Propolis soft capsule and preparation method thereof

Abstract

The invention belongs to the field of propolis soft capsules, and provides a propolis soft capsule and a preparation method thereof.The propolis soft capsule is prepared from, by weight, 30%-50% of propolis extract, 40%-60% of modified egg yolk phospholipid and 5%-15% of cosolvent; wherein the modified egg yolk phospholipid is partially hydrogenated, the sn-2 site of the modified egg yolk phospholipid is substituted by branched chain fatty acid, and the iodine value of the modified egg yolk phospholipid is shown in the specification; according to the invention, specifically modified egg yolk phospholipid is used as a core carrier, and the unique molecular structure of the egg yolk phospholipid can be efficiently self-assembled in an oil phase, so that the egg yolk phospholipid has excellent wrapping and solubilizing capabilities on various active components in propolis. The propolis extract can be stably loaded into an oil phase to form a transparent nano-dispersion with small average particle size and low polydispersity index, so that high-efficiency and high-loading oil dissolution of propolis is realized, and a stable nano-dispersion system is formed.

Description

Technical Field

[0001] This invention belongs to the field of propolis soft capsules, specifically a propolis soft capsule and its preparation method. Background Technology

[0002] Propolis is a fragrant, gummy solid formed by bees collecting resin from plant buds and tree trunks, mixing it with secretions from their mandibular glands and beeswax. It is rich in flavonoids, phenolic acids, terpenes, and other bioactive components, exhibiting significant antibacterial, anti-inflammatory, antioxidant, and immunomodulatory effects, and is widely used in health products and food. However, the widespread application of propolis is severely limited by its inherent physicochemical properties. The active ingredients in propolis, such as highly polar flavonoids, are insoluble in both water and common edible oils, resulting in low bioavailability and making it difficult to directly add to oil-based soft capsules, beverage emulsions, or functional oil products.

[0003] Regarding existing technologies, the inventors believe the following drawbacks exist: Chemical modifications such as esterification and etherification of propolis active ingredients introduce lipophilic groups. While this method can improve lipophilicity, the reaction process may destroy the natural active structure of propolis and may introduce chemical residues, which does not align with consumers' preference for natural products. Adding large amounts of surfactants / cosolvents presents safety dosage limitations; excessive addition may lead to unpleasant taste or gastrointestinal irritation, and long-term stability is poor, easily resulting in stratification and precipitation. Utilizing the amphiphilicity of phospholipids to form micelles or liquid crystal structures to encapsulate propolis active ingredients results in large particle sizes and poor physical stability, making them prone to aggregation and crystallization during storage. Some studies have explored hydrogenation of phospholipids to improve oxidative stability or enzymatic modification to replace the fatty acid at the sn-2 position. However, existing technologies often involve hydrogenation or replacement alone, and the replaced fatty acids are mostly straight-chain structures. Deep hydrogenation alone can lead to excessively high melting points of phospholipids and poor solubility in the oil phase. While replacing with straight-chain fatty acids can change the hydrophilic-hydrophobic balance, it still does not provide sufficient steric stability to the micelles and cannot solve the recrystallization and exudation problems of propolis active ingredients during long-term storage.

[0004] To address the problems raised in the background art, those skilled in the art have proposed a propolis soft capsule and its preparation method. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a propolis soft capsule and its preparation method. This addresses the shortcomings of existing technologies that involve hydrogenation or substitution, where the replaced fatty acids are mostly linear structures. Deep hydrogenation alone can lead to excessively high melting points of phospholipids and poor solubility in the oil phase. While simply replacing with linear fatty acids can alter the hydrophilic-hydrophobic balance, it still does not provide sufficient steric stability to the micelles, failing to solve problems such as recrystallization and exudation of propolis active ingredients during long-term storage.

[0006] A propolis soft capsule and its preparation method thereof, comprising the following raw materials by weight percentage: 30%-50% propolis extract, 40%-60% modified egg yolk phospholipids, and 5%-15% cosolvent; wherein the modified egg yolk phospholipids are partially hydrogenated egg yolk phospholipids with branched-chain fatty acids substituted at the sn-2 position, and their iodine value is [value missing]. .

[0007] Preferably, the branched fatty acid is phytic acid, and the substitution rate of the phytic acid at the sn-2 position is 15%-25%.

[0008] Preferably, the co-solvent is at least one of oleic acid or medium-chain triglycerides.

[0009] Preferably, the raw materials further include 1%-5.5% functional additives, which are fat-soluble active ingredients and antioxidants.

[0010] Preferably, the fat-soluble active ingredient is at least one of vitamin E, vitamin A, phytosterols or phytosterol esters, and the antioxidant is at least one of rosemary extract and ascorbyl palmitate.

[0011] A method for preparing propolis soft capsules as described in any one of claims 1-5, characterized by comprising the following steps:

[0012] S1: Prepare modified egg yolk phospholipids by providing egg yolk phospholipid raw materials and sequentially performing partial hydrogenation and enzymatic transesterification treatments to obtain the modified egg yolk phospholipids.

[0013] S2: Mixing and homogenizing. The modified egg yolk phospholipids, propolis extract and cosolvent obtained in step S1 are mixed in proportion and homogenized to form a stable nano-dispersion.

[0014] S3: Filling and molding: Fill the contents obtained in step S2 into soft capsule shells and seal them.

[0015] Preferably, the partial hydrogenation treatment specifically involves: in an inert organic solvent, in the presence of a catalyst, introducing hydrogen gas into an egg yolk phospholipid solution to carry out a reaction, and controlling the iodine value of the resulting partially hydrogenated egg yolk phospholipid to be... The enzymatic transesterification process specifically involves mixing the partially hydrogenated egg yolk phospholipids with ethyl phytate in a non-aqueous medium, adding sn-1,3 specific lipase to catalyze the reaction, and introducing branched fatty acids.

