Preparation method of origanum vulgare essential oil loaded shellac-sodium caseinate composite nanoparticles
Shellac-sodium caseinate composite nanoparticles were prepared by antisolvent precipitation and sodium caseinate modification, which solved the problems of low stability and encapsulation efficiency of oregano essential oil and achieved efficient and stable loading and delivery of hydrophobic bioactive substances.
Patent Information
- Application Number
- CN202511433699.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-09
- Publication Date
- 2025-12-19
AI Technical Summary
Oregano essential oil is difficult to use effectively in food due to its high volatility and poor stability. Single shellac nanoparticles tend to aggregate in the acidic environment of the stomach and have low encapsulation efficiency, so it is necessary to improve their stability and encapsulation efficiency under acidic conditions.
Oregano essential oil was loaded into shellac using an antisolvent precipitation method, and the shellac surface was modified with sodium caseinate to form shellac-sodium caseinate composite nanoparticles, which improved encapsulation efficiency and stability.
The prepared composite nanoparticles have uniform particle size, good stability in acidic environment, enhanced antioxidant and antibacterial properties, and improved encapsulation efficiency by 24.46%. The materials are safe and environmentally friendly, and the method is simple and easy to operate.
Smart Images
Figure CN121154581A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of preparation technology of functional composite nanoparticles that can be used to load essential oils, and specifically to a shellac-sodium caseinate composite nanoparticle loaded with oregano essential oil and its preparation method. Background Technology
[0002] Oregano essential oil (OEO) is a plant essential oil extracted from oregano (oregano herb) of the Lamiaceae family. It is typically a pale yellow liquid and a promising natural bioactive compound. Its main components are carvacrol and thymol, exhibiting significant antibacterial, antioxidant, and anti-inflammatory activities. However, oregano essential oil suffers from high volatility, poor stability, easy oxidation, and difficulty in preservation, limiting its direct application in food. Nanoencapsulation offers advantages such as simple preparation processes, good biocompatibility, and high drug loading capacity. It protects the essential oil from the effects of air, light, heat, and humidity, improves its solubility in water, enhances its physical and chemical stability, and prevents adverse reactions such as volatilization or oxidation. Therefore, nanoencapsulation technology is an effective and feasible method.
[0003] Shellac, a novel delivery material, is a natural resin secreted by female lacquer beetles on trees and is an animal-derived natural biopolymer. Due to its excellent biocompatibility, it can be used in the biomedical and food industries to manufacture nanoparticles. Shellac's unique molecular structure consists of polyhydroxy aliphatic and cyclic terpene acids linked by ester bonds, giving the compound both hydrophobic and hydrophilic properties. This dual nature allows shellac to aggregate under acidic conditions and dissolve only in alkaline solutions or lipophilic solvents, making it possible to form food-grade nanoparticles via antisolvent precipitation to protect and deliver bioactive substances, thus making it an ideal material for encapsulating and transporting hydrophobic substances. Although shellac has the potential as a nanocarrier, single shellac nanoparticles still have some limitations as a delivery carrier for hydrophobic bioactive substances: the encapsulation efficiency and redispersibility of shellac nanoparticles are generally low, and they are prone to irreversible aggregation under conditions such as gastric acid (pH 1.2), which limits its application as an ideal carrier. However, this can be overcome by adding stabilizers.
[0004] Protein modification strategies are an excellent choice for adjusting the dispersibility and stability of drug loading systems. Sodium caseinate, prepared by neutralizing casein precipitated from skim milk with NaOH and then spray drying, is an amphiphilic, water-soluble protein complex. Due to its non-toxic, natural, and mild properties, sodium caseinate is often used to design nanoparticle delivery systems with good dispersibility and stability. Surface modification with sodium caseinate significantly affects the high salt sensitivity, low redispersibility, acid instability, and improved encapsulation efficiency of oregano essential oil in shellac nanoparticles. Therefore, developing and exploring sodium caseinate-modified shellac nanoparticles loaded with oregano essential oil to further improve their encapsulation efficiency and stability when loaded with essential oil is of great significance for expanding the delivery of hydrophobic bioactive substances. Summary of the Invention
[0005] The purpose of this invention is to provide a method for preparing shellac-sodium caseinate composite nanoparticles loaded with oregano essential oil. The method utilizes an antisolvent precipitation method to load oregano essential oil into shellac, and employs a protein modification strategy to modify the shellac surface with sodium caseinate, thereby obtaining shellac-sodium caseinate composite nanoparticles loaded with oregano essential oil. The prepared composite nanoparticles have a uniform particle size distribution, high encapsulation efficiency for oregano essential oil, good stability under acidic conditions, and stronger antioxidant and antibacterial properties. This invention provides a novel carrier material for encapsulating and delivering hydrophobic bioactive substances in related technical fields.
