A method for preparing seabuckthorn seed oil double emulsion-microcapsule powder

Abstract

The present application relates to the technical field of microencapsulation, and discloses a preparation method of seabuckthorn seed oil double emulsion-microcapsule powder, which comprises the following steps: 1) mixing seabuckthorn seed oil with sodium caseinate-phosphate buffer solution to prepare a single emulsion; mixing fucoidan-phosphate buffer solution with the single emulsion to prepare a double emulsion; 2) adding malt dextrin or a malt dextrin / β-cyclodextrin mixture to the double emulsion, and freeze-drying to obtain seabuckthorn seed oil double emulsion-microcapsule powder. The present application is based on secondary emulsification+microencapsulation technology, and by optimizing the types, proportions and microencapsulation process types of raw materials, a kind of seabuckthorn seed oil double emulsion-microcapsule powder is prepared, which can significantly improve the storage stability and bioavailability of seabuckthorn seed oil. The seabuckthorn seed oil double emulsion-microcapsule powder of the present application is especially suitable for preparing enteric products, and after taking, it can ensure that seabuckthorn seed oil is mainly digested and absorbed in the intestinal digestion stage.

Description

Technical Field

[0001] This invention relates to the field of microencapsulation technology, and in particular to a method for preparing a bilayer emulsion-microcapsule powder of sea buckthorn seed oil. Background Technology

[0002] Sea buckthorn (Hippophae rhamnoides L.) is a plant used for both food and medicine. Sea buckthorn seeds, a major byproduct of the sea buckthorn processing industry, contain approximately 10% oil. Sea buckthorn seed oil is rich in polyunsaturated fatty acids, such as alpha-linolenic acid (ALA), and has good nutritional and health benefits. However, the polyunsaturated fatty acids in sea buckthorn seed oil lead to its unstable physicochemical properties, making it susceptible to the effects of light, heat, and yang energy, generating a series of oxides such as aldehydes and ketones. This results in a short shelf life and the formation of unpleasant odors, affecting the sensory quality and nutritional value of the product. Furthermore, functional oils, due to their inherent instability and hydrophobic properties, have low absorption and utilization rates in the body. To overcome the numerous limitations in the processing, storage, and human utilization of sea buckthorn seed oil, it is urgent to design a stable delivery system for sea buckthorn seed oil to further expand and promote its application in the food and pharmaceutical fields.

[0003] Emulsions and microcapsules are two commonly used delivery systems for functional oils. Their raw materials and excipients comply with food and pharmaceutical regulations, exhibiting strong adaptability. Through formulation and process adjustments, they can meet the needs of different products, possessing characteristics such as stability and industrial feasibility. Emulsions, in particular, involve mixing oils with one or more polyelectrolyte layers and homogenizing them using strong external mechanical energy to form a micron or nano-sized emulsion containing single or multiple layers of electrolyte-encapsulated oil particles. This is a high-encapsulation technology commonly used for stabilizing oils rich in polyunsaturated fatty acids. However, limitations such as short shelf life and stringent storage conditions typically restrict their development as nutritional supplements or pharmaceutical products. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing a bilayer emulsion-microcapsule powder of sea buckthorn seed oil. First, the invention compares the effects of different wall materials (protein and polysaccharides) and raw material ratios on the stability of the sea buckthorn seed oil emulsion, selecting suitable raw materials and ratios to obtain a stable bilayer emulsion. Then, maltodextrin or a maltodextrin / β-cyclodextrin mixture is added to prepare microcapsule powder. By comparing the effects of different maltodextrin addition amounts and different mixing ratios of maltodextrin and β-cyclodextrin on the stability of sea buckthorn seed oil, a suitable microcapsule formulation is determined. In summary, the sea buckthorn seed oil microcapsule powder prepared by this invention through secondary emulsification + microencapsulation technology improves the storage stability and bioavailability of sea buckthorn seed oil. This method has advantages such as simple operation and high encapsulation efficiency, making it suitable for preparing enteric-coated / intestinal-targeted release products.

[0005] The specific technical solution of this invention is as follows: First, this invention provides a method for preparing a bilayer emulsion-microcapsule powder of sea buckthorn seed oil, which includes the following steps: 1) Preparation of sea buckthorn seed oil bilayer emulsion: Under neutral conditions, sea buckthorn seed oil was mixed with sodium caseinate (NaCS)-phosphate buffer solution at a mass ratio of 5:95-15:85. After shearing and homogenization, a sodium caseinate monolayer emulsion with a particle size of 250-350 nm, a PDI of 0.1-0.25, and a ζ-potential of -45 to -30 mV was formed. Fucoidan (FUC)-phosphate buffer solution at a mass ratio of 1:8-10 was added to the monolayer emulsion. The mixture was stirred, and the pH was adjusted to 5.0-5.5. After shearing, a sea buckthorn seed oil bilayer emulsion with a particle size of 350-500 nm, a PDI of 0.12-0.38, and a ζ-potential of -50 to -35 mV was obtained. The core of the sea buckthorn seed oil bilayer emulsion is sea buckthorn seed oil, and the outer layer is a sodium caseinate-fucose polysaccharide composite layer. The hydrophilic groups of the fucoidan are exposed on the outer surface and in contact with the aqueous phase, forming a stable emulsion system.

[0006] 2) Preparation of microcapsule powder: Add 10-15 wt% maltodextrin or a mixture of 50-70 wt% maltodextrin / β-cyclodextrin to the bilayer emulsion of sea buckthorn seed oil and stir to form a homogeneous emulsion system. Pre-freeze at -90 to -70℃ for 20-30 h, freeze-dry at -60 to -40℃ for 70-80 h, thaw, grind into powder, and sieve to obtain sea buckthorn seed oil bilayer emulsion-microcapsule powder.

[0007] Compared with existing technologies that only perform single emulsification, double emulsification, or direct microencapsulation of sea buckthorn seed oil, the double emulsion-microcapsule powder of sea buckthorn seed oil obtained by the present invention through double emulsification → microencapsulation can further significantly improve the storage stability and bioavailability of sea buckthorn seed oil, and is especially suitable for the preparation of enteric-coated / intestinal-targeted products (with higher release during intestinal digestion).

