Preparation method and application of mussel oil

Abstract

The invention relates to the technical field of marine organism grease preparation, in particular to a preparation method and application of mussel oil. The invention discloses a preparation method of mussel oil, which comprises the following steps: pretreating mussels to obtain homogenate, adding magnetic compound enzyme for enzymolysis, extracting, purifying and aminating to obtain aminated mussel oil; emulsifying mussel oil, assembling the emulsified mussel oil, chitosan and hyaluronic acid layer by layer through electrostatic interaction to form a multi-layer wrapper, adding calcium chloride and sodium alginate for crosslinking, and performing ultrafiltration, sterilization and filtration to obtain the electric field sensitive gel. According to the method, through an innovative process of multilayer film wrapping and gel crosslinking, the stability of the mussel oil in the mask is remarkably improved, the shelf life of the mask is effectively prolonged, and a reliable technical path is provided for development of high-quality mussel oil related products in the field of cosmetics.

Description

Technical Field

[0001] This invention relates to the field of marine biological oil preparation technology, and in particular to a method for preparing mussel oil and its application. Background Technology

[0002] Marine biological resources, due to their unique nutritional components and bioactive substances, exhibit enormous development potential in fields such as medicine and cosmetics. Mussels, as a high-yield and widely distributed economic shellfish, are rich in nutrients such as polyunsaturated fatty acids, proteins, vitamins, and minerals, especially omega-3 long-chain polyunsaturated fatty acids. Their oil is an important source for extracting high-quality marine biological oils. Effective extraction and high-value utilization of these oils can significantly enhance the added value of marine biological resources.

[0003] Chinese patent CN116875368A discloses a method for preparing mussel oil. The method involves saponifying crushed mussel meat with an ethanol solution of potassium hydroxide, filtering to remove residue, and then extracting, acidifying, re-extracting, and concentrating under vacuum to obtain alkaline-extracted mussel oil. While the alkaline extraction method is cheaper than supercritical fluid extraction, it requires more extraction and acidification steps compared to ethanol extraction. Furthermore, the alkaline conditions of the potassium hydroxide ethanol solution may damage some heat-sensitive components. Mussel oil is rich in omega-3 long-chain polyunsaturated fatty acids, whose unsaturated double bonds are easily oxidized by oxygen, light, and temperature, leading to increased acid value and flavor deterioration. No antioxidant strategies are mentioned, and exposure to air during vacuum concentration may accelerate lipid oxidation.

[0004] Chinese patent CN110100907A discloses a method for preparing mussel lipid microencapsulated products. First, oil is extracted from freeze-dried mussel powder using supercritical carbon dioxide extraction. Second, β-cyclodextrin and soluble starch are dissolved in hot water in a specific ratio to prepare a wall material solution. Next, soybean lecithin and Tween-80 are added to the wall material solution and mixed thoroughly. Then, oil is added in a certain proportion, and the mixture is stirred to obtain an emulsion. The emulsion is subjected to multiple high-pressure homogenization processes. Finally, the microencapsulated powder product is obtained by spray drying. Although the supercritical extraction method can retain more polyunsaturated fatty acids, the strong diffusivity of carbon dioxide during extraction may lead to the loss of some small-molecule fatty acids. While supercritical extraction avoids organic solvent residues, it cannot completely remove heavy metals or algal metabolites from mussel lipids. Furthermore, the patent does not mention a heavy metal removal process, which could pose a product safety risk if the raw mussels grow in polluted waters. The wall material, composed of β-cyclodextrin and soluble starch, can form an encapsulation structure, but neither of them has antioxidant activity. When the microcapsule wall material is damaged, the internal oils lack antioxidant protection.

[0005] Chinese patent CN1304988A discloses three methods for preparing mussel oil: one, extraction after processing of mussel meat by boiling in alkaline water; two, extraction and processing after reflux extraction with acetone; and three, extraction and processing after extraction using supercritical carbon dioxide under specific pressure, temperature, and time conditions. Among these methods, boiling in alkaline water may cause saponification of unsaturated fatty acids due to the alkaline environment and high temperature, damaging their structural integrity. Repeated reflux in the acetone method may lead to degradation of heat-sensitive components. Supercritical carbon dioxide extraction, due to its long extraction time, not only increases energy costs but may also result in the loss of polar components in the oil due to prolonged exposure to carbon dioxide.

[0006] Furthermore, in the field of cosmetic masks, mussel oil is widely used due to its rich content of active ingredients. However, in masks prepared with existing technology, mussel oil often faces key problems. On the one hand, the lack of an efficient encapsulation and stabilization system means that mussel oil is easily exposed to air and moisture, causing its unsaturated double bonds to oxidize rapidly and leading to the inactivation of active ingredients. On the other hand, traditional mask matrices cannot achieve controlled release of mussel oil, resulting in a large amount of leakage of active ingredients in the early stages of storage or instant release during use. This not only prevents the product from maintaining its efficacy but also significantly shortens its shelf life due to the premature loss of effective ingredients. Moreover, the product is prone to deterioration such as layering and off-odors during storage, which severely limits its industrial application in the cosmetics field.

[0007] In conclusion, with the increasing demand from consumers for natural, safe, and efficient products, and the deepening of the concepts of green chemistry and sustainable development, there is an urgent need to develop a mussel oil preparation method that balances high extraction rate, low energy consumption, and high safety, while also addressing the issues of controlled release and shelf life of mussel oil in cosmetic masks. This would overcome the shortcomings of existing technologies, achieve green and high-value production of mussel oil, and meet the application needs of multiple fields such as pharmaceuticals and cosmetics. Summary of the Invention

[0008] To address the aforementioned problems, the present invention aims to provide a method for preparing mussel oil and its application. The method for preparing mussel oil specifically includes the following steps: S001, add magnetic complex enzyme to mussel homogenate, adjust pH, enzymatically hydrolyze, separate with a magnet, collect the solution to obtain mussel enzymatic hydrolysate; S002, an extractant was added to the mussel enzymatic hydrolysate for extraction, and the extracted organic phases were combined; S003, nitrogen gas is introduced, rotary evaporation is performed, and rosemary extract is added to obtain crude mussel oil; S004: After heating the crude mussel oil, add ultrapure water and stir. After standing and separating the layers, collect the upper layer, add sodium hydroxide solution to neutralize it, and wash with ultrapure water until neutral. S005, add activated clay, stir, filter, vacuum distill, filter again to obtain refined mussel oil; S006, refined mussel oil and ethanolamine are added to a three-necked flask, nitrogen gas is introduced and heated, and the mixture is stirred until dissolved. Cyclohexylcarbodiimide and 4-dimethylaminopyridine are added. After the reaction, ethyl acetate is added for extraction. The organic phases are combined, washed and distilled under reduced pressure to obtain aminated mussel oil, denoted as mussel oil.