[0006] To achieve the above objectives, the following steps are included:
[0007] (1) Weigh 2g of shellac and dissolve it in 50mL of ethanol solution. Use sonication to completely dissolve it and change the water in time to prevent the temperature from getting too high.
[0008] (2) Centrifuge the solution obtained in step (1) at 4000 rpm / min and 25°C for 10 min to remove undissolved shellac.
[0009] (3) Add a certain amount of oregano essential oil and Tween 80 to the solution obtained in step (2) and stir continuously to mix them thoroughly.
[0010] (4) Dissolve sodium caseinate of different masses in 100 mL of deionized water, stir thoroughly to ensure complete dissolution, and prepare sodium caseinate solutions of different concentrations.
[0011] (5) Using a syringe, add 10 mL of oregano oil / shellac solution obtained in step (3) dropwise to a series of sodium caseinate solutions of different concentrations obtained in step (4). Stir magnetically for 40 min at 600 rpm. Use a rotary evaporator to evaporate the ethanol in the sample at 40°C and dilute to 100 mL with deionized water.
[0012] (6) The solution obtained in step (5) is centrifuged at 1500 rpm / min and 25°C for 10 min to remove the larger shellac aggregates, thus obtaining an aqueous dispersion of shellac-sodium caseinate composite nanoparticles loaded with oregano essential oil. The composite nanoparticles are then freeze-dried.
[0013] The present invention also includes the following features:
[0014] The ethanol solution in step (1) is anhydrous ethanol, and the ultrasonic time is 40 min.
[0015] In step (3), the amounts of oregano oil and Tween 80 added were 0.5g and 2.86g, respectively, with mass ratios of 1:4 and 10:7 to shellac, and the stirring time was 2h.
[0016] In step (4), the mass of sodium caseinate is 0.05g, 0.1g, 0.15g, and 0.2g, respectively, the concentration is 0.05%-0.2% (w / v), and the stirring time is 2h.
[0017] The syringe used in step (5) has an inner diameter of 0.42 mm.
[0018] The beneficial effects of this invention are:
[0019] 1. The shellac used in this invention is a natural biopolymer with good biocompatibility, is biodegradable, and is non-toxic.
[0020] 2. The sodium caseinate used in this invention is an amphiphilic water-soluble protein complex. Due to its non-toxic, natural and mild properties, sodium caseinate surface modification can improve the high salt sensitivity, low redispersibility and acid instability of shellac nanoparticles, and improve the encapsulation efficiency of active substances.
[0021] 3. This invention uses oregano essential oil, which has antibacterial and antioxidant activities, as a hydrophobic bioactive substance, and shellac-sodium caseinate as a carrier. It employs an antisolvent precipitation method to prepare shellac-sodium caseinate composite nanoparticles loaded with oregano essential oil. The materials are safe and environmentally friendly, and the method is simple and easy to operate. At the same time, it also provides a theoretical basis for loading antibacterial and antioxidant active substances.
[0022] 4. The encapsulation rate of oregano essential oil by the composite nanoparticles prepared in this invention is 24.46% higher than that of single shellac nanoparticles, and the stability, antibacterial and antioxidant activities of the prepared composite nanoparticles are significantly improved. Attached Figure Description
[0023] Appendix Figure 1 Scanning electron microscope images of the nanoparticles prepared in comparative examples and Examples 1-4.
[0024] Appendix Figure 2 Particle size and zeta potential of nanoparticles prepared in comparative examples and Examples 1-4
[0025] Appendix Figure 3 Encapsulation efficiency of oregano essential oil by nanoparticles prepared in comparative examples and Examples 1-4
[0026] Appendix Figure 4 The changes in particle size and zeta potential of the nanoparticles prepared in the comparative example and Example 2 at different pH values.