[0008] In order to ensure the preparation of sea buckthorn seed oil bilayer emulsion-microcapsule powder with the above-mentioned technical effects, the key points of the preparation method of the present invention are as follows: (1) Screening of protein types in monolayer emulsion and polysaccharide types in bilayer emulsion: The present invention first uses proteins and polysaccharides to perform secondary emulsification of sea buckthorn seed oil. The present invention found through experiments that different proteins and polysaccharides have a significant impact on the properties of sea buckthorn seed oil bilayer emulsion. In the monolayer emulsification test, this invention found that the monolayer emulsion prepared with sodium caseinate (NaCS) had the smallest particle size, PDI, and higher absolute zeta potential value compared to soy protein isolate (SPI) and pea protein isolate (PPI). (A higher absolute zeta potential value indicates a stronger electrostatic repulsion between oil droplets, which can overcome the molecular attraction between droplets and energy barriers, thus preventing flocculation and reducing the tendency for droplet stratification. Smaller particle size and lower PDI indicate a more uniform molecular weight distribution and greater stability in the emulsion system.) This indicates that sodium caseinate is more stable than the other two proteins in emulsifying sea buckthorn seed oil. Furthermore, this invention found that in secondary emulsification, for the specific protein sodium caseinate, the bilayer emulsion prepared with fucoidan (FUC) had the smallest particle size and PDI value compared to sodium alginate (SA) and chitosan (CTS), indicating the most uniform particle size distribution. Therefore, sodium caseinate and fucoidan are the most suitable wall material combination for sea buckthorn seed oil emulsion.

[0009] (2) Optimization of protein concentration in monolayer emulsion and polysaccharide concentration in bilayer emulsion: This invention compared the preparation of sea buckthorn seed oil monolayer emulsions with different concentrations of sodium caseinate (NaCS)-phosphate buffer solution. The results showed that when the concentration was less than 2.5%, the particle size of the emulsion gradually decreased, and the PDI value generally showed a downward trend. This is because at lower concentrations, the added polyelectrolyte concentration was insufficient to cover the entire droplet surface, causing adjacent droplets to aggregate by sharing the same polyelectrolyte, resulting in a larger particle size and a larger PDI. When the concentration increased to 2.5-3.0%, sodium caseinate could just completely cover the oil droplet surface without flocculation. At this time, the repulsive force between the droplets was greater than their attractive force, and the interaction force between the droplets reached a steady-state equilibrium, thus obtaining a stable monolayer emulsion. However, when the concentration continued to increase (greater than 3.0%), dissipative flocculation occurred, increasing the particle size of the emulsion. Therefore, the preferred sodium caseinate concentration range was 2.5-3.0%.

[0010] This invention compares the preparation of bilayer emulsions of sea buckthorn seed oil using fucoidan-phosphate buffer solutions of different concentrations. The results show that an inappropriate fucoidan concentration can lead to flocculation or stratification of the emulsion, reducing its stability. At low concentrations (<0.3%), the fucoidan is insufficient to completely cover the monolayer emulsion droplets, making it easy for polymer bridges to form between adjacent droplets, resulting in aggregation and increased particle size. When the concentration increases to 0.4%, the monolayer emulsion is completely covered by fucoidan, increasing the repulsive force between droplets, making aggregation less likely, and reducing particle size. When the concentration continues to increase (>0.4%), excess unadsorbed fucoidan leads to increased osmotic stress within the system, potentially causing droplet aggregation and further increasing the emulsion particle size.

[0011] (3) Screening of microcapsule wall materials: This invention selects maltodextrin or a mixture of maltodextrin and β-cyclodextrin as the microcapsule wall material. This invention found that using maltodextrin or a mixture of maltodextrin and β-cyclodextrin has better effects. Maltodextrin is a mixture of linear or branched oligosaccharides with low degree of polymerization. It has good water solubility, low sweetness, and moderate viscosity, and mainly acts as a filler, thickener, and carrier. β-cyclodextrin molecules are cylindrical, hydrophilic on the outside and hydrophobic on the inside, with a unique cavity structure. It is suitable for embedding hydrophobic small molecules with a high embedding rate, which can compensate for the relatively low embedding rate of maltodextrin and its disadvantage of being easily digested and absorbed by the human body to provide heat. However, its solubility is low. Therefore, a mixture of maltodextrin and β-cyclodextrin can be preferred to prepare powder microcapsules.

[0012] (4) Optimization of the amount of wall material used in microcapsule powder: When using pure maltodextrin, as the amount of maltodextrin increases, the total oil content and surface oil content of the microcapsule powder gradually decrease, while the encapsulation rate gradually increases. However, the total oil content remains almost unchanged or even shows a downward trend after the amount of maltodextrin added exceeds 10%. Therefore, the optimal amount of maltodextrin added is 10-15%. Under the condition of adding a mixture of maltodextrin and β-cyclodextrin with a total amount of 10-15%, and within the mass ratio of maltodextrin to β-cyclodextrin of 9:1-5:5, this invention found that when the mass ratio exceeds 7:3, the total oil content and encapsulation rate of the powder microcapsules remain basically unchanged. Therefore, the preferred mass ratio of maltodextrin to β-cyclodextrin is 7:3-5:5.

[0013] (5) Determination of process route: This invention compared two process routes, "assembly before emulsification" and "secondary emulsification," to prepare bilayer sea buckthorn seed oil. The results showed that the bilayer emulsion obtained by "secondary emulsification" had smaller particle size, PDI value, and lower ζ-potential, while the bilayer emulsion prepared by "assembly before emulsification" had larger particles, higher PDI value, and higher ζ-potential. This indicates that the emulsion prepared by this route has larger particles, uneven particle size distribution, and unstable properties. After being placed at room temperature for one week, the emulsion prepared by the "assembly before emulsification" route experienced severe demulsification, with oil overflowing to form a thicker oil layer. In contrast, the emulsion prepared by the "secondary emulsification" route did not experience severe demulsification, and the upper layer of oil accumulation was not obvious, thus exhibiting better stability. The reason for the difference in emulsion stability caused by the two preparation methods may be that sodium caseinate and fucoidan lost their good amphiphilicity after assembly and could not be firmly adsorbed onto the surface of oil droplets, leading to demulsification and oil precipitation during storage. Therefore, the "secondary emulsification" process is a suitable method for preparing sea buckthorn seed oil double-layer emulsion and then preparing powder microcapsules.