[0016] Preferably, in the partial hydrogenation treatment, the catalyst is palladium on carbon or rhodium triphenylphosphine chloride, and the amount used is 0.5%-2.0% of the egg yolk phospholipid content. The reaction temperature is 40-60℃, and the hydrogen pressure is 2-4 bar. In the enzymatic transesterification treatment, the lipase is immobilized Lipozyme TL IM, the reaction temperature is 50-60℃, the reaction time is 12-24 hours, and the molar ratio of the partially hydrogenated egg yolk phospholipid to ethyl phytate is 1:1.5 to 1:3.0.

[0017] A composition of propolis soft capsule contents, characterized in that it is prepared by the method described in any one of claims 6-8.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention utilizes specifically modified egg yolk phospholipids as a core carrier. Their unique molecular structure allows for efficient self-assembly in the oil phase, exhibiting excellent encapsulation and solubilization capabilities for various active ingredients in propolis. This allows for the stable loading of propolis extract into the oil phase, forming transparent nano-dispersions with small average particle size and low polydispersity index. This achieves efficient, high-loading oil solubility of propolis, resulting in a stable nano-dispersion system.

[0020] This invention achieves excellent long-term physical stability by modifying the branched-chain fatty acids in phospholipids to introduce strong steric hindrance and moderate saturation through partial hydrogenation, resulting in micelles or nanoparticles with extremely high kinetic stability and solving the problems of layering and precipitation.

[0021] This invention significantly improves the oxidative stability of products and effectively protects active ingredients through a synergistic antioxidant system composed of lipid-soluble antioxidants and free radical scavengers.

[0022] This invention utilizes gentle hydrogenation and enzymatic modification, without introducing harmful chemical residues. The entire system does not rely on large amounts of synthetic surfactants or organic solvents, resulting in a final product with clearly defined, safe, and controllable components. This meets market demands for clean-label and natural health products, ensuring product safety and natural attributes, and aligning with health-conscious consumption trends.

[0023] The present invention provides a method for preparing modified egg yolk phospholipids with clear process parameters and endpoint control indicators. The steps are clear, reproducibility is good, the preparation process is controllable, and it is suitable for industrial production. Detailed Implementation

[0024] The embodiments of the present invention will be described in further detail below with reference to examples. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0025] Example 1: This invention provides a propolis soft capsule and its preparation method, including the following steps: 1. Preparation of modified egg yolk phospholipids: Partial hydrogenation, in a 500 mL high-pressure reactor equipped with mechanical stirring, a hydrogen inlet valve, a pressure gauge, and a temperature control system, 100.0 g of egg yolk phospholipid powder and 300 mL of anhydrous tetrahydrofuran dried through 4A molecular sieves are added. Dissolution and catalysis: stirring is started at 300 rpm, and the temperature is raised to 45°C to completely dissolve the phospholipids. After the system is purged with nitrogen three times, 1.0 g of dried palladium on carbon catalyst is added. Hydrogenation reaction: the gas in the reactor is purged with hydrogen three times again, and then hydrogen is introduced and the pressure is maintained at 3.0 bar. The reaction temperature is raised to 50°C, and the reaction is started. Endpoint control: every 45 minutes, about 1 mL of reaction solution is taken out through the sampling valve, the catalyst is quickly filtered out, and the iodine value is determined according to the method of "GB / T 5532-2008 Determination of Iodine Value of Animal and Vegetable Oils". When the results of two consecutive measurements are stable at 100°C, the iodine value is determined. After approximately 5.5 hours of reaction, hydrogen flow was stopped, and the mixture was cooled to room temperature. Post-treatment: The reaction solution was filtered through a 0.45 μm polytetrafluoroethylene (PTFE) membrane to completely remove the catalyst. The filtrate was transferred to a rotary evaporator and evaporated at a water bath of 40°C and a vacuum of -0.095 MPa to remove tetrahydrofuran, yielding a pale yellow, waxy, partially hydrogenated egg yolk phospholipid. The iodine value was measured to be... Store in a nitrogen-protected desiccator for later use.

[0026] Enzymatic transesterification to introduce branched-chain fatty acids: In a 250 mL stoppered conical flask, weigh 50.0 g of the partially hydrogenated egg yolk phospholipids and add 42.7 g of ethyl phytate at a molar ratio of 1:2.0. Add 100 mL of anhydrous tert-butanol containing 5 g of pre-activated 3A molecular sieve as the reaction medium. Add 10% (by weight) of immobilized lipase LipozymeTL IM to the system. After sealing the conical flask, place it in a constant temperature shaking incubator at 55°C and a shaking frequency of 200 rpm to start the reaction. The reaction proceeds for 18 hours. During this period, the reaction progress can be judged by monitoring the shift of phosphatidylcholine spots using thin-layer chromatography. After the reaction is complete, filter using a Buchner funnel to separate the immobilized enzyme. Remove tert-butanol from the filtrate by rotary evaporation. Dissolve the crude product in a small amount of chloroform and load it onto a 200-300 mesh silica gel column with a column volume of approximately 500 mL. Gradient elution was employed: unreacted ethyl phytate was first washed away with chloroform / methanol (95:5, v / v), and then the ratio was adjusted to chloroform / methanol (85:15, v / v) to elute the target product. The fractions containing the target product were collected, combined, and then rotary evaporated and dried under vacuum to obtain a white solid modified egg yolk phospholipid.