[0027] Appendix Figure 5 The changes in particle size and zeta potential of nanoparticles prepared in the comparative example and Example 2 under different salt ion concentrations are shown in the appendix. Figure 6 Antioxidant capacity of nanoparticles prepared in Comparative Example and Example 2 Detailed Implementation
[0028] Comparative Example
[0029] Step 1: Weigh 2g of shellac and dissolve it in 50mL of anhydrous ethanol solution. Sonicate for 40min to completely dissolve it, and change the water in time to prevent the temperature from getting too high.
[0030] Step 2: Centrifuge the shellac solution at 4000 rpm / min and 25°C for 10 min to remove undissolved shellac.
[0031] Step 3: Add 0.5g of oregano essential oil and 2.86g of Tween 80 dropwise to the solution obtained after centrifugation, and stir continuously for 2 hours to ensure thorough mixing.
[0032] Step 4: Using a syringe with an inner diameter of 0.42 mm, add 10 mL of oregano oil / shellac solution dropwise to 100 mL of deionized water. Stir magnetically for 40 min at 600 rpm. Use a rotary evaporator to evaporate the ethanol in the sample at 40 °C, and then bring the volume to 100 mL with deionized water.
[0033] Step 5: Centrifuge the obtained dispersion at 1500 rpm / min and 25℃ for 10 min to remove the larger shellac aggregates, thus obtaining an aqueous dispersion of shellac nanoparticles loaded with oregano essential oil. Freeze-dry to obtain shellac nanoparticles loaded with oregano essential oil.
[0034] Example 1
[0035] Step 1: Weigh 2g of shellac and dissolve it in 50mL of anhydrous ethanol solution. Sonicate for 40min to completely dissolve it, and change the water in time to prevent the temperature from getting too high.
[0036] Step 2: Centrifuge the shellac solution at 4000 rpm / min and 25°C for 10 min to remove undissolved shellac.
[0037] Step 3: Add 0.5g of oregano essential oil and 2.86g of Tween 80 dropwise to the solution obtained after centrifugation, and stir continuously for 2 hours to ensure thorough mixing.
[0038] Step 4: Dissolve 0.05g of sodium caseinate in 100mL of deionized water and stir thoroughly until completely dissolved to prepare a 0.05% (w / v) sodium caseinate solution.
[0039] Step 5: Using a syringe with an inner diameter of 0.42 mm, add 10 mL of oregano oil / shellac solution dropwise to 100 mL of 0.05% (w / v) sodium caseinate solution, with a shellac to sodium caseinate mass ratio of 8:1. Stir magnetically at 600 rpm for 40 min, then evaporate the ethanol from the sample using a rotary evaporator at 40°C, and bring the volume to 100 mL with deionized water.
[0040] Step 6: Centrifuge the obtained dispersion at 1500 rpm / min and 25℃ for 10 min to remove the larger shellac aggregates, thus obtaining an aqueous dispersion of shellac-sodium caseinate composite nanoparticles loaded with oregano essential oil. Freeze-dry to obtain shellac-sodium caseinate composite nanoparticles loaded with oregano essential oil.
[0041] Example 2
[0042] Step 1: Weigh 2g of shellac and dissolve it in 50mL of anhydrous ethanol solution. Sonicate for 40min to completely dissolve it, and change the water in time to prevent the temperature from getting too high.
[0043] Step 2: Centrifuge the shellac solution at 4000 rpm / min and 25°C for 10 min to remove undissolved shellac.
[0044] Step 3: Add 0.5g of oregano essential oil and 2.86g of Tween 80 dropwise to the solution obtained after centrifugation, and stir continuously for 2 hours to ensure thorough mixing.
[0045] Step 4: Dissolve 0.1g of sodium caseinate in 100mL of deionized water and stir thoroughly until completely dissolved to prepare a 0.1% (w / v) sodium caseinate solution.