[0014] Preferably, in step 1), the method for preparing sea buckthorn seed oil includes: after screening, washing and drying sea buckthorn seeds, extracting them using supercritical CO2, and then encapsulating them with urea to obtain sea buckthorn seed oil.

[0015] Preferably, in step 1), the α-linolenic acid content of the sea buckthorn seed oil is 40-45%.

[0016] Preferably, in step 1), when preparing the sodium caseinate monolayer emulsion, the shearing speed is 6000-10000 rpm and the time is 2-5 min.

[0017] Preferably, in step 1), when preparing the sea buckthorn seed oil bilayer emulsion, the shearing speed is 10000-15000 rpm and the time is 3-7 min; the homogenization pressure is 35-45 MPa and the number of times is 2-5.

[0018] Preferably, in step 2), the particle size of the sea buckthorn seed oil bilayer emulsion-microcapsule powder obtained after sieving is 500-1000 nm.

[0019] Preferably, in step 2), the total oil content of sea buckthorn seed oil in the sea buckthorn seed oil bilayer emulsion-microcapsule powder is ≥15%, and the encapsulation rate is ≥74%.

[0020] The sea buckthorn seed oil bilayer emulsion-microcapsule powder prepared by the method of the present invention has a high sea buckthorn seed oil content and encapsulation rate.

[0021] Secondly, the present invention provides a sea buckthorn seed oil bilayer emulsion-microcapsule powder obtained by the above preparation method.

[0022] Furthermore, the present invention provides an application of the above-mentioned sea buckthorn seed oil bilayer emulsion-microcapsule powder in the preparation of enteric-coated / intestinal-targeted release foods or enteric-coated / intestinal-targeted release drugs.

[0023] The sea buckthorn seed oil bilayer emulsion-microcapsule powder prepared by the method of the present invention is particularly suitable for the preparation of enteric-coated (or intestinal-targeted release) foods or enteric-coated (or intestinal-targeted release) drugs. After taking the medicine, the amount of sea buckthorn seed oil released during the oral and gastric digestion stages is relatively low, while the amount of oil released during the intestinal digestion stage is relatively high, thereby ensuring that the sea buckthorn seed oil is fully digested and absorbed by the intestine.

[0024] Preferably, in an in vitro simulated digestive fluid, the oil release amount of the sea buckthorn seed oil bilayer emulsion-microcapsule powder is ≤0.4% in the oral digestion stage, ≤47% in the gastric digestion stage, and ≥84% in the intestinal digestion stage.

[0025] Finally, the present invention provides a method for improving the stability or bioavailability of sea buckthorn seed oil, which includes the following steps: 1) Preparation of sea buckthorn seed oil bilayer emulsion: Under neutral conditions, sea buckthorn seed oil was mixed with sodium caseinate (NaCS)-phosphate buffer solution at a mass ratio of 5:95-15:85. After shearing and homogenization, a sodium caseinate monolayer emulsion with a particle size of 250-350 nm, a PDI of 0.1-0.25, and a ζ-potential of -45 to -30 mV was formed. Fucoidan (FUC)-phosphate buffer solution at a mass ratio of 1:8-10 was added to the monolayer emulsion. The mixture was stirred, and the pH was adjusted to 5.0-5.5. After shearing, a sea buckthorn seed oil bilayer emulsion with a particle size of 350-500 nm, a PDI of 0.12-0.38, and a ζ-potential of -50 to -35 mV was obtained.

[0026] 2) Preparation of microcapsule powder: Add 10-15 wt% maltodextrin or a mixture of 50-70 wt% maltodextrin / β-cyclodextrin to the bilayer emulsion of sea buckthorn seed oil and stir to form a homogeneous emulsion system. Pre-freeze at -90 to -70℃ for 20-30 h, freeze-dry at -60 to -40℃ for 70-80 h, thaw, grind into powder, and sieve to obtain sea buckthorn seed oil bilayer emulsion-microcapsule powder.

[0027] On the one hand, the method of this invention can significantly improve the storage stability of sea buckthorn seed oil; on the other hand, the method of this invention can significantly improve the bioavailability of sea buckthorn seed oil. The reason for this may be that in the bilayer emulsion-microcapsule powder structure of sea buckthorn seed oil, NaCS and FUC assemble to form a stable and dense complex. The hydrophobic groups of NaCS are bound to the sea buckthorn seed oil and then encapsulated by the complex. Simultaneously, FUC enhances mucus penetration. The loose and porous structure formed by freeze-drying maltodextrin or maltodextrin-β-cyclodextrin complex allows for rapid release of small-diameter oil particles after reconstitution, increasing the cell contact area and thus significantly improving its bioavailability.

[0028] Compared with the prior art, the beneficial effects of the present invention are: (1) Based on the secondary emulsification + microencapsulation technology, this invention has prepared a double emulsion-microcapsule powder of sea buckthorn seed oil by optimizing the types of raw materials, the proportion of raw materials and the type of microencapsulation filler, which can significantly improve the storage stability and bioavailability of sea buckthorn seed oil. This method has the advantages of simple operation, low equipment requirements and high encapsulation rate.