[0009] Mussel oil is encapsulated in a multilayer membrane of hyaluronic acid and chitosan, utilizing the electrostatic interaction between the two to achieve layer-by-layer assembly. This process balances the encapsulation efficiency of oil-soluble components with the release performance during iontophoresis, resulting in a mussel oil-containing gel mask. The specific steps include: S101, mix mussel oil with Tween-80, dilute with phosphate buffer, and then emulsify by ultrasonication to obtain mussel oil emulsion; S102, slowly add an equal volume of chitosan solution and mix, stir, centrifuge, discard the supernatant, and wash twice with phosphate buffer. S103, add an equal volume of hyaluronic acid solution, stir, centrifuge, discard the supernatant, and wash twice with phosphate buffer. S104, slowly add an equal volume of chitosan solution and mix, stir, centrifuge, discard the supernatant, wash twice with phosphate buffer to obtain chitosan-hyaluronic acid-chitosan-encapsulated mussel oil, which is recorded as a multi-layer encapsulated material; S105, add calcium chloride solution, stir, add an equal volume of sodium alginate solution, add calcium chloride solution dropwise for cross-linking, ultrafilter, sterilize and filter to obtain mussel oil gel mask.

[0010] The mussel homogenate solution is obtained by pretreating mussels through washing, desalination, and homogenization steps, specifically including the following steps: S201. The fresh mussel meat is rinsed, drained, soaked in sodium chloride solution, and then rinsed again to obtain clean mussel meat. S202, clean mussel meat is added to a phosphate buffer solution containing antioxidants and homogenized to obtain a mussel homogenate.

[0011] The magnetic composite enzyme is prepared by co-precipitation of magnetic iron oxide nanoparticles, followed by silanization and glutaraldehyde activation. Neutral protease and lipase are then covalently immobilized on the surface of a magnetic carrier. The composite enzyme consists of neutral protease and lipase in a mass ratio of 2:1. The preparation method of the magnetic composite enzyme specifically includes the following steps: S301, weigh out ferric chloride hexahydrate and ferrous chloride tetrahydrate, dissolve them in ultrapure water, introduce nitrogen gas, and stir; S302 is slowly added to an ammonia solution. After the reaction, the precipitate is separated by a magnet, washed and dried to obtain iron oxide nanoparticles. S303: Fe3O4 nanoparticles were weighed and dispersed in anhydrous ethanol, sonicated, and then 3-aminopropyltriethoxysilane was added and stirred. After separation by magnet, washing and drying, amino-functionalized Fe3O4 was obtained. S304: Aminofunctionalized iron oxide is dispersed in phosphate buffer, glutaraldehyde solution is added, the reaction is carried out, and the mixture is separated by a magnet and washed to obtain glutaraldehyde-activated iron oxide carrier. S305: Weigh neutral protease and lipase, dissolve them in phosphate buffer, stir, add glutaraldehyde to activate the carrier iron(III) oxide, react, separate by magnet, wash, disperse in glutaraldehyde-containing phosphate buffer, crosslink, separate by magnet, wash, and obtain magnetic complex enzyme.

[0012] In step S001, the amount of magnetic complex enzyme added is 0.5% of the mussel homogenate, the pH is adjusted to 7.5, and the enzymatic hydrolysis conditions are 50 °C in a water bath, 200 rpm / min, for 2 h. The magnetic separation is performed by letting the mixture stand in a magnetic rack for 5 min.

[0013] The extractant is composed of n-hexane and ethanol containing antioxidants in a volume ratio of 3:1. The antioxidants are composed of 0.1% vitamin E and 0.05% citric acid.

[0014] In step S002, the extractant is subjected to two extraction processes. The first extraction is 3 to 5 times the weight of the mussel meat, and the second extraction is 2 to 4 times the weight of the mussel meat. The extraction conditions are 35 ℃, 300 W ultrasound, and 40 min. After each extraction, centrifugation is performed at 15000 r / min for 15 min.

[0015] In step S003, the rotary evaporation conditions are 40 °C and -0.09 MPa, and the amount of rosemary extract added is 0.05%.

[0016] In step S004, the temperature is raised to 60 °C, the amount of ultrapure water added is 5-9%, the stirring conditions are 150 rpm / min for 30 min, and the concentration of sodium hydroxide solution is 0.1 M.

[0017] In step S005, the amount of activated clay added is 1-3%, the stirring conditions are 70 ℃, 300 rpm / min, 1h, the vacuum distillation conditions are 120 ℃, nitrogen, -0.09 MPa, 2h, and filtration is performed using a 0.22μm filter membrane.

[0018] In step S006, the molar ratio of mussel oil to ethanolamine is 1:1.2, the temperature is heated to 50 °C, the molar ratios of dicyclohexylcarbodiimide and 4-dimethylaminopyridine to ethanolamine are 0.5~1.5 and 0.05~0.15, respectively, the reaction conditions are 50 °C for 7 h, the amount of ethyl acetate added is 2 times the volume, the extraction process is performed 3 times, the washing process is to first wash twice with 1 volume of 10% citric acid aqueous solution, and then wash with ultrapure water until neutral, the vacuum distillation conditions are 40 °C and -0.09 MPa.

[0019] In step S101, the amount of Tween-80 added is 0.5%, the phosphate buffer is diluted to 100 mL, the sonication conditions are 300 W for 15 min, and the concentration of the phosphate buffer is 1X.

[0020] In steps S102 to S104, the concentrations of hyaluronic acid and chitosan are 1% and 1.5%, respectively, the stirring conditions are 200 rpm / min and 30 min, and the centrifugation conditions are 4 ℃, 15000 r / min and 15 min.