[0046] Step 5: Using a syringe with an inner diameter of 0.42 mm, add 10 mL of oregano oil / shellac solution dropwise to 100 mL of 0.1% (w / v) sodium caseinate solution, with a shellac to sodium caseinate mass ratio of 4:1. Stir continuously with magnetic stirring at 600 rpm for 40 min. Evaporate the ethanol from the sample using a rotary evaporator at 40°C, and then bring the volume to 100 mL with deionized water.
[0047] Step Six: Centrifuge the obtained dispersion at 1500 rpm / min and 25°C for 10 min to remove the larger shellac aggregates, thus obtaining an aqueous dispersion of shellac-sodium caseinate composite nanoparticles loaded with oregano essential oil. Freeze-dry to obtain shellac-sodium caseinate composite nanoparticles loaded with oregano essential oil.
[0048] Example 3
[0049] Step 1: Weigh 2g of shellac and dissolve it in 50mL of anhydrous ethanol solution. Sonicate for 40min to completely dissolve it, and change the water in time to prevent the temperature from getting too high.
[0050] Step 2: Centrifuge the shellac solution at 4000 rpm / min and 25°C for 10 min to remove undissolved shellac.
[0051] Step 3: Add 0.5g of oregano essential oil and 2.86g of Tween 80 dropwise to the solution obtained after centrifugation, and stir continuously for 2 hours to ensure thorough mixing.
[0052] Step 4: Dissolve 0.15g of sodium caseinate in 100mL of deionized water and stir thoroughly until completely dissolved to prepare a 0.15% (w / v) sodium caseinate solution.
[0053] Step 5: Using a syringe with an inner diameter of 0.42 mm, add 10 mL of oregano oil / shellac solution dropwise to 100 mL of 0.15% (w / v) sodium caseinate solution, with a shellac to sodium caseinate mass ratio of 8:3. Stir magnetically at 600 rpm for 40 min, then evaporate the ethanol from the sample using a rotary evaporator at 40°C, and bring the volume to 100 mL with deionized water.
[0054] Step Six: Centrifuge the obtained dispersion at 1500 rpm / min and 25°C for 10 min to remove the larger shellac aggregates, thus obtaining an aqueous dispersion of shellac-sodium caseinate composite nanoparticles loaded with oregano essential oil. Freeze-dry to obtain shellac-sodium caseinate composite nanoparticles loaded with oregano essential oil.
[0055] Example 4
[0056] Step 1: Weigh 2g of shellac and dissolve it in 50mL of anhydrous ethanol solution. Sonicate for 40min to completely dissolve it, and change the water in time to prevent the temperature from getting too high.
[0057] Step 2: Centrifuge the shellac solution at 4000 rpm / min and 25°C for 10 min to remove undissolved shellac.
[0058] Step 3: Add 0.5g of oregano essential oil and 2.86g of Tween 80 dropwise to the solution obtained after centrifugation, and stir continuously for 2 hours to ensure thorough mixing.
[0059] Step 4: Dissolve 0.2g of sodium caseinate in 100mL of deionized water and stir thoroughly until completely dissolved to prepare a 0.2% (w / v) sodium caseinate solution.
[0060] Step 5: Using a syringe with an inner diameter of 0.42 mm, add 10 mL of oregano oil / shellac solution dropwise to 100 mL of 0.2% (w / v) sodium caseinate solution, with a shellac to sodium caseinate mass ratio of 2:1. Stir magnetically at 600 rpm for 40 min, then evaporate the ethanol from the sample using a rotary evaporator at 40°C, and bring the volume to 100 mL with deionized water.
[0061] Step Six: Centrifuge the obtained dispersion at 1500 rpm / min and 25°C for 10 min to remove the larger shellac aggregates, thus obtaining an aqueous dispersion of shellac-sodium caseinate composite nanoparticles loaded with oregano essential oil. Freeze-dry to obtain shellac-sodium caseinate composite nanoparticles loaded with oregano essential oil.
[0062] Scanning electron microscope images of the composite nanoparticles prepared in comparative examples and Examples 1-4 are shown below. Figure 1 As can be seen from the figure, the composite nanoparticles prepared in Example 2 are relatively uniformly distributed and do not exhibit agglomeration.
[0063] Performance testing:
[0064] 1. Test the particle size and zeta potential of the nanoparticles prepared in comparative examples and Examples 1-4.