[0027] Preparation and shaping of soft capsule contents: Premixing: The following components were accurately weighed into a 100mL jacketed glass reactor according to the following weight percentages: propolis extract (ethanol extract, total flavonoid content 52.1%), 40.0 g; modified egg yolk phospholipids, 52.0 g; medium-chain triglycerides (MCT, C8:C10=60:40), 8.0 g. The reactor was stirred, and 60℃ circulating water was introduced through the jacket to stabilize the system temperature at 60±2℃. Stirring was continued for 30 minutes until all solids were completely melted, forming a homogeneous dark brown viscous liquid. High-pressure homogenization: The premix was transferred to the feed tank of a high-pressure homogenizer, maintaining the material temperature at 55-60℃. The homogenization pressure was set to 600 bar, and cyclic homogenization was performed. After the first and second homogenizations, samples were observed under a microscope, showing that large particles were broken down. A total of 5 cycles were performed. Finally, a homogeneous, transparent, bright reddish-brown, and highly fluid nano-dispersion was obtained. The average particle size (Z-Average) was determined to be 95 nm and the polydispersity index (PDI) was 0.18 using a Malvern laser particle size analyzer. Filling and drying: The homogenized contents were transferred to the hopper of a soft capsule machine, maintaining the liquid temperature at 50±2℃. Standard oval gelatin capsules (gelatin:glycerol:water = 40:20:40) were used for capsule compression at room temperature (25℃) and ambient humidity (40%). The capsule contents were 500 mg / capsule. The compressed wet capsules were dried in a rotary dryer at 20-25℃ and 20-25% humidity with circulating airflow for 24 hours until the capsule weight reached a constant. The finished soft capsules were then obtained.

[0028] Example 2: This example is basically the same as the previous example, except that a propolis soft capsule and its preparation method include the following steps: Preparation of modified egg yolk phospholipids: Partial hydrogenation, in a 500 mL high-pressure reactor equipped with mechanical stirring, a hydrogen inlet valve, a pressure gauge and a temperature control system, 100.0 g of egg yolk phospholipid powder and 300 mL of anhydrous tetrahydrofuran dried through 4A molecular sieves are added. Dissolution and catalysis, stirring is started at 300 rpm, and the temperature is raised to 45°C to completely dissolve the phospholipids. After the system is purged with nitrogen three times, 1.0 g of dried palladium on carbon catalyst is added. Hydrogenation reaction, the gas in the reactor is purged with hydrogen three times again, and then hydrogen is introduced and the pressure is maintained at 3.0 bar. The reaction temperature is raised to 50°C and the reaction is started. Endpoint control, every 45 minutes, about 1 mL of reaction solution is taken out through the sampling valve, the catalyst is quickly filtered out, and the iodine value is determined according to the method of "GB / T 5532-2008 Determination of Iodine Value of Animal and Vegetable Oils". When the results of two consecutive measurements are stable at After approximately 5.5 hours of reaction, hydrogen flow was stopped, and the mixture was cooled to room temperature. Post-treatment: The reaction solution was filtered through a 0.45 μm polytetrafluoroethylene (PTFE) membrane to completely remove the catalyst. The filtrate was transferred to a rotary evaporator and evaporated at a water bath of 40°C and a vacuum of -0.095 MPa to remove tetrahydrofuran, yielding a pale yellow, waxy, partially hydrogenated egg yolk phospholipid. The iodine value was measured to be... Store in a nitrogen-protected desiccator for later use.

[0029] Enzymatic transesterification to introduce branched-chain fatty acids: In a 250 mL stoppered conical flask, weigh 50.0 g of the partially hydrogenated egg yolk phospholipids and add 42.7 g of ethyl phytate at a molar ratio of 1:2.0. Add 100 mL of anhydrous tert-butanol containing 5 g of pre-activated 3A molecular sieve as the reaction medium. Add 10% (by weight) of immobilized lipase LipozymeTL IM to the system. After sealing the conical flask, place it in a constant temperature shaking incubator at 55°C and a shaking frequency of 200 rpm to start the reaction. The reaction proceeds for 18 hours. During this period, the reaction progress can be judged by monitoring the shift of phosphatidylcholine spots using thin-layer chromatography. After the reaction is complete, filter using a Buchner funnel to separate the immobilized enzyme. Remove tert-butanol from the filtrate by rotary evaporation. Dissolve the crude product in a small amount of chloroform and load it onto a 200-300 mesh silica gel column with a column volume of approximately 500 mL. Gradient elution was employed: unreacted ethyl phytate was first washed away with chloroform / methanol (95:5, v / v), and then the ratio was adjusted to chloroform / methanol (85:15, v / v) to elute the target product. The fractions containing the target product were collected, combined, and then rotary evaporated and dried under vacuum to obtain a white solid modified egg yolk phospholipid.

[0030] The preparation and shaping of the soft capsule contents, premixed, consist of: 35.0 g propolis extract, 50.0 g modified egg yolk lecithin, 10.0 g oleic acid, 3.0 g d-α-tocopherol (vitamin E), 1.5 g rosemary extract (20% oxalic acid content), and 0.5 g ascorbyl palmitate. Key operational sequence: First, dissolve the modified egg yolk lecithin, oleic acid, vitamin E, and ascorbyl palmitate at 60°C. Then add the propolis extract and stir until dissolved. Finally, add the rosemary extract; as it is a solid powder and may affect viscosity, continue stirring until completely dispersed and uniform. This sequence helps stabilize the active ingredients.

[0031] High-pressure homogenization: The premixed material was transferred to the feed tank of a high-pressure homogenizer, maintaining the material temperature at 55-60℃. The homogenization pressure was set to 600 bar, and cyclic homogenization was performed. After the first and second homogenizations, samples were observed under a microscope, revealing that large particles had been broken down. A total of 5 cycles were performed. The final product was a uniform, transparent, bright reddish-brown, and highly fluid nano-dispersion. Using a Malvern laser particle size analyzer, the average particle size (Z-Average) was 105 nm, and the polydispersity index (PDI) was 0.2. Filling and drying: The homogenized contents were transferred to the hopper of a soft capsule machine, maintaining the liquid temperature at 50±2℃. Standard oval gelatin capsules (gelatin:glycerol:water = 40:20:40) were used for capsule compression at room temperature (25℃) and ambient humidity (40%). The capsule contents were 500 mg / capsule. The pressed wet capsules are dried in a rotary dryer at 20-25℃ and 20-25% humidity under circulating airflow for 24 hours until the capsule weight is constant. The finished soft capsules are then obtained.