[0021] In step S105, the concentration of calcium chloride solution is 10 mM, the concentration of sodium alginate solution is 0.5%, the volume of calcium chloride solution is 10% of the mixture, the stirring conditions are 25 ℃, 200 rpm / min, 10 min, the crosslinking conditions are 25 ℃, 30 min, an ultrafiltration membrane with a molecular weight cutoff of 100 kDa is used, and 0.22 μm sterile filtration is performed.

[0022] In step S201, the water is rinsed three times with clean water, the concentration of sodium chloride solution is 1%, the soaking time is 15 minutes, and ultrapure water is used for rinsing twice.

[0023] In step S202, phosphate buffer containing antioxidants is added at a material-to-liquid ratio of 1:2. The antioxidants are composed of ethylenediaminetetraacetic acid (EDTA) and tea polyphenols, both of which have a concentration of 0.05%. Homogenization is performed by homogenizing the tissue in an ice-water bath for 3 minutes using a tissue homogenizer.

[0024] In step S301, the molar ratio of ferric ions to ferrous ions in ferric chloride hexahydrate and ferrous chloride tetrahydrate is 2:1, and the stirring conditions are nitrogen and 800 rpm / min.

[0025] In step S302, the concentration of the ammonia solution is 5%, the reaction conditions are 80 °C, nitrogen, 800 rpm / min, 1 h, and the product is washed three times with ultrapure water and anhydrous ethanol. The drying conditions are 60 °C, -0.09 MPa, 12 h.

[0026] In step S303, the concentration of iron oxide nanoparticles is 10 mg / mL, the mixture is sonicated for 30 min, the volume-to-mass ratio of 3-aminopropyltriethoxysilane to iron oxide nanoparticles is 2:1, the mixture is stirred for 24 h, washed 5 times with anhydrous ethanol, and dried under the conditions of 60 ℃, -0.09 MPa, and 8 h.

[0027] In step S304, the concentration of amino-functionalized iron(III) oxide is 10 mg / mL, the concentration of glutaraldehyde solution is 2.5%, the reaction conditions are 800 rpm / min for 3 h, and the mixture is washed three times with phosphate buffer. In step S305, the concentrations of neutral protease and lipase are 4 mg / mL and 2 mg / mL, respectively. The stirring conditions are 4℃, 300 rpm / min, and 30 min. The reaction conditions are 4℃, 300 rpm / min, and 12 h. The sample is washed 5 times with phosphate buffer. The concentration of glutaraldehyde in the glutaraldehyde-containing phosphate buffer is 0.25%. The crosslinking conditions are 4℃, 300 rpm / min, and 2 h.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention prepares magnetic iron oxide nanoparticles via co-precipitation and immobilizes neutral protease and lipase to form a magnetic composite enzyme. This enzyme can be rapidly separated by a magnet, avoiding the cumbersome steps of traditional centrifugation or filtration, improving enzyme reusability, and reducing production costs. Compared to existing technologies, traditional free enzyme hydrolysis requires additional purification steps and suffers from high enzyme loss, while magnetically immobilized enzymes achieve "immediate separation via magnetization," improving hydrolysis efficiency. Simultaneously, by combining pretreatment with ethylenediaminetetraacetic acid and tea polyphenols, metal ion oxidation is inhibited, free radicals are scavenged, and the enzymatic conversion rate of mussel protein and fat is improved. 2. This invention utilizes a composite extractant to extract mussel oil and a multi-stage purification process to improve oil purity. The extractant consists of n-hexane and ethanol containing antioxidants, namely vitamin E and citric acid. Compared to traditional single organic solvents, this improves extraction efficiency while inhibiting oil oxidation and reducing peroxide value. Environmentally, ethanol is recyclable, reducing organic solvent pollution and meeting green chemistry requirements. The use of activated clay adsorption combined with vacuum distillation enhances impurity removal, resulting in a clear oil color and lower acid value. The addition of rosemary extract provides superior natural antioxidant effects compared to synthetic antioxidants, extending the product's stability. 3. This invention achieves a performance breakthrough through molecular design in the chemical structure modification of mussel oil. By combining mussel oil with ethanolamine and using a dicyclohexylcarbodiimide / 4-dimethylaminopyridine catalytic system, a directed amination reaction is carried out, introducing amino groups into the mussel oil molecule. This successfully breaks the limitation of its single oil solubility, transforming the oil-soluble component into an amphiphilic molecule with amphiphilic properties, thus improving its water solubility. Traditional mussel oil, originally only suitable for oil-soluble systems, can now be used as a key ingredient in the preparation of stable oil-in-water emulsions after structural optimization, expanding its application to emerging fields such as cosmetics and health products. For example, it can be used to develop highly moisturizing emulsions in cosmetics, and to improve the bioavailability of nutrients in health products. Furthermore, the reaction conditions are relatively mild. Compared to high-temperature amidation, these mild conditions effectively avoid the damage to the molecular structure caused by high temperatures, reduce the degradation of unsaturated fatty acids, increase the retention rate of double bonds, and reduce the loss rate of active ingredients. This efficient protection of active ingredients not only ensures the nutritional and functional value of the product but also reduces energy costs, aligning with the development trend of green chemistry and providing technical feasibility for large-scale industrial production. 4. This invention utilizes a multilayer membrane structure for efficient encapsulation and an electric field-responsive release mechanism to produce a gel mask containing mussel oil with intelligent release characteristics through electric field-sensitive encapsulation technology. Based on layer-by-layer self-assembly technology, using positively charged chitosan and negatively charged hyaluronic acid as building blocks, a three-layer structure of chitosan-hyaluronic acid-chitosan is prepared through alternating deposition via electrostatic interactions. Compared with traditional single-layer membrane encapsulation, this improves encapsulation efficiency. The mussel oil droplets encapsulated by the multilayer membrane have uniform particle size, falling within the ideal nanoscale range, effectively avoiding agglomeration caused by uneven particle size. This system maintains good dispersibility, enhances its stability, and reduces sedimentation or stratification, providing reliable protection for the long-term storage and transportation of mussel oil. A semi-interpenetrating network gel prepared by crosslinking sodium alginate and calcium chloride is used as an intelligent carrier. This system exhibits unique controllable release characteristics under electric field stimulation. When a certain electric field strength is applied, the charge distribution of the multilayer membrane structure inside the gel changes, and the interlayer electrostatic repulsion effect is enhanced, promoting the precise release of the encapsulated mussel oil. On-demand release can be achieved by adjusting the electric field parameters, and the release rate can be linearly adjusted with the electric field strength to achieve targeted delivery. This breaks through the technical bottleneck of traditional packaging materials having no environmental responsiveness and provides an innovative solution for the targeted delivery of functional components. 5. This invention utilizes multi-layer membrane encapsulation technology to isolate mussel oil from direct contact with air and moisture, inhibiting the oxidative deterioration of unsaturated fatty acids. Simultaneously, the semi-interpenetrating network structure formed by the cross-linking of sodium alginate and calcium chloride further fixes the active ingredients, reducing leakage and deterioration during storage. Compared to traditional mussel oil masks that are prone to delamination and rapid loss of active ingredients, the gel mask prepared by this patent significantly slows down the deterioration process, greatly extends the product's shelf life, and improves its marketability and safety. 6. This invention innovates the entire process of enzymatic hydrolysis-extraction-modification-encapsulation, achieving multi-dimensional breakthroughs in process efficiency, product function, and application scenarios while maintaining the high activity of mussel oil. In particular, it is significantly superior to existing technologies in terms of controlled release, green production, and preparation of multifunctional materials, and has the potential for industrial-scale application. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments.

