulsions of P. daiquiri (particle size (a), PDI (b), and ζ-potential (c)); Figure 6 This is a picture of the physical appearance of Dai Dai Hua Pickering Emulsion when stored; Figure 7 Thermal stability diagram of Pickering emulsion of Daphne daphne (particle size (a), PDI (b) and ζ-potential (c)); Figure 8 pH stability diagram of Pickering emulsion of P. daidaihua (particle size (a), PDI (b) and ζ-potential (c)); Figure 9 The physical appearance of Pickering emulsion of Daidaihua at different pH values ​​is shown; Figure 10 Storage stability graphs of the Pickering emulsion of daidaihua at high ionic strength (particle size (a), PDI (b) and ζ-potential (c)); Figure 11 This is a diagram of the physical appearance of Dai Dai Hua Pickering emulsion stored at high ionic strength; Figure 12 This is a graph of the emulsion encapsulation efficiency of the Daidaihua Pickering emulsion; Figure 13 This is a graph showing the antimicrobial activity of Daidaihua Pickering emulsion; Figure 14 Graph showing the antioxidant activity of Pickering's emulsion of Datura strychnifolia. DETAILED DESCRIPTION

[0017] The present application will be further described below in the form of specific embodiments. Those skilled in the art will be able to implement the present application based on these descriptions. In addition, the embodiments of the present application involved in the following description are generally only embodiments of a portion of the present application, rather than all embodiments. Therefore, based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making any creative efforts should fall within the scope of protection of the present application.

[0018] In the following examples, the unit of mass volume concentration is g / mL. Example 1: Preparation of Daidaihua Pickering Emulsion

[0019] The preparation of the Daidaihua Pickering emulsion of this embodiment comprises the following steps: Crude starch was dissolved in water at a ratio of 1:5 (w / v) to form a suspension. The pH of the starch slurry was adjusted to 9.0 using 1 M NaOH, then gently stirred for 30 minutes and allowed to stand overnight. The suspension, which had been allowed to stand overnight, was neutralized to a pH of 7.0 using 1 M HCl. The suspension was then washed three times with deionized water to remove residual NaCl. The washed starch was dried in an oven at 45°C for 24 hours. After drying, it was pulverized into a powder and passed through a 100-mesh sieve for storage for further use. The resulting product is referred to as refined starch.

[0020] Refined starch was dispersed in distilled water to prepare a 10% (w / v) starch suspension. The suspension was continuously stirred in a 35°C water bath while adjusting the pH to 8.5. 2-Octenylsuccinic anhydride (v / v = 0.2) diluted in anhydrous ethanol was slowly added in multiple additions over 2 hours, maintaining a constant pH of 8.5. The reaction was terminated by adjusting the pH to 6.5 with 1 M HCl. Subsequently, esterified sweet potato starch was obtained by centrifugation at 2,000 rpm for 5 minutes. The obtained esterified sweet potato starch was washed three times with deionized water and three times with 70% ethanol. The starch was freeze-dried and stored until further use.

[0021] Esterified sweet potato starch was prepared into a 2% (w / v) starch suspension and maintained in a boiling water bath at 100°C for 30 minutes for complete gelatinization. After cooling the starch paste to 50°C, it was sonicated for 10 minutes using an ultrasonic disruptor. Ultrasonication was performed at an amplitude of 80%, with an on-time of 5 seconds and a rest time of 3 seconds. Immediately after sonication, the starch slurry was dropwise added to anhydrous ethanol and rapidly precipitated under magnetic stirring to re-extract the starch. The precipitate, obtained by centrifugation at 2,000 rpm for 5 minutes at 4°C, was nano-esterified sweet potato starch. The nano-esterified sweet potato starch was then freeze-dried in a freeze dryer.

[0022] Daisy flower essential oil was completely dissolved in 5% (w / v) soybean oil at concentrations of 1%, 2%, and 3% (w / v), respectively, as the oil phase. This oil phase was mixed with deionized water (as the aqueous phase) and 5% (w / v) nanoesterified sweet potato starch was added. The mixture was homogenized at 10,000 rpm for 3 minutes. The homogenized mixture was then sonicated at 480 W for 15 minutes. Homogenization was performed in an ice bath to obtain a uniform and stable nanoemulsion and minimize the adverse effects of high temperature on emulsion stability and the activity of the essential oil.