[0032] Example 3: Based on Example 1, a propolis soft capsule and its preparation method include the following steps: partial hydrogenation treatment using a 250 mL micro high-pressure reactor equipped with a magnetic stirrer, electric heating mantle, pressure sensor, hydrogen inlet valve, exhaust valve, and sampling tube. Accurately weigh 50.0 g of egg yolk phospholipid raw material and place it in the inner liner of the reactor. Add 150 mL of dried anhydrous toluene as a solvent. Start stirring and gradually raise the temperature to 40°C to completely dissolve the egg yolk phospholipid and form a homogeneous solution. Add 0.40 g (0.8% of the phospholipid content) of triphenylphosphine rhodium chloride as a homogeneous catalyst to the solution. Perform three pressurization-depressurization cycles with high-purity nitrogen to replace the air inside the reactor.

[0033] Hydrogenation reaction and endpoint control: After displacement, hydrogen gas is introduced until the pressure inside the reactor stabilizes at 2.0 bar. The reaction temperature is maintained at 40±1℃, and the reaction is started on a timer. Iodine value monitoring: Every 2 hours after the start of the reaction, approximately 0.5 mL of the reaction solution is taken through the built-in sampling tube. The sample is immediately filtered through a short section of neutral alumina column to remove the catalyst, and the filtrate is collected in a pre-weighed vial. Iodine value is determined using the Wijs method: The filtered sample is dissolved in 10 mL of chloroform, and 25.00 mL of Wijs reagent (iodine monochloride glacial acetic acid solution) is added. The reaction is carried out precisely in the dark for 1 hour. Then, 20 mL of potassium iodide solution (150 g / L) and 100 mL of distilled water are added, and titrated with standardized sodium thiosulfate standard solution (0.1 mol / L) until the blue color of the starch indicator disappears. A blank experiment is performed simultaneously. The real-time iodine value is calculated using a formula. When the iodine values ​​measured at two consecutive sampling points are respectively... and When the trend stabilizes, the reaction is considered to have reached its endpoint. The total reaction time is approximately 10.5 hours. Immediately close the hydrogen valve, stop heating, cool the reactor to below 25°C, and slowly release the pressure.

[0034] Post-processing: The entire reaction solution was transferred to a flask. To decompose and remove the homogeneous rhodium catalyst, 5 mL of ethanolamine was added to the solution, and the mixture was stirred at 40 °C for 1 hour. Subsequently, the reaction solution was poured into a separatory funnel and washed twice with 100 mL of dilute hydrochloric acid (1 mol / L), followed by one wash with 100 mL of saturated saline solution to remove ethanolamine and residual acid. The organic phase (toluene layer) was dried over anhydrous sodium sulfate and filtered. The filtrate was rotary evaporated at 40 °C and -0.09 MPa vacuum to remove toluene, yielding a pale yellow solid partially hydrogenated egg yolk phospholipid. The iodine value was retested using the aforementioned method. ,conform to Upper limit requirements for the range. The product is placed in a nitrogen-filled desiccator for later use.

[0035] Enzymatic transesterification: Reactant preparation and feeding: Weigh 30.0 g of the partially hydrogenated egg yolk phospholipids mentioned above. Based on a molar ratio of 1:1.6, accurately calculate and weigh the required amount of ethyl phytate: 0.0395 mol * 1.6 * 310 g / mol ≈ 19.6 g. Place both into a 250 mL round-bottom flask equipped with a magnetic stirrer. Add 60 mL of anhydrous tert-butanol to the flask. Simultaneously add 3 g of activated 3Å molecular sieve. Place the flask in a 60℃ constant-temperature oil bath and start stirring (300 rpm) to completely dissolve / disperse the solid. Add immobilized lipase Lipozyme TL IM to the system at 12% of the total reactant mass, i.e., 5.95 g. Catalytic reaction and process monitoring: Install a reflux condenser at the flask mouth to prevent solvent evaporation. Continuously stir the reaction at 60±1℃. Thin-layer chromatography monitoring: Samples were taken using capillary tubes at 6, 9, and 12 hours of reaction. The samples were spotted onto silica gel GF254 plates and developed using chloroform / methanol / glacial acetic acid / water (50:30:8:4, v / v). After drying, the samples were developed using phosphomolybdic acid ethanol solution and ninhydrin ethanol solution, respectively. After 12 hours of reaction, TLC results showed a significant reduction in the phosphatidylcholine (PC) spot, with a new main spot (modified PC) appearing at a slightly lower Rf value, and no obvious purple-red spot observed with ninhydrin. The reaction was deemed essentially complete. After the reaction, the mixture was cooled to room temperature. The mixture was filtered through a sintered glass funnel to recover the immobilized enzyme (which could be washed twice with tert-butanol and dried for reuse). The filtrate and washings were combined. The filtrate was concentrated to near dryness by rotary evaporation (40℃, -0.085 MPa).

[0036] The preparation and filling of the propolis soft capsule contents were performed as follows: Each component was weighed using a balance with an accuracy of 0.01 g and placed in a 100 mL mixing tank with a temperature-controlled jacket, according to the following weight percentages: propolis extract (total flavonoid content 50.5%): 45.0 g; modified egg yolk phospholipids prepared above: 45.0 g; medium-chain triglycerides (MCT, C8 / C10=50 / 50): 8.0 g; phytosterol esters (derived from pine wood, esterification rate ≥96%): 2.0 g. The mixing tank was stirred, and circulating hot water was introduced through the jacket to raise the material temperature to 60±2℃ and maintain this temperature. Stirring was continued at this temperature for 45 minutes. During this time, the solid modified egg yolk phospholipids and phytosterol esters were observed to gradually melt and integrate with the liquid MCT and propolis extract, forming a homogeneous, dark brown, slightly viscous, transparent liquid mixture. The stirring speed could be gradually increased to 200 rpm to enhance mixing.