[0029] (2) The sea buckthorn seed oil double emulsion-microcapsule powder prepared by the method of the present invention is particularly suitable for the preparation of enteric (or intestinal targeted release) food or enteric (or intestinal targeted release) medicine. After taking the medicine, the amount of sea buckthorn seed oil released in the oral cavity and stomach digestion stage is relatively low, while the amount of oil released in the intestinal digestion stage is high, thereby ensuring that the sea buckthorn seed oil is fully digested and absorbed by the intestine. Attached Figure Description

[0030] Figure 1 Particle size, PDI (a), and ζ-potential (b) of monolayer emulsions prepared from different protein solutions are shown. The addition amounts of NaCS, PPI, and SPI were 2.5%. Note: Different letters indicate significant differences between groups. p <0.05); Figure 2 Particle size, PDI (a), and ζ-potential (b) of monolayer emulsions prepared for NaCS solutions of different concentrations were analyzed. Note: Different letters indicate significant differences between groups. p <0.05); Figure 3 Particle size, PDI (a), and ζ-potential (b) of bilayer emulsions prepared from different polysaccharide solutions; Note: Different letters indicate significant differences between groups. p <0.05); Figure 4 Particle size, PDI (a), and ζ-potential (b) of bilayer emulsions prepared from FUC solutions of different concentrations; Note: Different letters indicate significant differences between groups. p <0.05); Figure 5 Changes in particle size (a), ζ-potential (b), and ALA-EE content (c) of sea buckthorn seed oil during single and double emulsion storage. Figure 6 For maltodextrin (ac) and maltodextrin+ β - Comparison of the physicochemical properties of powder microcapsules using a cyclodextrin mixture (7:3, mass ratio) (df) as a filler; Figure 7 The changes in POV value and ALA-EE content of crude oil and powder microcapsules during room temperature storage; Figure 8 The fatty acid release rate of sea buckthorn seed oil in powdered microcapsules during simulated intestinal digestion; Figure 9 Cytotoxicity test of steady-state samples (a) and apparent permeability coefficient (Papp) of sea buckthorn seed oil (b); Note: Different letters indicate significant differences between groups ( p <0.05); Figure 10 This example compares the particle size, PDI, ζ-potential (a & b), and morphological stability (c) of emulsions prepared from sea buckthorn seed oil using sodium caseinate (NaCS) and fucoidan (FUC) as raw materials, respectively, through pre-assembly followed by emulsification (Scheme A) and secondary emulsification (Scheme B); Note: Different letters indicate significant differences between groups. p <0.05); Figure 11 This is a transmission electron microscope image of the bilayer emulsion prepared by scheme B (secondary emulsification) in Example 2. Detailed Implementation

[0031] The present invention will be further described below with reference to embodiments.

[0032] Example 1 (1) Preparation of sea buckthorn seed oil emulsion (1.1) Preparation of wall material solution After being screened, cleaned, and dried, sea buckthorn seeds are used to extract sea buckthorn seed oil (with an α-linolenic acid content of about 30%) using supercritical CO2 extraction. The sea buckthorn seed oil with α-linolenic acid (about 42%) is then encapsulated and enriched with urea as raw material.

[0033] Prepare 10x phosphate buffer solutions (pH 7.0) and thoroughly dissolve soy protein isolate (SPI), pea protein isolate (PPI), sodium caseinate (NaCS), chitosan (CTS), sodium alginate (SA), and fucoidan (FUC) at room temperature to prepare protein-phosphate buffer solutions (1-4 wt%) or polysaccharide-phosphate buffer solutions (0.1-0.5 wt%) of different types at specific concentrations.

[0034] (1.2) Preparation of sea buckthorn seed oil emulsion Preparation of monolayer emulsions: At pH 7.0, 10 wt% sea buckthorn seed oil was mixed with 90 wt% of different types of protein-phosphate buffer solutions of specific concentrations, and the mixture was sheared at 12,000 rpm for 5 min. Then, it was homogenized three times under high pressure at 40 MPa to form monolayer emulsions. The emulsification effects of different types of proteins on sea buckthorn seed oil were compared, the most suitable protein was screened, and the concentration of protein-phosphate buffer solution was further optimized.

[0035] Preparation of bilayer emulsion: Different types of polysaccharide-phosphate buffer solutions of specific concentrations were added to the monolayer emulsion at a mass ratio of 1:8. The mixture was magnetically stirred at 200 rpm for 30 min, and the pH was adjusted to 5.0 to prepare the bilayer emulsion. Then, the mixture was sheared at 8000 rpm for 3 min to obtain the bilayer emulsion.

[0036] (1.3) Determination of emulsion particle size distribution and surface ζ-potential The particle size of the emulsion was determined using a nanoparticle size analyzer and a zeta potential analyzer. The relative refractive index and absorbance were set to 1.590 and 0.001, respectively, and the test temperature was 25 °C. Emulsion storage stability analysis was also performed. (1.4) Analysis of emulsion storage stability The appearance changes of the emulsion at room temperature (26 °C) for 0 and 14 days were recorded, and the particle size, ζ-potential and PDI of the emulsion were measured. At the same time, the changes in fatty acid content of the oil after demulsification were measured by gas chromatography.

[0037] (1.5) Determination of fatty acid and linolenic acid content in sea buckthorn seed oil First, sea buckthorn seed oil was ethylated. A 1% (w / w) KOH catalyst was dissolved in 95% ethanol and poured into a three-necked flask. Sea buckthorn seed oil was added at a molar ratio of ethanol to sea buckthorn seed oil of 8:1. The mixture was magnetically stirred at 200 rpm for a certain period and refluxed at 70°C for 1.5 h. After the reaction was complete, the mixture was cooled to room temperature. The cooled mixture was poured into a separatory funnel to remove the lower reaction layer. The upper oil layer was collected and continuously washed with an equal volume of hot saturated NaCl solution until the lower aqueous phase was clear and transparent. The upper oil layer was collected, and an appropriate amount of petroleum ether was added to dissolve it. Anhydrous sodium sulfate was added for dehydration and drying. After filtration, the petroleum ether was removed by vacuum rotary evaporation to obtain sea buckthorn seed oil fatty acid ethyl ester.