[0030] Example 1 The preparation of a mussel enzymatic hydrolysate specifically includes the following steps: Weigh 5.4 g of ferric chloride hexahydrate and 2.5 g of ferrous chloride tetrahydrate, dissolve them in 100 mL of ultrapure water, purge with nitrogen gas, and stir. Ammonia solution was slowly added, and after the reaction, the precipitate was separated by a magnet, washed and dried to obtain iron oxide nanoparticles. 1 g of iron oxide nanoparticles were weighed and dispersed in 100 mL of anhydrous ethanol. The mixture was sonicated, and 2 mL of 3-aminopropyltriethoxysilane was added and stirred. The mixture was then separated by a magnet, washed, and dried to obtain amino-functionalized iron oxide. 0.5 g of amino-functionalized iron(III) oxide was dispersed in 50 mL of phosphate buffer, 10 mL of glutaraldehyde solution was added, the reaction was carried out, and the mixture was separated by a magnet and washed to obtain the glutaraldehyde-activated iron(III) oxide carrier. Weigh 0.2 g of neutral protease and 0.1 g of lipase, dissolve them in 50 mL of phosphate buffer, stir, add glutaraldehyde to activate the carrier iron oxide, react, separate by magnet, wash, disperse in 50 mL of glutaraldehyde-containing phosphate buffer, crosslink, separate by magnet, wash, and obtain magnetic complex enzyme. Rinse the fresh mussel meat, drain it, soak it in sodium chloride solution, and then rinse it again to obtain clean mussel meat. Add 1 g of mussel meat to phosphate buffer containing antioxidants, homogenize it in an ice water bath for 3 minutes using a tissue homogenizer at a material-to-liquid ratio of 1:2, and obtain mussel homogenate. Magnetic complex enzyme was added to the mussel homogenate, the pH was adjusted, and enzymatic hydrolysis was performed. The magnetic complex enzyme was separated by a magnet, and the solution was collected to obtain the mussel enzymatic hydrolysate, which was designated as test sample 1.

[0031] Example 2 The preparation of a refined mussel oil specifically includes the following steps: Extraction was performed twice with an extractant added to the mussel enzymatic hydrolysate prepared in Example 1, and the two organic phases were combined. The extractant added in the first extraction was 4 times the mass of the mussel meat, and the extractant added in the second extraction was 3 times the mass of the mussel meat. Nitrogen gas is introduced, the mixture is rotary evaporated, and rosemary extract is added to obtain crude mussel oil. After heating the crude mussel oil, add 7% ultrapure water and stir. After standing and separating the layers, collect the upper layer, add sodium hydroxide solution to neutralize it, and wash with ultrapure water until neutral. Add 2% activated clay, stir, filter, vacuum distill, filter again to obtain refined mussel oil, which is sample 2.

[0032] Example 3 The preparation of a mussel oil-containing gel mask involves, firstly, preparing aminated mussel oil via a chemical method; secondly, assembling multiple layers of encapsulated mussel oil through electrostatic interaction; and finally, preparing a gel mask with a semi-interpenetrating network structure via sodium alginate-calcium chloride crosslinking. The specific steps include: The refined mussel oil obtained in Example 2 and ethanolamine were added to a three-necked flask, heated after purging with nitrogen, and stirred until dissolved. Cyclohexylcarbodiimide and 4-dimethylaminopyridine were then added, wherein the molar ratios of dicyclohexylcarbodiimide and 4-dimethylaminopyridine to ethanolamine were 1.0 and 0.1, respectively. After the reaction, ethyl acetate was added for extraction. The organic phases were combined, washed, and distilled under reduced pressure to obtain mussel oil. Mussel oil was mixed with Tween-80, diluted with phosphate buffer, and then ultrasonically emulsified to obtain a mussel oil emulsion. Slowly add an equal volume of chitosan solution, mix, stir, centrifuge, discard the supernatant, and wash twice with phosphate buffer. Add an equal volume of hyaluronic acid solution, stir, centrifuge, discard the supernatant, and wash twice with phosphate buffer. Slowly add an equal volume of chitosan solution, mix, stir, centrifuge, discard the supernatant, and wash twice with phosphate buffer to obtain chitosan-hyaluronic acid-chitosan-encapsulated mussel oil, which is denoted as a multilayer encapsulated material. Add calcium chloride solution, stir, add an equal volume of sodium alginate solution, add calcium chloride solution dropwise for cross-linking, ultrafilter, sterilize and filter to obtain a gel mask containing mussel oil, denoted as test sample 3.

[0033] Comparative Example 1 The difference from Example 1 is that a free complex enzyme was used instead of a magnetic complex enzyme to prepare mussel enzymatic hydrolysate. The free complex enzyme was composed of neutral protease and lipase in a mass ratio of 2:1. After enzymatic hydrolysis, the mixture was centrifuged at 15,000 rpm for 15 min, the supernatant was collected, filtered, and the mussel enzymatic hydrolysate was obtained, which was designated as control 1.