[0037] High-pressure homogenization to form nano-dispersions: The premixed material was transferred to the insulated feed tank of a high-pressure homogenizer, maintaining the material temperature at 55-60℃. The homogenization pressure was set to 600 bar. The homogenizer was started, allowing the material to circulate through the homogenization valve. Homogenization process monitoring: After the first and third homogenization cycles, a drop sample was taken and placed on a glass slide for observation under a 400x optical microscope. A small number of incompletely dispersed micro-molecular particles were visible in the initial sample; after the third homogenization, the field of view showed a largely uniform background with no obvious particles. A total of 5 homogenization cycles were performed. The final homogenized sample was then measured using a dynamic light scattering particle size analyzer. The results showed an average hydrodynamic diameter of 108 nm and a polydispersity index of 0.21, indicating the formation of a narrowly distributed nano-dispersion. This dispersion appeared clear and transparent reddish-brown under natural light.

[0038] For soft capsule filling and post-processing, the aforementioned nano-dispersion was transferred to the storage tank of the soft capsule pelletizing machine. The tank was equipped with an insulation layer, and the temperature was set at 50±2℃ to prevent the viscosity of the contents from increasing. An oval-shaped, opaque gelatin sheet was selected. The pelletizing machine mold was adjusted to set the content filling amount of each capsule to 500 mg. The pelletizing operation was carried out in a clean room with an ambient temperature of 25±2℃ and a relative humidity of <40%. The pressed wet capsules were conveyed into a shaping drum via a conveyor belt. The shaped capsules were then transferred to a dynamic drying tunnel. The drying conditions were: temperature 22-25℃, humidity 20-25%, and drying time 24 hours. During this period, samples were taken and weighed periodically until the weight loss of the capsules tended to stabilize. After drying, the capsules were inspected and polished to obtain the finished propolis soft capsules of Example 3. The finished capsules had a smooth appearance, uniform color, and no leakage or deformation.

[0039] Comparative Example 1: In a mixing tank preheated to 60°C, 40.00 g of propolis extract, 52.00 g of ordinary egg yolk lecithin, and 8.00 g of MCT were added sequentially. Stirring was started, with an initial speed set at 100 rpm. The system temperature was maintained at 60±1°C using jacketed circulating water. Process observation and recording: 0-5 minutes: MCT rapidly wetted the egg yolk lecithin powder, forming a pale yellow paste. When the propolis extract was added, most of it adhered to the stir bar shaft and tank walls, not immediately dissolving into the oil phase. 6-20 minutes: With continued stirring, the mixture turned an uneven dark brown, becoming a completely opaque, turbid suspension. Visual inspection revealed no signs of transparency. Undispersed propolis particles could be observed moving with the streamlines behind the stir bar. 21-30 minutes: The stirring speed was increased to 200 rpm in an attempt to improve mixing. However, the turbidity of the system did not improve, and the adhering material to the tank walls remained. Stirring was stopped, and samples were taken for analysis. Initial Sample Analysis: Microscopic Examination: Place a drop of the mixture on a glass slide, cover with a coverslip, and observe immediately. The field of view reveals: numerous sharp-edged, dark brown to black irregular solid particles (propolis extract) ranging in size from 10-50 μm; and light yellow, translucent spheres or aggregates (phospholipid micelles / vesicles) ranging in size from 2-10 μm; both exist independently and do not form a homogeneous phase. Appearance: After standing for 1 minute, signs of particle deposition begin to appear at the bottom of the sample vial.

[0040] High-pressure homogenization was performed, transferring all the premixed material to the insulated feed tank (55℃) of a high-pressure homogenizer. The homogenization pressure was set to 600 bar, and cyclic homogenization was carried out, planned for 5 cycles. After the first homogenization: the mixture changed from a coarse suspension to a uniform, milky-white emulsion similar to a latte, completely opaque. Microscopic observation showed that large propolis particles were mechanically broken down, reducing their size to 1-5 μm, but still remaining in solid form; phospholipids formed a large number of more uniform submicron-sized (0.5-2.0 μm) emulsion droplets. After the third homogenization: there was no significant change in appearance, maintaining the milky-white color. Laser particle size analysis showed a bimodal distribution: the main peak was at 800 nm (polydispersity index PDI=0.35), corresponding to phospholipid micelles / droplets; the secondary peak was at 3 μm (broad distribution), corresponding to undissolved propolis solid particles. After the fifth homogenization: the particle size distribution did not change significantly. The system is a thermodynamically unstable emulsion-suspension mixture. Because the contents are an opaque, heterogeneous system with a risk of solid particle sedimentation, and its rheological properties do not meet the requirements of soft capsule machines for homogeneous, stable, and solid-free contents, the filling process was discontinued. Approximately 30 mL of homogenized sample was placed in a 50 mL stoppered transparent glass graduated cylinder and observed statically in a 25.0℃ incubator. After 2 hours: clear stratification appeared. The upper layer was a pale yellow, slightly turbid oil layer (approximately 30% of the total volume), and the lower layer was a dark brown, opaque emulsion layer (approximately 70% of the total volume), with a smooth interface. After 24 hours: the lower layer further separated, with a thick, dark brown precipitate of approximately 5 mm thickness forming at the bottom of the graduated cylinder. Gently inverting the graduated cylinder caused the precipitate to adhere to the bottom and be difficult to resuspend.

[0041] Conclusion: Using unmodified common egg yolk phospholipids, it is impossible to solubilize propolis extract to form a stable oil-phase nanodispersion. The process produces an unstable emulsion that eventually separates and precipitates.