[0038] The content of fatty acid ethyl esters in sea buckthorn seed oil was determined by gas chromatography (GC). The content of ethyl linolenic acid (ALA-EE) represents the content of linolenic acid in sea buckthorn seed oil. 200 mg of fatty acid ethyl esters from sea buckthorn seed oil was weighed and diluted to 2 mL with n-heptane. After thorough mixing, anhydrous sodium sulfate was added for dehydration. The mixture was filtered through a 0.22 μm microporous membrane and transferred to a gas chromatographic vial for analysis. GC conditions: flame ionization detector, detector temperature 240 ℃, injection port temperature 230 ℃; SH-2560 column (100 m × 0.25 mm × 0.2 μm); temperature program: initial 100 ℃, hold for 4 min, increase to 240 ℃ at a rate of 2.9 ℃ / min, hold for 15 min; split mode: split (100:1); control mode: constant linear velocity (21 cm / s); carrier gas: nitrogen; fuel gas: hydrogen; combustion gas oxidizer: high-purity air; injection volume: 1 μL. According to formula (1), the ALA-EE content in fatty acid ethyl esters of sea buckthorn seed oil was calculated using the area normalization method. Where A represents the peak area of ​​ALA-EE. This is the sum of the peak areas of all fatty acid ethyl esters (excluding solvent peaks).

[0039] (2) Preparation of powder microcapsules Preparation of powder microcapsules by freeze-drying: A certain proportion of maltodextrin or a mixture of maltodextrin and β-cyclodextrin as wall material filler is added to the emulsion, poured into a petri dish, pre-frozen in a -80 ℃ freezer for 24 h, and freeze-dried in a -50 ℃ vacuum freeze dryer for 72 h until the sample temperature returns to room temperature. The block microcapsules are then pulverized into powder using a grinder and sieved to obtain sea buckthorn seed oil powder microcapsules.

[0040] (2.2) Screening of powder microcapsule wall material concentration or ratio 5–15 wt% of maltodextrin powder or maltodextrin powder mixed with β-cyclodextrin in different proportions were added to single-layer emulsions and double-layer emulsions, respectively. After magnetic stirring at 200 rpm for 1 h, a stable and homogeneous emulsion system was formed. The system was then immediately subjected to freeze pretreatment. The physicochemical properties of the prepared powder microcapsules, such as surface oil content and encapsulation efficiency, were investigated.

[0041] (2.3) Total oil content, surface oil content, and encapsulation efficiency of the powder microparticles After vortexing 3 g of sample for 10 minutes with 30 mL of extraction solvent (hexane:isopropanol = 1:1, v / v), centrifuge at 4500 rpm for 10 min at room temperature, collect the supernatant, filter through a 0.22 μm filter membrane, add 30 mL of extraction solvent to the remaining filter residue, and repeat the above steps until the filter residue is white to ensure complete oil extraction. Add anhydrous sodium sulfate to the collected supernatant for dehydration, remove the anhydrous sodium sulfate, and then rotary evaporate to remove the solvent. The weight of the remaining oil in the bottle is the total oil weight m1. The formula for calculating the total oil content (2) is as follows: Add 30 mL of hexane to 3 g of powder microcapsules and mix for 1 min. Quickly filter the solvent using a Buchner funnel. Rinse the powder microcapsules twice with 10 mL of hexane. Collect the filtrate and remove the solvent by rotary evaporation. The weight of the remaining oil in the bottle is the surface oil m2. The formula for calculating the surface oil content (3) is as follows: The embedding rate is expressed as a percentage (%), and the calculation formula (4) is as follows: (2.4) Storage stability test of powder microcapsules Accelerated storage experiments were conducted in an oven at 40 ℃. The powdered microcapsules and the raw oil were placed in the oven and stored in a sealed container for 4 weeks. The peroxide value (POV) and ALA content of the raw oil and powdered microcapsules were measured every 7 days. The method for determining the peroxide value (POV) of sea buckthorn seed oil was as follows: The determination was performed according to the method specified in GB 5009.227-2023, "National Food Safety Standard - Determination of Peroxide Value in Food".

[0042] (3) Results and Discussion (3.1) Effects of different proteins on the properties of sea buckthorn seed oil monolayer emulsion In emulsion systems, a larger absolute value of the zeta potential indicates a stronger electrostatic repulsion between oil droplets. This repulsion can overcome the molecular attraction between droplets and energy barriers, thus preventing emulsion flocculation and reducing the tendency for droplet stratification. Smaller emulsion particle size and lower PDI indicate a more uniform molecular weight distribution and greater stability of the emulsion system. Sea buckthorn seed oil monolayer emulsions were prepared using 2.5% sodium caseinate (NaCS), soy protein isolate (SPI), and pea protein isolate (PPI) solutions, respectively. The particle size distribution and zeta potential are shown below. Figure 1As shown, the monolayer emulsion prepared by sodium caseinate (NaCS)-phosphate buffer solution has the smallest particle size (274.47 nm), PDI (0.254), and a relatively high absolute value of ζ-potential (-40.53 mV), indicating that the monolayer emulsion prepared by sodium caseinate (NaCS)-phosphate buffer solution is more stable than the monolayer emulsions prepared by the other two protein-phosphate buffer solutions. Therefore, sodium caseinate was selected as the suitable protein for sea buckthorn seed oil.

[0043] (3.2) Effect of different concentrations of sodium caseinate (NaCS) on the stability of monolayer emulsions Six different concentrations (1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%) of NaCS-phosphate buffer solution were selected to prepare sea buckthorn seed oil monolayer emulsions, and their particle size, zeta potential, and PDI value were investigated. Figure 2 ).Depend on Figure 2 It can be seen that within the range of 1.0% to 4.0%, the particle size of the emulsion first decreases and then increases. When the NaCS addition is 2.5%-3.0%, the particle size is the smallest (266.88-276.02 nm), and the PDI value is 0.185-0.188. At this point, NaCS completely covers the surface of the oil droplets, and the repulsive force between the emulsion particles is greater than their attractive force, thus obtaining a stable monolayer emulsion. However, when the NaCS addition is low, it is insufficient to cover the entire surface of the oil droplets, causing adjacent droplets to aggregate by sharing the same polyelectrolyte, resulting in larger and non-uniform particle sizes. When the NaCS addition exceeds 3.0%, the excessive NaCS will cause mutual repulsion between emulsion particles, resulting in dissipative flocculation and increasing the particle size of the emulsion. The PDI value first increases and then continues to decrease when the NaCS addition is greater than 3.0%, while the ζ-potential continues to decrease with the increase of NaCS addition, remaining stable at -41.5 mV. In summary, 2.5-3.0% NaCS is the optimal concentration for monolayer emulsions, with 3.0% being the best concentration.