[0023] Through different steps, S (refined raw sweet potato starch), OS (esterified sweet potato starch), NS (nano-sweet potato starch), and ONS (nano-esterified sweet potato starch) were prepared. They were loaded with 1%, 2%, and 3% (w / v) of Datura essential oil, respectively. Table 1 shows the different formulations for preparing the emulsions.

[0024] Table 1. Composition and addition ratio Example 2: Preparation of Daidaihua Pickering Emulsion

[0025] The difference between this embodiment and embodiment 1 is that in the Daisy Pickering emulsion, the mass volume concentration of the solid colloidal particles is 3%, the mass volume concentration of the Daisy essential oil is 1%, and the mass volume concentration of the carrier oil is 4%. Example 3: Preparation of Daidaihua Pickering Emulsion

[0026] The difference between this embodiment and embodiment 1 is that in the Daisy Pickering emulsion, the mass volume concentration of the solid colloidal particles is 8%, the mass volume concentration of the Daisy essential oil is 3%, and the mass volume concentration of the carrier oil is 6%. Performance test 1. Particle size and potential determination of emulsion

[0027] The particle size, PDI and ζ-potential of the emulsion were measured by Malvern particle size analyzer ( Figure 1 ).

[0028] See also Figure 1 , it was found that the droplet size of the prepared Daphne daphne Pickering emulsions was all at the nanometer level (Z-average <500 nm). With increasing Daphne daphne essential oil content, the droplet size and PDI increased, while the ζ-potential decreased. Compared with the OS emulsion, the ONS emulsion had smaller droplet size and lower PDI (p < 0.05), indicating better system homogeneity. Among them, the 1% ONS-P emulsion had the smallest average particle size (240.87 nm) and the lowest PDI (0.14). The absolute values ​​of the ζ-potential of all emulsions were above 30 mV, indicating sufficient electrostatic stability; in particular, the 1% ONS-P emulsion had the highest absolute value of ζ-potential (37.60 mV), indicating stronger electrostatic repulsion and excellent emulsification stability. Performance test 2, emulsion microstructure analysis

[0029] Transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) were used to observe the microstructure of the emulsion. Figure 2 and 3 ).

[0030] CLSM observations showed that the emulsion droplet size gradually increased with increasing amounts of daisy essential oil. The 3% OS-P emulsion showed noticeable droplet aggregation and uneven distribution, likely due to insufficient interfacial electrostatic repulsion. In contrast, the 3% ONS-P emulsion exhibited smaller droplets and a more uniform distribution, demonstrating excellent dispersion stability. TEM observations further confirmed that the daisy Pickering emulsion droplets exhibited a uniformly dispersed spherical structure. The emulsion system prepared with ONS (i.e., OSNP) exhibited smaller droplet size, more uniform dispersion, and greater stability compared to the OS system. This is primarily attributed to the uniform distribution of ONS at the oil-water interface and the strong interfacial film formed by the highly substituted OSA lipophilic groups, which effectively inhibited droplet aggregation, successfully encapsulating the organic phase and providing an ideal carrier for the stable storage of active ingredients. Performance test 3, emulsion type test

[0031] The nanoemulsion was determined to be of the type using the dropping method. Figure 4 ).

[0032] Testing revealed that all prepared daisy flower Pickering emulsions were oil-in-water (O / W) emulsions. The emulsion droplets dispersed evenly in deionized water but agglomerated in soybean oil, demonstrating good water compatibility. Furthermore, the nanoparticles effectively stabilized the oil-water interface, forming a stable Pickering emulsion system. Performance test 4, storage stability test

[0033] The emulsion was placed in a sealed container and stored in the dark at room temperature (about 25±1°C) for 28 days. The appearance of the emulsion was observed regularly (0 d, 7 d, 14 d, 21 d, 28 d). Figure 5 ).