[0034] Comparative Example 2 The preparation of mussel oil differs from that in Example 2 in that it uses single-component n-hexane extraction and simple filtration purification, without the addition of antioxidants. Specifically, it includes the following steps: Rinse the fresh mussel meat, drain it, soak it in sodium chloride solution, and then rinse it again to obtain clean mussel meat. Add 1 g of mussel meat to phosphate buffer and homogenize it in an ice water bath for 3 min using a tissue homogenizer at a material-to-liquid ratio of 1:2 to obtain mussel homogenate. Hexane was added to the mussel homogenate and extracted twice, and the two organic phases were combined. The hexane added in the first extraction was 4 times the mass of the mussel meat, and the hexane added in the second extraction was 3 times the mass of the mussel meat. Nitrogen gas was introduced, the mixture was rotary evaporated, and then filtered to obtain mussel oil, which is also known as reference standard 2.

[0035] Comparative Example 3 The preparation of a mussel oil gel differs from Embodiment 3 in that, firstly, a single layer of chitosan encapsulating mussel oil is obtained through a natural polymer method, and finally, gel is prepared by replacing sodium alginate-calcium chloride with gelatin. Specifically, the preparation includes the following steps: The refined mussel oil obtained in Example 2 was mixed with Tween-80, diluted with phosphate buffer, and then ultrasonically emulsified to obtain a mussel oil emulsion. Slowly add an equal volume of chitosan solution, mix, stir, centrifuge, discard the supernatant, and wash twice with phosphate buffer to obtain monolayer chitosan-encapsulated mussel oil. Mussel oil encapsulated in a single layer of chitosan was slowly added to an equal volume of gelatin solution at 50 °C. The mixture was stirred thoroughly and then cooled to room temperature to obtain reference product 3.

[0036] Comparative Example 4 The preparation of a mussel oil-containing facial mask specifically includes the following steps: The refined mussel oil obtained in Example 2 was mixed with 5% glycerol and 0.3% xanthan gum, and 100 mL of ultrapure water was added. The mixture was stirred at 50 °C and 300 rpm for 30 min and then filtered to obtain reference standard 4.

[0037] Experimental Example 1 This experimental example measures the enzymatic conversion rate and residual enzyme activity of Example 1 and Comparative Example 1, specifically including the following steps: The isolated enzymes were collected and the enzymatic hydrolysis experiment was repeated three times under the same conditions. The enzymatic conversion rate and enzyme activity residual rate were recorded each time.

[0038] 1. Determination of enzymatic conversion rate: The enzymes collected and isolated in Example 1 and Comparative Example 1 were used to repeat the enzymatic hydrolysis experiment three times under the same conditions. The content of fatty acids and amino acids in test sample 1 and control sample 1 was measured each time.

[0039] Determination of fatty acid content: Take 2 mL of test sample 1 and reference sample 1 respectively and place them in a stoppered test tube. Add 2 mL of 2% sulfuric acid methanol solution, vortex to mix, place in a 70℃ constant temperature water bath, heat under reflux for 1 h, and remove the test tube every 15 min to gently shake. Cool to room temperature, add 2 mL of n-hexane, shake vigorously for 1 min, let stand for 15 min, transfer the upper n-hexane phase to another clean stoppered test tube, blow dry with nitrogen at 40℃, immediately add 1 mL of n-hexane, vortex until dissolved, filter through a 0.22 μm filter membrane to obtain the fatty acid methyl ester sample to be analyzed. Take the mixed standard solution of fatty acid methyl esters and dilute it with n-hexane to prepare a series of standard working solutions with mass concentrations of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 5.0 mg / mL and 10.0 mg / mL, respectively. These are the standard working solutions of fatty acid methyl esters to be analyzed. The fatty acid methyl ester samples and the standard working solutions of the fatty acid methyl esters to be analyzed were injected into the gas chromatograph, with three replicates for each sample. The gas chromatographic column was an HP-INNOWAX capillary column, 30m × 0.25mm × 0.25μm; the carrier gas was high-purity helium; the flow rate was 1.0mL / min; the split ratio was 10:1; the injection port temperature was 250℃; the detector temperature was 280℃; the temperature program was as follows: initial temperature 150℃, hold for 2 min; increase to 220℃ at 5℃ / min, hold for 15 min.

[0040] A standard curve was plotted with the concentration of fatty acid methyl ester standard working solution as the abscissa and the corresponding peak area as the ordinate, and a linear regression equation was obtained. The fatty acid content was calculated based on fresh mussel meat, and the results are shown in Table 1-1.

[0041] Table 1-1 Fatty acid content

[0042] As shown in Table 1-1, the enzymatic hydrolysis system of Example 1 has a stable effect on fatty acid composition, and the products from the three hydrolysis cycles are highly consistent, indicating that its enzyme-substrate interaction mode is controllable, and the immobilization effect of the magnetic composite enzyme improves the reproducibility of the reaction. In Comparative Example 1, the free composite enzyme exhibits poor hydrolytic stability; with increasing hydrolysis cycles, its fatty acid enrichment capacity significantly decreases, possibly due to the ease of inactivation and difficulty in recovery of the free enzyme, leading to large fluctuations in reaction efficiency. In Example 1, the total amount of EPA and DHA remained stable at 3.8%~3.9% during the three hydrolysis cycles, accounting for over 50% of the total fatty acids. EPA / DHA are the core functional components of mussel oil. Example 1's ability to stably retain a high content of functional fatty acids indicates that its enzymatic hydrolysis process can directionally enrich nutrients and avoid the loss of active ingredients. In Comparative Example 1, the total amount of EPA and DHA plummeted from 0.6% to 0.1% during the three hydrolysis cycles, with DHA completely disappearing in the third hydrolysis. In free enzyme systems, enzyme activity decreases sharply with repeated exposures, failing to continuously catalyze the hydrolysis of triglycerides into functional fatty acids. Instead, enzyme inactivation may lead to substrate residue and product decomposition, resulting in nutrient waste. Comparison shows that the magnetic complex enzyme in Example 1 is significantly superior to the free complex enzyme in Comparative Example 1 in terms of enzymatic stability, functional component enrichment, and process sustainability. It can stably produce mussel oil products with high EPA / DHA content, supports enzyme reuse, reduces industrial costs, and provides a reliable process route for the standardization and functionalization development of mussel oil.