[0065] Test Method: The particle size distribution of nanoparticles was measured using a Zeta Sizer Nano ZS90 particle size analyzer. Before testing, the aqueous dispersion of nanoparticles was diluted with deionized water to 1 mg / mL to minimize multiple scattering effects. The test temperature was 25℃, the equilibration time was 60 s, and the refractive indices of the aqueous phase and nanoparticles were 1.33 and 1.53, respectively. The nanoparticle dispersion was placed in a particle size cuvette and a potentiometer cuvette, and its particle size and Zeta potential were measured, repeated three times.
[0066] Test results are as follows Figure 2 As can be seen from the figure, compared with the comparative example, the average particle size of Examples 1-4 has increased. This is due to the large amount of sodium caseinate adhering to the surface of the shellac polymer. Among them, Example 2 has the smallest increase in particle size, the highest potential, and more uniform and stable dispersion.
[0067] 2. Test the encapsulation efficiency of the nanoparticles prepared in the comparative examples and Examples 1-4 for oregano essential oil.
[0068] Test method: The aqueous dispersion of nanoparticles was mixed with an equal volume of anhydrous ethanol and shaken vigorously. The mixture was then centrifuged at 10,000 rpm for 10 min. This extraction process was repeated three times. The supernatant was collected and diluted 10-fold with ethanol. The absorbance was measured at 276 nm using a UV-Vis spectrophotometer (UV-2700). A standard curve for oregano essential oil was prepared to determine the content of free oregano essential oil.
[0069] Oregano essential oil encapsulation rate (%) = (Total oregano essential oil content - Free oregano essential oil content) / Total oregano essential oil content × 100
[0070] Test results are as follows Figure 3 As can be seen from the figure, the encapsulation rate of oregano essential oil in the comparative example was relatively low. Compared with the comparative example, the encapsulation rates of Examples 1-4 were significantly higher than those in the comparative example. This indicates that the addition of sodium caseinate as a surface modifier can improve the encapsulation efficiency of oregano essential oil and better complete the encapsulation of oregano essential oil.
[0071] 3. Test the pH stability of the nanoparticles prepared in the comparative example and Example 2.
[0072] Test method: The pH of the nanoparticle dispersion was adjusted to 3-8 using 0.1M hydrochloric acid and 0.1M sodium hydroxide solution, and the average particle size and zeta potential of the nanoparticles were measured.
[0073] Test results are as follows Figure 4 As shown in the figure, the composite nanoparticles prepared in Example 2 have a smaller particle size under alkaline conditions, indicating excellent alkaline pH stability. Under acidic conditions, the composite nanoparticles prepared in Example 2 exhibit a positive potential because sodium caseinate is primarily positively charged. At the isoelectric point of sodium caseinate (pH 4.6), the particle size of the composite nanoparticles changes drastically, with significant aggregation. The zeta potential changes from positive to negative, and all positive and negative charges on the composite nanoparticles are neutralized, making them the most unstable and thus enabling the formation of larger aggregates. Besides the isoelectric point, the composite nanoparticles prepared in Example 2 show even better stability at acidic pH (pH 3), with no aggregation, indicating that surface modification with sodium caseinate can improve the acid instability of single shellac nanoparticles.
[0074] 4. Test the salt ion stability of the nanoparticles prepared in the comparative example and Example 2.
[0075] Test method: Different masses of sodium chloride were added to the nanoparticle dispersion to make the sodium chloride concentrations 0, 100 mmol / L, 150 mmol / L and 200 mmol / L, and the average particle size and zeta potential of the nanoparticles were measured.
[0076] Test results are as follows Figure 5 As shown in the figure, when the sodium chloride concentration exceeds 0.1 M, it can induce complete aggregation of the shellac nanoparticles prepared in the comparative example, indicating that single shellac nanoparticles are highly sensitive to salt. With increasing salt ion concentration, the particle size of the composite nanoparticles prepared in Example 2 slightly increases. Even when the sodium chloride concentration reaches 0.2 M, the particle size of the composite nanoparticles is only 341.50 ± 2.15 nm, while the potentials are all higher than those of the comparative example, indicating that surface modification with sodium caseinate can further improve the salt ion stability of single shellac nanoparticles.