[0044] (3.3) Effect of different polysaccharides on the stability of bilayer emulsions Bilayer emulsions exhibit higher encapsulation efficiency than monolayer emulsions. Based on a stable monolayer emulsion, bilayer emulsions were prepared by adding different secondary polymers. The effects of sodium alginate (SA), chitosan (CTS), and fucoidan (FUC) on the stability of the bilayer emulsions are as follows: Figure 3 As shown.

[0045] The results showed that only the bilayer emulsion prepared by NaCS-FUC had the smallest particle size (355.23 nm) and the lowest PDI value (0.317), indicating that the emulsion particles were uniformly dispersed and the system was stable. Although sodium caseinate molecules are negatively charged overall, there are still local positively charged regions on their surface. These regions combine with the negatively charged groups of fucoidan through electrostatic interactions, causing sodium caseinate to bond to the cross-linking network of fucoidan, forming a dense sodium caseinate-fucose self-assembled complex. CTS is positively charged and has a large molecular weight, exhibiting high rigidity and strength. It forms a complex with the negatively charged sodium caseinate through strong electrostatic attraction. Therefore, the bilayer emulsion prepared by NaCS-CTS has the largest particle size (567.01 nm) and a ζ-potential close to zero, with a PDI value of 0.65, indicating that the emulsion is unstable. SA is a weakly anionic polysaccharide that encapsulates monolayer emulsion particles by forming a gel network. The particle size (435.76 nm), zeta potential (-45.47 mV), and PDI value (0.53) of the NaCS-SA bilayer emulsion are significantly higher than those of the NaCS-FUC bilayer emulsion. Therefore, NaCS-FUC is selected as the most suitable protein-polysaccharide wall material combination for bilayer emulsions.

[0046] (3.4) Effect of different polysaccharide concentrations on the stability of bilayer emulsions Inappropriate polysaccharide concentrations can lead to flocculation or stratification of emulsions, reducing emulsion stability. For example... Figure 4 As shown, when the FUC concentration is low (<0.3%), it is insufficient to completely cover the monolayer emulsion droplets, and polymer bridges easily form between adjacent droplets, leading to aggregation and an increase in emulsion particle size. When the FUC concentration is between 0.3% and 0.4%, the monolayer emulsion is completely covered by FUC, the repulsive force between emulsion droplets increases, aggregation is less likely, particle size decreases, and the PDI value and ζ-potential are also lower, indicating that the emulsion particles are more uniform and stable, especially when the FUC addition is 0.4%. When the FUC concentration continues to increase (>0.4%), the excess unadsorbed FUC leads to an increase in osmotic stress within the system, which may cause droplet aggregation, thereby increasing the emulsion particle size. Therefore, 0.4 wt% is the optimal FUC solution concentration for bilayer emulsions.

[0047] (3.5) Results of emulsion storage stability analysis After 7 days of storage at room temperature (26 °C), the separation of monolayer and bilayer emulsions was not obvious. In the monolayer emulsion, slight demulsification at the top led to oil precipitation and aggregation, resulting in a top emulsion layer with increased particle size and zeta potential. In the bilayer emulsion, the introduction of FUC enhanced the protection of oil droplets and protein molecules. Although both particle size and zeta potential increased, compared to the monolayer emulsion, the bilayer emulsion showed less change in particle size, lower zeta potential, and higher ALA-EE retention rate after 14 days of storage. Figure 5This indicates that bilayer emulsions are more stable than monolayer emulsions.

[0048] (3.6) Screening results of preparation methods for powder microcapsules Maltodextrin and β-cyclodextrin can reduce the hygroscopicity of powdered oils, enhance the rehydration ability of dried samples, and promote the formation of more ideal particle morphology in powdered oils. Different proportions of maltodextrin or a mixture of maltodextrin and β-cyclodextrin were added to prepared monolayer or bilayer emulsions, thoroughly mixed, and then freeze-dried to prepare powder microcapsules. The freeze-dried powder microcapsules of monolayer and bilayer emulsions were named monolayer powder and bilayer powder, respectively. Figure 6 As shown: As the concentration of maltodextrin increases, the total oil content and surface oil content of the microcapsule powder gradually decrease, while the encapsulation efficiency gradually increases. However, the total oil content of the monolayer powder remains almost unchanged after the maltodextrin addition exceeds 10%. Under the same concentration of maltodextrin addition, the oil encapsulation efficiency and surface oil content of the bilayer powder are slightly higher. Therefore, the optimal maltodextrin addition for preparing both monolayer and bilayer emulsion powder microcapsules is 15%.

[0049] When a mixture of maltodextrin and β-cyclodextrin (15 wt%) was added, the total oil content, surface oil content, and encapsulation efficiency varied with the ratio of the two components. When the mass ratio of maltodextrin to β-cyclodextrin was greater than 7:3, the total oil content (23.01–24.32%) and encapsulation efficiency (88.49–93.55%) remained unchanged, while the surface oil content continuously decreased to 1.5–2.38%. In conclusion, adding a mixture of maltodextrin and β-cyclodextrin at a mass ratio of 15% (7:3) is the optimal filler for preparing single-layer and double-layer powder microcapsules of sea buckthorn seed oil.

[0050] (3.7) Results of the storage stability test of powder microcapsules The peroxide value (POV) of oils is an indicator of the degree of oxidation of oils. Changes in the POV and ALA-EE values ​​of sea buckthorn seed oil and two types of powdered microcapsules are shown below. Figure 7As shown in the figure, the initial POV value was low and the ALA-EE content was high, indicating that the oil was relatively fresh and less oxidized. With increasing time, the POV value increased significantly, while the relative ALA-EE content gradually decreased. On the 7th day of storage, the POV value of sea buckthorn seed oil increased significantly to 1.75 g / 100g, and then rose slowly. The POV values ​​of monolayer powder (0.84 g / 100g → 1.92 g / 100g) and double-layer powder (0.72 g / 100g → 1.64 g / 100g) increased slowly throughout the storage period, with the double-layer powder consistently showing the lowest POV value. During storage, the relative ALA-EE content in sea buckthorn seed oil decreased most significantly (38.21% → 29.12%), ALA oxidation slowed in monolayer powder (38.28% → 32.42%), and ALA oxidation was slowed in double-layer powder (40.86% → 36.96%). This demonstrates that powdered microcapsules can effectively prevent the oxidation of sea buckthorn seed oil and improve the stability of unsaturated fatty acids.