[0043] Determination of amino acid content: Transfer 5 mL of test sample 1 and reference sample 1 to a clean hydrolysis tube, add 10 mL of 6 mol / L hydrochloric acid solution, gently shake to mix the solution evenly, slowly purge with nitrogen for 30 s, immediately seal the hydrolysis tube, place it in a 110 ℃ constant temperature drying oven, hydrolyze for 24 h, allow it to cool naturally to room temperature, transfer it to a flask of a rotary evaporator, and rotary evaporate at 40 ℃ and -0.09 MPa to obtain the sample to be derivatized; Add 1 mL of 0.1 mol / L borax buffer to the mixed solution of the sample to be derivatized and the amino acid standard, and sonicate to completely dissolve the residue. Then add 200 μL of 6-aminoquinoline-N-hydroxysuccinimide carbamate derivatizing reagent and 200 μL of acetonitrile, and vortex thoroughly. Transfer to a 10 mL stoppered test tube, place in a 60 ℃ water bath and stir for 10 min. Cool to room temperature, and dilute to 10 mL with ultrapure water. Vortex again to mix, filter through a 0.22 μm filter membrane, and collect the filtrate in a sample vial to obtain the stock solution of the amino acid sample and amino acid standard to be analyzed. Take six 10 mL volumetric flasks and add 0.01 mL, 0.05 mL, 0.1 mL, 0.5 mL, 1 mL, and 5 mL of amino acid standard stock solution, respectively. Dilute to the mark with ultrapure water to prepare a series of standard working solutions with concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, and 500 μg / mL. Vortex for 3 min to obtain the standard working solution of the amino acid to be analyzed. The amino acid samples and standard working solutions to be analyzed were injected into a high-performance liquid chromatograph (HPLC), with three replicates for each sample. The chromatographic column was a C18, 250 mm × 4.6 mm, 5 μm. Mobile phase A was 0.1 mol / L sodium acetate buffer at pH 4.95, and mobile phase B was 40% acetonitrile aqueous solution. The mobile phase gradient was as follows: 0 min, 10% B phase; 0–11 min, 22% B phase; 11–16 min, 35% B phase; 16–20 min, 60% B phase; 20–25 min, 60% B phase; 25–26 min, 10% B phase. The flow rate was 1.0 mL / min. The column temperature was 37 ℃. The injection volume was 10 μL. The detection wavelength was 248 nm.

[0044] A standard curve was plotted with the concentration of the amino acid standard working solution as the abscissa and the corresponding peak area as the ordinate, and a linear regression equation was obtained. The amino acid content was calculated based on fresh mussel meat, and the results are shown in Table 1-2.

[0045] Table 1-2 Amino acid content

[0046] As shown in Tables 1-2, the content of each amino acid in the three enzymatic hydrolysis data of Example 1 showed small fluctuations. The coefficients of variation for glycine, alanine, glutamic acid, and taurine were 5.8%, 4.5%, 2.5%, and 4.5%, respectively, while the coefficients of variation in Comparative Example 1 were 63%, 71%, 63%, and 71%, respectively, indicating significant fluctuations. In the three enzymatic hydrolysis of Example 1, glutamic acid remained stable at 2.0%–2.1%, and taurine remained stable at 1.2%–1.3%, with these two key amino acids accounting for over 40% of the total amino acids. In the three enzymatic hydrolysis of Comparative Example 1, glutamic acid plummeted from 1.0% to 0.2%, a loss of 80%, and taurine decreased from 0.5% to 0.0%, a complete loss. The comparison shows that the magnetic complex enzyme in Example 1 is significantly better than the free complex enzyme in Comparative Example 1 in terms of amino acid stability, retention of functional components, and process sustainability. It can stably produce enzymatic hydrolysates with high content of glutamic acid and taurine, ensuring flavor and efficacy. It can support the reuse of enzymes, reduce industrial costs, and provide a reliable technical path for the in-depth development of mussel protein.

[0047] 2. Determination of enzyme activity residual rate: The enzymes collected and isolated in Example 1 and Comparative Example 1 were subjected to repeated enzymatic digestion experiments 2 and 3 times under the same conditions, and the activities of neutral protease and lipase were measured.

[0048] Neutral protease activity assay: Weigh 10 mg of tyrosine, dissolve it in 0.1 mol / L hydrochloric acid and bring the volume to 100 mL to obtain a tyrosine standard solution. Take 6 test tubes and add 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mL of the tyrosine standard solution respectively. Add ultrapure water to 1.0 mL, then add 5 mL of 0.4 mol / L sodium carbonate solution and 1 mL of 33% Folin-Ciocalteu reagent. After mixing well, place in a 40℃ water bath for 20 min to obtain the tyrosine standard working solution. Take 1 mL of the magnetic complex enzyme solution prepared in Example 1, the free complex enzyme in Comparative Example 1, and the enzyme collected after reuse, respectively, add 5 mL of 1% casein solution, react in a 37℃ water bath for 10 min, add 2 mL of 0.4 mol / L trichloroacetic acid solution to terminate the reaction, let stand for 10 min, filter, take 1 mL of filtrate, add 5 mL of 0.4 mol / L sodium carbonate solution and 1 mL of 33% Folin-Ciocalteu reagent in sequence, mix well, and place in a 40℃ water bath for 20 min to obtain the sample to be tested; The absorbance of the tyrosine standard working solution and the test sample was measured at a wavelength of 680 nm. Each sample was in triplicate. A standard curve was plotted with the concentration of the tyrosine standard working solution on the x-axis and the corresponding absorbance on the y-axis. The amount of tyrosine generated by enzymatic hydrolysis was calculated based on the tyrosine standard curve. One enzyme activity unit was defined as the amount of enzyme required to generate 1 μg of tyrosine per minute. The magnetic complex enzyme solution prepared in Example 1 and the free complex enzyme in Comparative Example 1 were used as their respective initial enzymes. The enzyme activity residual rate was calculated based on the ratio of enzyme activity after repeated use to the initial enzyme activity. The measurement results are shown in Tables 1-3.