[0077] 5. Test the antioxidant capacity of the nanoparticles prepared in the comparative example and Example 2.
[0078] Test method: Take 2 mL of freshly prepared nanoparticle dispersion and mix with an equal volume of DPPH ethanol solution (0.1 mmol / L), shake repeatedly to dissolve completely, let stand in the dark for 30 min, and measure the absorbance at a wavelength of 517 nm. The DPPH free radical scavenging rate is calculated by formula.
[0079]
[0080] Where A0 represents the absorbance after mixing DPPH with deionized water instead of the sample reaction solution, A1 represents the absorbance of the sample reaction solution, and A2 represents the absorbance of the sample without DPPH free radical solution.
[0081] Take 1 mL of freshly prepared nanoparticle dispersion and 3 mL of ABTS solution (absorbance at 734 nm wavelength is 0.70 ± 0.02 cm⁻¹). -1 Mix thoroughly, place the mixture in the dark for 30 minutes, measure the absorbance at a wavelength of 734 nm, and calculate the ABTS free radical scavenging rate using the formula.
[0082]
[0083] Where A0 represents the absorbance after mixing ABTS with deionized water instead of the sample reaction solution, A1 represents the absorbance of the sample reaction solution, and A2 represents the absorbance of the sample without ABTS free radical solution.
[0084] Test results are as follows Figure 6 As can be seen from the figure, Example 2 has higher free radical scavenging activity compared with the comparative example. This may be because Example 2 has a higher encapsulation rate of oregano essential oil, and the concentration of oregano essential oil is higher, resulting in stronger antioxidant capacity.
Claims
1. A method for preparing a shellac-sodium caseinate composite nanoparticle loaded with oregano essential oil, characterized by: The method comprises the following steps: (1) 2 g of shellac is weighed and dissolved in 50 mL of ethanol solution, and ultrasonic is used to completely dissolve it, and water is replaced in time to prevent the temperature from being too high; (2) The solution obtained in step (1) is centrifuged at 4000 rpm / min and 25 DEG C for 10 min, and the undissolved shellac is removed; (3) A certain mass of oregano essential oil and Tween 80 are added dropwise to the solution obtained in step (2), and stirring is continued to make them fully mixed; (4) Different masses of sodium caseinate are dissolved in 100 mL of deionized water, and fully stirred to make them completely dissolved, to prepare sodium caseinate solutions with different concentrations; (5) Using a syringe, 10 mL of the oregano essential oil / shellac solution obtained in step (3) is added dropwise to a series of sodium caseinate solutions with different concentrations obtained in step (4), and magnetic stirring is continued at a speed of 600 rpm for 40 min, and ethanol in the sample is evaporated using a rotary evaporator at 40 DEG C, and deionized water is used to make up to 100 mL; (6) The solution obtained in step (5) is centrifuged at 1500 rpm / min and 25 DEG C for 10 min, and the larger shellac aggregates generated are removed, to obtain a water dispersion of oregano essential oil-loaded shellac-sodium caseinate composite nanoparticles, and freeze-drying is used to obtain the composite nanoparticles.
2. A method of preparing a shellac-sodium caseinate composite nanoparticle loaded with oregano essential oil according to claim 1, characterized by: The ethanol solution in step (1) is anhydrous ethanol, and the ultrasonic time is 40 min.
3. A method of preparing a shellac-sodium caseinate composite nanoparticle loaded with oregano essential oil according to claim 1, characterized by: The mass of oregano essential oil and Tween 80 added in step (3) is 0.5 g and 2.86 g, and the mass ratio with shellac is 1:4 and 10:7 respectively, and the stirring time is 2 h.
4. A method of preparing a shellac-sodium caseinate composite nanoparticle loaded with oregano essential oil according to claim 1, characterized by: The mass of sodium caseinate in step (4) is 0.05 g, 0.1 g, 0.15 g and 0.2 g respectively, the concentration is 0.05%-0.2% (w / v), and the stirring time is 2 h.
5. A method of preparing a shellac-sodium caseinate composite nanoparticle loaded with oregano essential oil according to claim 1, characterized by: The inner diameter of the syringe used in step (5) is 0.42 mm.