[0051] (3.8) In vitro simulated digestion characteristics and sustained-release effect of steady-state samples As shown in Table 1, after the simulated oral digestion stage, there was no significant change or difference in the oil release rate of the emulsion and powder microcapsules. This may be due to the short oral digestion time and the presence of a polyelectrolyte layer on the surface of the oil, resulting in minimal oil release. After the simulated gastric digestion stage, due to drastic pH changes and pepsin hydrolysis, the stabilized emulsion and powder microcapsule samples showed varying degrees of oil droplet leakage. The oil release rate of the monolayer emulsion was higher than that of the bilayer emulsion, while the oil release rate of the powder microcapsules was lower than that of the corresponding emulsions. This indicates that the bilayer emulsion has a better oil protection effect, while the powder microcapsules can further reduce oil release in the upper digestive tract on the basis of the emulsion. After the simulated intestinal digestion, the polyelectrolyte layer on the surface of the oil was gradually completely digested, and a large amount of oil seeped out from all stabilized samples. The monolayer emulsion and monolayer powder had the highest oil release rates (91.60% and 89.81%, respectively), while the bilayer emulsion and bilayer powder had lower oil release rates, especially the bilayer powder, which had an oil release rate of only 86.06% after intestinal digestion.

[0052] In summary, powdered microcapsules have an additional protective layer compared to emulsions, resulting in better encapsulation and protection of sea buckthorn seed oil, allowing more sea buckthorn seed oil to reach the digestive tract for release and absorption.

[0053] Table 1: Oil release amount of different powder microcapsules at different digestion stages Note: Different letters indicate significant differences between groups (p<0.05).

[0054] During fat digestion, lipases hydrolyze fats, releasing fatty acids and monoglycerides. Simulating the fatty acid release rate of different samples during digestion directly reflects the digestion rate and progress of each sample. In simulated intestinal digestion, the fatty acid release rate of monolayer and bilayer powders was measured at different digestion times (0–120 min). The results are as follows: Figure 8 As shown. The sea buckthorn seed oil in the powder microcapsules was almost entirely digested at the end of the simulated intestinal digestion, indicating that the polyelectrolyte layer and the maltodextrin wall filler do not affect the intestinal digestion of sea buckthorn seed oil. The release of fatty acids from both single-layer and double-layer powders was rapid in the early stages of digestion (15-20 min), followed by a slow, relatively constant release rate. The fatty acid release curves of the steady-state samples were fitted using the Higuchi pharmacokinetic equation, and the R² values ​​for both were... 2 Both values ​​are greater than 0.95, indicating a good fit and suggesting that both have sustained-release properties.

[0055] The cytotoxicity test results of sea buckthorn seed oil and powder microcapsule samples are as follows: Figure 9 As shown. By Figure 9 It was found that neither crude oil nor microcapsule powder showed significant cytotoxicity within the given range. Therefore, 1 mg / mL was selected as the concentration of the powder microcapsule sample for subsequent cell transport and absorption experiments. A Caco-2 monolayer cell model was established in a Transwell 12-well cell culture plate, and the transport and absorption coefficient (Papp) of sea buckthorn seed oil in the samples was measured to evaluate the in vitro bioavailability of sea buckthorn seed oil in different samples. Figure 9 (b) Sea buckthorn seed oil is highly hydrophobic and difficult to disperse evenly in the culture medium. After 24 hours of culture, the oil aggregates at the top of the medium, reducing the efficiency of contact with the Caco-2 cell monolayer membrane, resulting in the lowest Papp value for the crude oil. The Papp values ​​of the powder microcapsule samples were all significantly increased, with the bilayer powder showing the highest Papp value. This may be because the loose porous structure formed by freeze-drying maltodextrin and β-cyclodextrin in the bilayer powder structure allows for rapid release of small-diameter oil particles after reconstitution, increasing the cell contact area. The outer layer of the FUC enhances mucus penetration, improving the transport rate, thus resulting in the highest bioavailability.

[0056] Example 2: Effect of different emulsification process routes on the stability of bilayer emulsions Option A (Assembly followed by emulsification process): At pH 5.5, 3.0 wt% sodium caseinate-phosphate buffer solution and 0.4 wt% fucoidan-phosphate buffer solution are mixed at a mass ratio of 8:1 and sheared and emulsified at 8000 rpm for 3 min to obtain a sodium caseinate-fucoidan bilayer shell emulsion; then, sea buckthorn seed oil is mixed with the sodium caseinate-fucoidan bilayer shell emulsion at a mass ratio of 1:9, sheared at 12000 rpm for 5 min, and homogenized 3 times under 40 MPa pressure to obtain a sea buckthorn seed oil bilayer emulsion.

[0057] Option B (Secondary Emulsification Process): At pH 7.0, 10 wt% sea buckthorn seed oil is mixed with 90 wt% of a 3.0 wt% sodium caseinate-phosphate buffer solution, and the mixture is sheared at 12,000 rpm for 5 min. Then, it is homogenized three times under high pressure at 40 MPa to form a monolayer emulsion. 0.4 wt% fucoidan-phosphate buffer solution is added to the monolayer emulsion (protein to polysaccharide mass ratio of 8:1), and the mixture is magnetically stirred at 200 rpm for 30 min. The pH is adjusted to 5.0, and then sheared at 8,000 rpm for 3 min to obtain a bilayer emulsion of sea buckthorn seed oil.

[0058] By comparing the data of the bilayer emulsions prepared by scheme A and scheme B ( Figure 10 The results showed that the bilayer emulsion obtained by scheme B had smaller particle size (375.23 nm), PDI value (0.272), and lower ζ-potential (-49.58 mV), while the emulsion prepared by scheme A had larger particles (439.26 nm), a PDI value (0.554), and a higher ζ-potential (-37.39 mV). This indicates that the emulsion prepared by scheme A had larger particles, uneven particle size distribution, and unstable properties. After standing at room temperature for one week, the emulsion prepared by scheme A experienced severe demulsification, with oil overflowing to form a thick oil layer. In contrast, the emulsion prepared by scheme B showed less severe demulsification and less significant oil accumulation in the upper layer, thus exhibiting better stability.