[0042] Comparative Example 2: Premixing Attempt. 40.00 g of propolis extract, 52.00 g of fully hydrogenated egg yolk phospholipid powder, and 8.00 g of MCT were added to a mixing tank. Stirring was started (100 rpm), and the jacket temperature was set to 60°C. 0-15 minutes: MCT showed extremely poor solubility for the fully hydrogenated phospholipids. Most of the phospholipid powder remained in its original granular state, suspended or settled at the bottom of the tank, with only a small amount swelling on the surface. The system exhibited a separation between solid powder and liquid; the stirrer could only agitate the upper liquid layer, resulting in a hardened layer at the bottom. 16-30 minutes: The stirring speed was increased to 300 rpm, and the temperature was maintained at 60°C. The hardened layer was partially broken up, but the overall consistency transformed into an extremely viscous, non-flowing paste. When picked up with a glass rod, a paste several centimeters long could be drawn without breaking. The propolis extract remained encapsulated in clumps and could not be dispersed. Sampling: Attempts were made to collect samples for microscopic observation, but the paste was too viscous to be used to prepare suitable slide samples.

[0043] Process Failure and Root Cause Analysis: Because the premix completely lacks fluid properties, it cannot be transported to the subsequent high-pressure homogenizer via pipelines or pumps. Forced attempts resulted in blockage of the delivery pipeline. Therefore, the entire preparation process was forced to terminate during the premixing stage. Root Cause Analysis: Fully hydrogenated egg yolk phospholipids, due to their extremely high saturation and melting point, exhibit extremely low solubility and dispersibility in MCT at a process temperature of 60°C, making it impossible to form micelles or liquid crystal structures. Its physical state is incompatible with the target oil phase carrier medium, completely losing its function of dissolving and carrying propolis extract.

[0044] Conclusion: When the iodine value is too low, the over-hydrogenated egg yolk phospholipids cannot form an effective solubilizing system under the process conditions, leading to process failure. This indicates that controlling the iodine value of egg yolk phospholipids within a certain range is crucial. Within a certain range, it is crucial to maintain the necessary physicochemical properties for its function as a carrier.

[0045] Comparative Example 3: Preparation of Modified Egg Yolk Lecithin: Partial hydrogenation was performed in a 500 mL high-pressure reactor equipped with a mechanical stirrer, hydrogen inlet valve, pressure gauge, and temperature control system. 100.0 g of egg yolk lecithin powder and 300 mL of anhydrous tetrahydrofuran dried through a 4A molecular sieve were added. Dissolution and catalysis were initiated by stirring at 300 rpm and heating to 45°C to completely dissolve the lecithin. After purging the system three times with nitrogen, 1.0 g of dried palladium-on-carbon catalyst was added. The hydrogenation reaction was then carried out by purging the reactor with hydrogen three more times, followed by introducing hydrogen and maintaining the pressure at 3.0 bar. The reaction temperature was raised to 50°C, and the reaction time was started. Endpoint control was achieved by sampling approximately 1 mL of the reaction solution every 45 minutes through a sampling valve. After rapid filtration to remove the catalyst, the iodine value was determined according to the method in GB / T 5532-2008 "Determination of Iodine Value of Animal and Vegetable Oils". The iodine value was determined when two consecutive measurements stabilized within a certain range. After approximately 5.5 hours of reaction, hydrogen flow was stopped, and the mixture was cooled to room temperature. Post-treatment: The reaction solution was filtered through a 0.45 μm polytetrafluoroethylene (PTFE) membrane to completely remove the catalyst. The filtrate was transferred to a rotary evaporator and evaporated at a water bath of 40°C and a vacuum of -0.095 MPa to remove tetrahydrofuran, yielding a pale yellow, waxy, partially hydrogenated egg yolk phospholipid. The iodine value was measured to be... Store in a nitrogen-protected desiccator for later use.

[0046] Enzymatic transesterification: In a reaction flask, add the partially hydrogenated egg yolk phospholipids and ethyl oleate (molar ratio 1:2.0). Add 100 mL of anhydrous tert-butanol and 5 g of 3 Å molecular sieve. Add 10% Lipozyme TL IM enzyme and react at 55 °C and 200 rpm for 18 hours.

[0047] The soft capsule contents were formulated and filled using the exact same formula and subsequent processes as in Example 1: 40% propolis extract, 52% of the aforementioned "linear modified phospholipids," and 8% MCT. Premixing at 60°C, homogenization at 600 bar five times, filling, and drying were performed. Initial product characterization: Appearance: The contents were translucent with a distinct milky-white halo (strong Tyndall effect), showing a significant visual difference from the "clear and transparent" appearance of Example 1. Particle size analysis: Average particle size 155 nm, polydispersity index (PDI) 0.28. The particle size distribution map showed a shoulder peak extending to the micrometer region. Accelerated stability comparison test.

[0048] Test Samples: 50 soft capsules each from Comparative Example 3 and Example 1 were placed in a 40°C incubator for storage. Observation and Testing: Day 0: As described above. Day 15: The contents of Comparative Example 3 capsules showed slight turbidity to the naked eye, while Example 1 showed no change. Day 30: The contents of Comparative Example 3 turned opaque milky white. Upon cutting the capsule open, the contents became less fluid and slightly viscous. Example 1 remained transparent. Day 60: After the contents of Comparative Example 3 were left to stand, fine white flocculent deposits were visible at the bottom of the capsules. Upon removing the contents, a fine granular feel was felt when rubbed between the fingers. The contents of Example 1 remained homogeneous. The average particle size of the Comparative Example 3 sample increased from 155 nm to >500 nm, with a very wide distribution (PDI>0.4); the particle size of the Example 1 sample increased from 95 nm to 110 nm (PDI~0.22).

[0049] Conclusion: While phospholipids modified with straight-chain fatty acids can achieve initial dispersion of propolis, the resulting micelles or nanostructures exhibit poor long-term physical stability and are prone to aggregation, maturation, or phase separation during storage, leading to product deterioration. This demonstrates that branched-chain fatty acids, due to their unique three-dimensional structure, can generate a stronger steric hindrance effect, which is a key structural feature endowing nanodispersions with excellent long-term storage stability.