[0034] The results showed that all ONS-stabilized emulsions showed no de-oiling during the 28-day storage period, demonstrating excellent storage stability. For example, the droplet size of 1% ONS-P increased only slightly from the initial 240.87 nm to 258.83 nm, the PDI increased slightly to 0.19, and the ζ-potential remained below -30 mV, indicating high electrostatic stability. In contrast, the OS-stabilized emulsion showed significant creaming and precipitation after 28 days of storage, with significant phase separation (see Figure 6 This further confirms that ONS can achieve long-term and stable encapsulation of daisy flower essential oil. Performance test 5. Thermal stability test

[0035] The freshly prepared emulsions were heated at 30 °C, 40 °C, 60 °C and 80 °C for 1 h, and then their particle size, PDI and ζ-potential ( Figure 7 ).

[0036] Experimental results show that the droplet size, PDI, and ζ-potential of ONS-stabilized emulsions remained relatively stable during heating from 20°C to 80°C, demonstrating excellent thermal stability. Although the droplet size and PDI of OS-stabilized emulsions increased at elevated temperatures, none of the emulsions showed significant delamination or instability after heating. This demonstrates that ONS effectively protects the oil phase from heat-induced aggregation and has promising potential for high-temperature applications. Performance Test 6: pH Stability

[0037] The pH of the freshly prepared emulsions was adjusted to 3, 5, 7, 9, and 11 using 1 M HCl or 1 M NaOH, and then their particle size, PDI, and ζ-potential were measured ( Figure 8 ).

[0038] The results showed that ONS stabilized emulsions exhibited good visual stability over a wide pH range of 3-11, with minimal changes in particle size, PDI, and ζ-potential, indicating excellent pH stability. In contrast, OS stabilized emulsions exhibited significant phase separation, flocculation, and de-oiling under strongly acidic conditions (pH 3 and 5), indicating poor stability ( Figure 9 ). This indicates that ONS can effectively resist the adverse effects of different pH environments. Performance Test 7: Ion Concentration Stability

[0039] Different concentrations of NaCl (50 mM, 100 mM, 150 mM and 200 mM) were added to the emulsion and allowed to stand for 24 h. The particle size, PDI and ζ-potential ( Figure 11 ).

[0040] The study found that when the NaCl concentration increased from 50 mM to 200 mM, the droplet size of the ONS-stabilized emulsion only increased slightly, with no significant stratification or de-oiling, demonstrating excellent salt ion stability. In contrast, the OS-stabilized emulsion exhibited significant phase separation, de-oiling, and starch granule precipitation at the same salt concentration. This suggests that ONS can more effectively resist the adverse effects of high-salt environments on emulsion stability, further supporting its potential for long-term stable encapsulation of active ingredients. Performance Test 8. Emulsion Encapsulation Efficiency Determination

[0041] The encapsulation efficiency was measured by UV spectrophotometer at a wavelength of 340 nm.

[0042] The Daidahlia Pickering emulsions prepared by ONS-P showed excellent encapsulation efficiency, with values ​​ranging from 79.33% to 89.00% ( Figure 12This demonstrates ONSP's ability to efficiently encapsulate active ingredients. However, as the amount of daisy essential oil increased, the EE decreased. In particular, at a 3% daisy essential oil loading, the EE of ONS-P (79.67%) was significantly higher than that of OS emulsion (67.33%, p < 0.05). This demonstrates the significant advantages of ONSP in terms of stability and active ingredient encapsulation. Performance Test 9. Determination of Antibacterial Activity

[0043] The antibacterial properties of the emulsion were investigated using the double-layer plate method ( Figure 13 ).

[0044] Pickering emulsions of daisy flower exhibited significant antibacterial activity, with varying susceptibility to different bacteria. The antibacterial effect increased with increasing essential oil concentration. In particular, the inhibition zone diameter of 3% ONS-P against Staphylococcus aureus reached 6.2 mm, significantly exceeding the maximum inhibition zone of EO-OS-P (4.7 mm), confirming the ability of ONS to enhance antibacterial activity by loading higher amounts of essential oil. However, the antibacterial effect against Escherichia coli was relatively weak. The lipophilic active ingredients in daisy flower essential oil exert their antibacterial effects by disrupting bacterial cell membranes, suggesting its broad application prospects in areas such as food preservation. Performance test 10, antioxidant activity determination