[0049] Lipase activity assay: Take 1 mL of the magnetic complex enzyme solution prepared in Example 1, the free complex enzyme in Comparative Example 1, and the enzyme collected after reuse, respectively, add 5 mL of 4% olive oil emulsion, react in a 37°C water bath for 15 min, add 4 mL of 95% ethanol, then add 3 drops of phenolphthalein indicator, and titrate with 0.05 mol / L sodium hydroxide standard solution until a faint red color appears. Record the volume of sodium hydroxide consumed. Each sample is repeated in triplicate.

[0050] The amount of fatty acid was directly calculated based on the amount of alkali consumed. One enzyme activity unit was defined as the amount of enzyme required to hydrolyze the substrate to produce 1 μmol of fatty acid per minute. The magnetic complex enzyme solution prepared in Example 1 and the free complex enzyme in Comparative Example 1 were used as their respective initial enzymes. The enzyme activity residual rate was calculated based on the ratio of enzyme activity after repeated use to the initial enzyme activity. The measurement results are shown in Tables 1-3.

[0051] Table 1-3 Enzyme activity residual rate

[0052] As shown in Tables 1-3, for neutral proteases, the initial enzyme activity residual rate of Example 1 reached 99.9%, and it maintained high activity even after three enzymatic hydrolysations. The residual rate decreased only slightly, decreasing by 1.1% in the first and second hydrolysations and by 0.9% in the second and third hydrolysations, indicating that the neutral protease in Example 1 has excellent stability and its activity is minimally affected by repeated use. In contrast, Comparative Example 1 had an initial residual rate of 55.8%, far lower than that of Example 1, and its activity decreased sharply, decreasing by 36.4% in the first and second hydrolysations and by 10.9% in the second and third hydrolysations, indicating that the neutral protease in Comparative Example 1 has poor stability and its activity is rapidly lost after repeated use. For lipases, the initial residual rate of Example 1 was 98.9%, and the activity residual rate showed a slow decreasing trend, decreasing by about 1-3% each time, demonstrating good stability after repeated use and strong enzyme activity retention. In contrast, Comparative Example 1 had an initial residual rate of 35.6%, and its activity continued to plummet, decreasing by 22.2% in the first and second hydrolysations and by 10% in the second and third hydrolysations, indicating that the lipase in Comparative Example 1 easily lost its activity upon repeated use and its enzyme stability was significantly insufficient.

[0053] In summary, the magnetic composite enzyme used in Example 1, with its high and stable enzyme activity residue rate, achieved stable and efficient enrichment of mussel oil enzymatic hydrolysis products. The process exhibited good repeatability and controllable product quality, making it suitable for continuous industrial production and ensuring the functionality and flavor of the mussel oil. The enzymatic hydrolysis process of Example 1 is significantly superior to Comparative Example 1 in terms of enzyme activity retention, product quality stability, and retention of functional components, demonstrating greater application value and development potential.

[0054] Experiment Example 2 This experimental example measures the extraction rate, peroxide value, acid value, and color of Example 2 and Comparative Example 2, specifically including the following steps: 1. Determination of extraction rate: Using mussel meat as the initial mass, the collected test sample 2 and control sample 2 were weighed separately. Each sample 3 was in parallel. Based on the ratio to the initial mass, the mussel oil extraction rate of Example 2 and Comparative Example 2 was calculated. The results are shown in Table 2.

[0055] 2. Determination of peroxide value: Weigh 3 g of test sample 2 and reference sample 2, and place them separately in 250 mL iodine flasks. Add 30 mL of a 2:3 mixture of chloroform and glacial acetic acid, shake to completely dissolve the sample, add 1.0 mL of saturated potassium iodide solution, tightly stopper the flask, gently shake for 0.5 min, place in the dark for 3 min, add 100 mL of ultrapure water, and immediately titrate with 0.01 mol / L sodium thiosulfate standard titration solution until the solution turns pale yellow. Add 1 mL of starch indicator and continue titrating until the blue color disappears as the endpoint. Record the volume of sodium thiosulfate standard titration solution consumed by the sample. Simultaneously perform a blank test by taking 20 mL of a 2:3 mixture of chloroform and glacial acetic acid and following the steps described above. Record the volume of sodium thiosulfate standard titration solution consumed in the blank test. Each sample is tested in triplicate.

[0056] Based on the difference between the volume of sodium thiosulfate standard titration solution consumed by the sample and the blank, the peroxide value was calculated using the mass fraction of iodine and the mass of the sample. The results are shown in Table 2.

[0057] 3. Determination of acid value: Weigh 5 g of test sample 2 and reference sample 2 and place them in 250 mL Erlenmeyer flasks respectively. Add 50 mL of 50% diethyl ether ethanol mixture to the Erlenmeyer flasks, shake to dissolve the sample, add 3 drops of phenolphthalein indicator, and titrate with 0.05 mol / L potassium hydroxide standard titration solution until the solution turns slightly red and does not fade within 15 seconds. Record the volume of potassium hydroxide standard titration solution consumed by the sample. Simultaneously perform a blank test. Take 50 mL of 50% diethyl ether ethanol mixture, follow the steps above, and record the volume of potassium hydroxide standard titration solution consumed in the blank test. Each sample is repeated in triplicate.

[0058] Based on the difference between the volume of potassium hydroxide standard titration solution consumed by the sample and the blank, the acid value was calculated using the mass fraction of potassium hydroxide and the sample mass. The results are shown in Table 2.

[0059] 4. Color determination: Take 5 mL of test sample 2 and reference sample 2 and inject them into cuvettes respectively, using a 40 mm optical path. Place the cuvettes into the sample cell of the colorimeter, adjust the yellow and red color plates so that the color of the sample in the instrument's field of view matches the color of the standard color glass slide, and record the yellow and red values. The measurement results are shown in Table 2.

[0060] Table 2. Extraction rate, peroxide value, acid value, and color of mussel oil.