[0050] Comparative Example 4: Raw Material Preparation and Precise Weighing. Use a clean 100mL glass beaker as the container, tare and zero it. Slowly add 35.00g of propolis extract using a special spatula. Replace the spatula and add 50g of modified egg yolk lecithin. Measure 14.00g of oleic acid using a graduated cylinder and slowly pour it along the beaker wall. After weighing, gently stir a few times with a glass rod to initially moisten the powder, and record the appearance of the raw materials.

[0051] Premixing and Dissolving: Place the beaker on a smart heating plate equipped with a magnetic stirrer and a constant-temperature water bath. Add a magnetic stir bar to the beaker. Turn on the water bath circulation and set the temperature to 60.0℃. Start stirring, with an initial speed of 200 rpm. As the temperature rises, the solid modified egg yolk phospholipids and propolis extract gradually soften. After about 5 minutes, increase the stirring speed to 400 rpm to enhance mass transfer and shear, preventing the material from sticking to the walls. The total mixing time is 30 minutes. During this time, pause stirring at 15 minutes and 25 minutes, and use a spatula to scrape the material adhering to the walls and bottom of the beaker to the center. The mixture becomes a homogeneous, dark reddish-brown, somewhat fluid, viscous liquid without visible particles. There are no undissolved solids at the bottom of the beaker.

[0052] High-pressure homogenization was used to form a dispersion using an APV-1000 high-pressure homogenizer equipped with a temperature-controlled feed tank. The feed tank was preheated to 55°C. All the mixture obtained in step 2 was transferred to the feed tank. The primary pressure setting was 600 bar, the secondary pressure setting was 50 bar, and the material temperature was controlled at 55-60°C via jacket cooling water. The homogenizer was started for circulation. Five cycles were planned. After the first and third cycles, approximately 2 mL of sample was taken from the sampling valve using a glass syringe. The sample was dropped onto a glass slide, covered with a coverslip, and observed under a conventional optical microscope (400x). After the first cycle, a small number of tiny globules <5 μm in size were visible; after the third cycle, the field of view was generally clean and uniform. After the fifth cycle, the final sample was taken. The contents were a clear, transparent reddish-brown color, visually indistinguishable from the initial sample of Example 2.

[0053] Using Malvern Zetasizer Nano ZS, the Z-Average particle size was 102 nm, and the polydispersity index (PDI) was 0.19, indicating that a nanodispersion was successfully formed.

[0054] Soft capsule filling and shaping / drying: Filling preparation: Quickly transfer the homogenized contents into the hopper of the soft capsule compression machine. The hopper is equipped with an insulation jacket, and the temperature is set to 50±1℃. Use the same gelatin coating formula as in Example 2 (gelatin:glycerin:water = 40:20:40, with 2% titanium dioxide added) and the same elliptical mold. The filling workshop temperature is 25±1℃, and the relative humidity is 35±5%. Adjust the equipment to ensure that parameters such as coating thickness, sealing temperature, and rolling speed are completely consistent with the production records of Example 2. Start the machine, and the pressed wet capsules are naturally cooled and solidified by the conveyor belt. Spread the wet capsules evenly on the drying tray and place them in a constant temperature and humidity drying chamber. Drying program: First stage, 25℃, 25% humidity, drying for 12 hours; Second stage, 25℃, 20% humidity, drying for 12 hours. Record the temperature and humidity of the drying chamber every hour. After drying, randomly select 20 capsules, weigh them, calculate the average weight, and confirm that the moisture has reached equilibrium.

[0055] Two sets of samples, each containing 50 particles (for day 0 testing), were immediately tested. The remaining samples were placed in a 60°C forced convection oven (Memmert UF110). Sampling points: On days 7, 14, 21, and 30, the corresponding set of samples (50 particles each) were removed from the oven.

[0056] Test Items and Operating Procedures: Peroxide Value (POV): Take 3 capsules from the same day's sample and wipe the surface clean with filter paper. Cut open the capsules and squeeze the contents completely into a 50mL stoppered conical flask. Add 30mL of glacial acetic acid-isooctane mixture (volume ratio 3:2) and vortex for 5 minutes until completely dissolved. Strictly follow Method 1 of GB 5009.227-2016 "Determination of Peroxide Value in Food". Add 1mL of saturated potassium iodide solution, let stand in the dark for 1 minute, immediately add 100mL of distilled water, and titrate with 0.01mol / L sodium thiosulfate standard solution until pale yellow. Add 1mL of starch indicator and continue titrating until the blue color disappears. Prepare a reagent blank simultaneously. Calculation of results: POV (meq / kg) = (V - V0) * C * 1000 / m, where V is the volume of sodium thiosulfate consumed by the sample (mL), V0 is the blank volume (mL), C is the concentration of sodium thiosulfate (mol / L), and m is the sample mass (g). Total flavonoid content and retention rate: Mix the contents of 3 capsules from the same sample. Accurately weigh approximately 0.1g of the mixed sample into a 10mL volumetric flask, dissolve and dilute to volume with 70% ethanol, and shake well. Accurately pipette 1.0mL into a 10mL stoppered test tube, add 4mL of 30% ethanol, mix well, and use as a blank reference solution. Separately, accurately pipette 1.0 mL into another 10 mL stoppered test tube, add 0.3 mL of 5% sodium nitrite solution, shake well, and let stand for 6 minutes; add 0.3 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 minutes; add 4 mL of 1 mol / L sodium hydroxide solution, and bring the volume to 10 mL with 30% ethanol, shake well, and let stand for 15 minutes. Measure the absorbance (A) at 510 nm using a 1 cm cuvette, with the blank solution as a reference. Calculate the total flavonoid content in the sample based on the rutin standard curve measured on the same day (concentration C vs. absorbance A, linear range 5-50 μg / mL, R²>0.999). Retention rate (%) = (total flavonoid content at this time point / total flavonoid content on day 0) * 100%.

[0057] Sensory evaluation: A panel of three trained assessors conducted the evaluation. Samples were randomly numbered and placed in a separate sensory evaluation room. Assessors cut open the capsules, smelled the contents, and recorded whether the odor was "normal propolis / plant scent," "odorless," "slightly oily," "obvious rancid," or "strongly rancid." The consensus of all three assessors was taken.