[0045] This performance test evaluated the antioxidant activity of the emulsion using the DPPH free radical scavenging method and the ABTS free radical scavenging method. Specifically, the DPPH free radical scavenging method involves mixing a freshly prepared 0.2 mmol / L DPPH ethanol solution with the emulsion and allowing the reaction to proceed for 30 minutes in the dark to ensure complete reaction. Subsequently, the absorbance at 517 nm was measured. The ABTS free radical scavenging method involves mixing a 7 mM ABTS solution with a 2.45 mM potassium persulfate solution in a 1:1 (v / v) ratio and allowing the reaction to proceed for 12 hours in the dark. Subsequently, the mixture is diluted with ethanol until its absorbance at 734 nm reaches 0.70 ± 0.02. The emulsion is then mixed with the ABTS working solution. The reaction is carried out at 25°C in the dark for 20 minutes, and the UV absorbance at 734 nm is recorded.

[0046] Daiyu Pickering Lotion can effectively preserve the antioxidant properties of Daiyu essential oil ( Figure 14The results showed that their scavenging abilities for both DPPH and ABTS free radicals were dose-dependent, increasing with increasing essential oil content. Specifically, 3% ONS-P achieved a DPPH free radical scavenging rate of 73.09% (p<0.05), significantly higher than the 62.77% achieved by 3% OS-P. Furthermore, the scavenging rates for ABTS free radicals exceeded 70%. This further confirms that OSNP-based Pickering nanoemulsions exhibit superior free radical scavenging ability when loaded with higher amounts of daisy flower essential oil, suggesting their potential for future applications.

Claims

1. A method for preparing a Daidaihua Pickering emulsion, characterized in that: Nano-esterified starch is used as solid colloidal particles, a mixed oil phase of daisy flower essential oil and carrier oil is used as a dispersed phase, and water is used as a continuous phase. Homogenization and ultrasonic treatment are performed so that the solid colloidal particles are coated on the surface of the oil droplets of the mixed oil phase to obtain a daisy flower Pickering emulsion; in the daisy flower Pickering emulsion, the mass volume concentration of the solid colloidal particles is 3-8%, the mass volume concentration of the daisy flower essential oil is 1-3%, and the mass volume concentration of the carrier oil is 4-6%.

2. The method for preparing the Pickering emulsion according to claim 1, wherein: The preparation steps of the nano-esterified starch include: esterifying refined starch with 2-octenylsuccinic anhydride under alkaline conditions to introduce amphiphilic groups on the surface of the refined starch to obtain esterified starch.

3. The method for preparing the Pickering emulsion according to claim 2, wherein: The preparation step of the nano-esterified starch further includes: gelatinizing the suspension of the esterified starch, cooling the suspension to make the system temperature lower than the gelatinization temperature, promoting the recrystallization of the esterified starch, and performing ultrasonic crushing to obtain the nano-esterified starch.

4. The method for preparing the Daidaihua Pickering emulsion according to claim 2 or 3, characterized in that: The refined starch is obtained by removing protein from crude starch through alkali washing, and the content of amylopectin in the crude starch is higher than that of amylose.

5. A Daidaihua Pickering emulsion, characterized in that The Daidaihua Pickering emulsion is prepared by the preparation method according to any one of claims 1 to 5.

6. The Pickering emulsion according to claim 5, characterized in that When the pH value of the Daidahlia Pickering emulsion is 11, the particle size is 280-310 nm, the polydispersity index is 0.25-0.35, and the potential is -30--40.

7. A Daidaihua Pickering emulsion according to claim 5 or 6, characterized in that: When the daisy flower Pickering emulsion is heated at 80° C. for 1 hour, its particle size is less than 300 nm, its polydispersity index is less than or equal to 0.2, and its potential is between -28 and -33.

8. The Pickering emulsion of claim 7, characterized in that: When the NaCl concentration of the Daidaihua Pickering emulsion is 200 mM, the particle size is 350-400 nm, the polydispersity index is ≤0.2, and the potential is -10--5.

9. The Pickering emulsion of claim 5, characterized in that: The particle size of the Pickering emulsion is 200-230 nm, the polydispersity index is 0.1-0.2, and the potential is -30--40.

10. Use of the Daidaihua Pickering emulsion according to any one of claims 5 to 9 in the preparation of food and medicine.

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