[0059] The difference in emulsion stability caused by the two preparation methods may be due to the loss of good amphiphilicity of sodium caseinate and fucoidan after assembly, making them unable to firmly adsorb onto the oil droplet surface, leading to demulsification and oil precipitation during storage. Therefore, Scheme B (secondary emulsification) is a suitable method for preparing sea buckthorn seed oil bilayer emulsions and subsequently for preparing powder microcapsules. Figure 11 Transmission electron microscopy (TEM) image of the bilayer emulsion prepared by scheme B (secondary emulsification).

Claims

1. A method for preparing a bilayer emulsion-microcapsule powder of sea buckthorn seed oil, characterized in that: include: 1) Preparation of sea buckthorn seed oil bilayer emulsion: Under neutral conditions, sea buckthorn seed oil was mixed with a sodium caseinate-phosphate buffer solution with a concentration of 2.5-3.0 wt% at a mass ratio of 5:95-15:

85. After shearing and homogenization, a sodium caseinate monolayer emulsion with a particle size of 250-350 nm, a PDI of 0.1-0.25, and a ζ-potential of -45 to -30 mV was formed. A fucoidan-phosphate buffer solution with a concentration of 0.3-0.4 wt% was added to the monolayer emulsion at a mass ratio of 1:8-10. The mixture was stirred, and the pH was adjusted to 5.0-5.

5. After shearing, a sea buckthorn seed oil bilayer emulsion with a particle size of 350-500 nm, a PDI of 0.12-0.38, and a ζ-potential of -50 to -35 mV was obtained. 2) Preparation of microcapsule powder: Add 10-15 wt% maltodextrin or a mixture of maltodextrin / β-cyclodextrin with 50-70 wt% maltodextrin to the bilayer emulsion of sea buckthorn seed oil and stir to form a homogeneous emulsion system. Pre-freeze, freeze-dry, thaw and take out, grind to powder, and sieve to obtain sea buckthorn seed oil bilayer emulsion-microcapsule powder.

2. The preparation method according to claim 1, characterized in that: In step 1), The method for preparing sea buckthorn seed oil includes: screening, washing, and drying sea buckthorn seeds, extracting them using supercritical CO2, and encapsulating them with urea to obtain sea buckthorn seed oil. The sea buckthorn seed oil contains 40-45% α-linolenic acid.

3. The preparation method according to claim 1, characterized in that: In step 1), the core of the emulsion particles in the bilayer emulsion of sea buckthorn seed oil is sea buckthorn seed oil, and the outer layer is a sodium caseinate-fucose polysaccharide composite layer. The hydrophilic groups of fucoidan are exposed on the outer surface and in contact with the aqueous phase to form a stable emulsion system.

4. The preparation method according to claim 1, characterized in that: In step 1), when preparing the sodium caseinate monolayer emulsion, the shearing speed is 6000-10000 rpm and the time is 2-5 min.

5. The preparation method according to claim 1 or 4, characterized in that: In step 1), when preparing the sea buckthorn seed oil bilayer emulsion, the shearing speed is 10000-15000 rpm and the time is 3-7 min; the homogenization pressure is 35-45 MPa and the number of times is 2-5.

6. The preparation method according to claim 1, characterized in that: In step 2), The pre-freezing temperature is -90 to -70°C, and the time is 20-30 hours; The freeze-drying temperature is -60 to -40°C, and the time is 70-80 hours; The particle size of the sea buckthorn seed oil bilayer emulsion-microcapsule powder obtained after sieving is 500-1000 nm. The total oil content of sea buckthorn seed oil in the bilayer emulsion-microcapsule powder is ≥15%, and the encapsulation rate is ≥74%.

7. A sea buckthorn seed oil bilayer emulsion-microcapsule powder obtained by the preparation method according to any one of claims 1-6.

8. The application of the sea buckthorn seed oil bilayer emulsion-microcapsule powder obtained by the preparation method according to any one of claims 1-6 or the sea buckthorn seed oil bilayer emulsion-microcapsule powder according to claim 7 in the preparation of enteric-coated / intestinal-targeted release foods or enteric-coated / intestinal-targeted release drugs.

9. The application as described in claim 8, characterized in that: In in vitro simulated digestive fluids, the oil release amount of the sea buckthorn seed oil bilayer emulsion-microcapsule powder is ≤0.4% in the oral digestion stage, ≤47% in the gastric digestion stage, and ≥84% in the intestinal digestion stage.

10. A method for improving the stability or bioavailability of sea buckthorn seed oil, characterized in that: include: 1) Preparation of sea buckthorn seed oil bilayer emulsion: Under neutral conditions, sea buckthorn seed oil was mixed with a sodium caseinate-phosphate buffer solution with a concentration of 2.5-3.0 wt% at a mass ratio of 5:95-15:

85. After shearing and homogenization, a sodium caseinate monolayer emulsion with a particle size of 250-350 nm, a PDI of 0.1-0.25, and a ζ-potential of -45 to -30 mV was formed. A fucoidan-phosphate buffer solution with a concentration of 0.3-0.4 wt% was added to the monolayer emulsion at a mass ratio of 1:8-10. The mixture was stirred, and the pH was adjusted to 5.0-5.

5. After shearing, a sea buckthorn seed oil bilayer emulsion with a particle size of 350-500 nm, a PDI of 0.12-0.38, and a ζ-potential of -50 to -35 mV was obtained. 2) Preparation of microcapsule powder: Add 10-15 wt% maltodextrin or a mixture of maltodextrin / β-cyclodextrin with 50-70 wt% maltodextrin to the bilayer emulsion of sea buckthorn seed oil and stir to form a homogeneous emulsion system. Pre-freeze, freeze-dry, thaw and take out, grind to powder, and sieve to obtain sea buckthorn seed oil bilayer emulsion-microcapsule powder.

Images