[0058] The embodiments of the present invention are given for illustrative and descriptive purposes. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention. In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. "A plurality of" means two or more, unless otherwise explicitly specified.

[0059] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0060] Table 1. Effects of different phospholipids on the dispersion effect and product performance of propolis

[0061] Table 2. Preparation process parameters and results of modified egg yolk phospholipids

[0062] Table 3 Comparison of product stability under accelerated oxidation conditions

[0063] The results in Table 1 show that a transparent, nanoscale, and long-term physically stable propolis oil-soluble system can only be obtained when the phospholipids simultaneously meet the conditions of "partial hydrogenation (iodine value 35-45)" and "substitution of the sn-2 position by branched-chain fatty acids such as phytic acid". Both the ordinary phospholipid Comparative Example 1 and the over-hydrogenated phospholipid Comparative Example 2 failed to achieve the purpose of this invention, proving that the parameter range is necessary. Although the linear fatty acid modification in Comparative Example 3 achieved initial dispersion, the long-term stability of the product was significantly inferior to that of the branched-chain fatty acid modified Example 1, demonstrating that the branched structure is crucial.

[0064] Table 2 shows that Example 1, using a Pd / C catalyst and higher temperature and pressure, achieved a lower iodine value; Example 3, using a homogeneous rhodium catalyst and lower temperature and pressure, achieved a higher iodine value. This indicates that, Within this target range, various catalytic systems and reaction conditions can be used, demonstrating that this range is not a single, stringent point in the process, but rather a range with industrial operational flexibility. Enzymatic transesterification: Example 1, using a higher substrate ratio and a longer reaction time, achieved a higher substitution rate; Example 3, using a lower substrate ratio and a shorter reaction time, achieved a lower substitution rate. This indicates that the substitution rate range of "15%-25%" can be achieved through conventional methods such as adjusting reactant ratios and reaction time, and is not difficult to control.

[0065] Table 3 shows the results regarding peroxide value: Comparative Example 4 had a POV as high as 31.6 meq / kg on day 30, far exceeding the conventional limit for rancidity of food oils (usually ≤10 meq / kg), while Example 2 only had 4.9 meq / kg, representing a reduction in oxidation by approximately 85%. In Comparative Example 4, the core functional component of propolis (total flavonoids) degraded by approximately 21% on day 30, while in Example 2 it only degraded by approximately 5%, demonstrating a more than fourfold improvement in the protection of active ingredients. Comparative Example 4 developed a noticeable unpleasant odor after 14 days and became severely rancid by day 30, completely losing its commercial value; Example 2, on the other hand, maintained good sensory quality throughout. The addition of an antioxidant system composed of vitamin E, rosemary extract, and ascorbyl palmitate plays a decisive and irreplaceable role in ensuring the product's shelf-life stability, efficacy, and acceptability.

Claims

1. A propolis soft capsule, characterized in that, It is composed of the following raw materials by weight percentage: propolis extract 30%-50%, modified egg yolk phospholipids 40%-60%, and cosolvents 5%-15%; wherein, the modified egg yolk phospholipids are partially hydrogenated egg yolk phospholipids with branched-chain fatty acids substituted at the sn-2 position, and their iodine value is [value missing]. .

2. The propolis soft capsule according to claim 1, characterized in that: The branched fatty acid is phytic acid, and the substitution rate of phytic acid at the sn-2 position is 15%-25%.

3. The propolis soft capsule according to claim 2, characterized in that: The co-solvent is at least one of oleic acid or medium-chain triglycerides.

4. The propolis soft capsule according to claim 1, characterized in that: The raw materials also include 1%-5.5% functional additives, which are fat-soluble active ingredients and antioxidants.

5. The propolis soft capsule according to claim 4, characterized in that, The fat-soluble active ingredient is at least one of vitamin E, vitamin A, phytosterols or phytosterol esters, and the antioxidant is at least one of rosemary extract and ascorbyl palmitate.

6. A method for preparing propolis soft capsules as described in any one of claims 1-5, characterized in that, Includes the following steps: S1: Prepare modified egg yolk phospholipids by providing egg yolk phospholipid raw materials and sequentially performing partial hydrogenation and enzymatic transesterification treatments to obtain the modified egg yolk phospholipids. S2: Mixing and homogenizing. The modified egg yolk phospholipids, propolis extract and cosolvent obtained in step S1 are mixed in proportion and homogenized to form a stable nano-dispersion. S3: Filling and molding: Fill the contents obtained in step S2 into soft capsule shells and seal them.

7. The preparation method according to claim 6, characterized in that, In step S1, the partial hydrogenation treatment specifically involves: in an inert organic solvent, in the presence of a catalyst, introducing hydrogen gas into an egg yolk phospholipid solution to carry out the reaction, and controlling the iodine value of the resulting partially hydrogenated egg yolk phospholipid to be... The enzymatic transesterification process specifically involves mixing the partially hydrogenated egg yolk phospholipids with ethyl phytate in a non-aqueous medium, adding sn-1,3 specific lipase to catalyze the reaction, and introducing branched fatty acids.

8. The preparation method according to claim 7, characterized in that, In the partial hydrogenation treatment, the catalyst is palladium on carbon or triphenylphosphine rhodium chloride, and the amount used is 0.5%-2.0% of the egg yolk phospholipid content. The reaction temperature is 40-60℃, and the hydrogen pressure is 2-4 bar. In the enzymatic transesterification treatment, the lipase is immobilized Lipozyme TL IM, the reaction temperature is 50-60℃, the reaction time is 12-24 hours, and the molar ratio of the partially hydrogenated egg yolk phospholipid to ethyl phytate is 1:1.5 to 1:3.

0.

9. A composition of propolis soft capsule contents, characterized in that, It is prepared by any one of claims 6-8.

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