[0061] As shown in Table 2, Example 2 achieved an extraction rate of 10.1%, while Comparative Example 2 only achieved 4.9%. This is because Example 2 uses the enzymatic hydrolysate from Example 1, which fully releases the oil components in the mussels. Subsequent multi-stage extraction and purification processes also contribute to oil enrichment. In contrast, Comparative Example 2 uses single-stage hexane extraction without enzymatic assistance, resulting in insufficient oil release and a low extraction rate, demonstrating the significant advantage of Example 2 in oil extraction efficiency. The peroxide value of Example 2 is only 0.02 g / 100 g, while that of Comparative Example 2 is 0.81 g / 100 g. The nitrogen inhalation, addition of rosemary extract, and multiple purification processes in Example 2 effectively inhibit and remove oil oxidation products. Comparative Example 2 lacks antioxidant measures and simple filtration is insufficient for impurity removal, leading to a higher accumulation of oxidation products. This indicates that the process of Example 2 better ensures the oxidative stability of mussel oil and prolongs product stability. The acid value of Example 2 is 0.4 mg / g, while that of Comparative Example 2 is as high as 4.1 mg / g. The purification steps in Example 2, including water washing and neutralization, activated clay adsorption, and vacuum distillation, effectively remove free fatty acids. Comparative Example 2, lacking these purification steps, has a higher residue of free fatty acids, indicating that the process in Example 2 improves the acid value quality of mussel oil and reduces undesirable flavors and potential health risks. Example 2 has a yellow value of 6 and a red value of 2, while Comparative Example 2 has a yellow value of 37 and a red value of 10. The activated clay decolorization and vacuum distillation deodorization processes in Example 2 remove pigments and impurities, resulting in a lighter oil color. Comparative Example 2, lacking a decolorization step, has a higher residue of pigments, leading to a darker color. This demonstrates that the process in Example 2 improves the appearance of mussel oil, facilitating product application.

[0062] In summary, Example 2, through its enzymatic hydrolysis, multi-stage extraction, multi-step purification, and antioxidant addition process, comprehensively outperforms the single extraction and simple filtration process of Comparative Example 2 in terms of mussel oil extraction rate, peroxide value, acid value, and color. This fully demonstrates that reasonable process optimization can significantly improve the extraction efficiency and quality of mussel oil, providing an effective technical path for the industrial production of mussel oil and the development of high-quality products.

[0063] Experimental Example 3 This experimental example measures the stability, encapsulation rate, and release rate of the oil-in-water emulsion in Example 3 and Comparative Example 3, specifically including the following steps: 1. Determination of the stability of oil-in-water emulsions: 5 mL of the mussel oil emulsion obtained in Example 3 and Comparative Example 3 were transferred to centrifuge tubes, with each sample in triplicate. The tubes were centrifuged at 3000 r / min for 10 min to observe the emulsion stratification. The stratification rate was calculated based on the ratio of the volume of the upper clear liquid to the total emulsion volume. The results are shown in Table 3-1.

[0064] Table 3-1 Stability of Oil-in-Water Emulsions

[0065] As shown in Table 3-1, the multi-layer encapsulation process using chitosan-hyaluronic acid-chitosan and the sodium alginate-calcium chloride semi-interpenetrating network process effectively encapsulates oil droplets through electrostatic assembly via charge interaction, while the semi-interpenetrating network further stabilizes the emulsion structure, significantly reducing the probability of oil droplet aggregation. Only 0.3% stratification occurred after centrifugation, demonstrating superior stability. In contrast, the single-layer chitosan encapsulation and gelatin gelation process resulted in a weak barrier and a loose gelatin network, leading to easy aggregation and floating of oil droplets due to Brownian motion. This resulted in 25.7% stratification after centrifugation, reflecting poor stability and a high risk of emulsion demulsification. A low stratification rate indicates greater stability during storage and transportation, preventing product failures caused by oil-water stratification, such as uneven gelation and precipitation of active ingredients, making it suitable for high-end formulation development. The multi-layer encapsulation and semi-interpenetrating network in Example 3 enhances oil droplet dispersibility through electrostatic layer-by-layer assembly and constructs a physical barrier through the semi-interpenetrating network to inhibit oil droplet aggregation. This provides a replicable technical path for the long-lasting, uniform, and high-quality development of mussel-containing oil gels.

[0066] 2. Determination of package rate: Weigh 3g of test sample 3 and reference sample 3 separately, with each sample 3 in parallel. Add 5 mL of methanol, sonicate, centrifuge at 3000 r / min for 5 min, and take the solution to obtain the wrapped mussel oil. The total mussel oil was determined by the mussel oil emulsion before wrapping.

[0067] The fatty acid and amino acid content of the coated mussel oil and total mussel oil were determined according to the method for determining fatty acid and amino acid content in Experiment Example 1. The coating rate was calculated by the ratio of coated mussel oil to total mussel oil. The results are shown in Table 3-2.

[0068] Table 3-2 Encapsulation rate of mussel oil

[0069] As shown in Table 3-2, the fatty acid encapsulation rate of Example 3 reached 97.9%, while that of Comparative Example 3 was only 50.0%, with the former's encapsulation efficiency being 1.96 times that of the latter. The amino acid encapsulation rates of Example 3 and Comparative Example 3 were 98.4% and 48.8%, respectively, with a ratio of 2.02. This indicates that Example 3 adopted a three-layer electrostatic assembly structure of chitosan-hyaluronic acid-chitosan. Chitosan is positively charged, and hyaluronic acid is negatively charged. Through electrostatic attraction between positive and negative charges, they adsorb layer by layer to form a sandwich-like encapsulation membrane. This structure can spatially encapsulate mussel oil droplets, reducing oil droplet leakage. At the same time, the physical barrier effect of the multilayer membrane is stronger, which can effectively prevent fatty acids and amino acids from escaping from the encapsulation system. Comparative Example 3 only used a single layer of chitosan encapsulation. The single-layer membrane is thin, loosely structured, and lacks the cross-linking effect of charge complementarity. The contact barrier between the oil droplets and the external environment is weak, and the encapsulated components are easily leaked due to Brownian motion or system disturbance, ultimately resulting in a low encapsulation rate. Furthermore, in Example 3, the sodium alginate-calcium chloride crosslinking forms a semi-interpenetrating network. This network structure has uniform porosity and good elasticity, which can fix multilayered oil droplets within the gel matrix, further reducing oil droplet aggregation and component loss. This, combined with the multilayer encapsulation, provides dual protection and synergistically improves encapsulation efficiency. In contrast, the gelatin gel in Comparative Example 3 easily forms a loose network structure at room temperature, with large pores and poor stability. It cannot effectively fix single-layered oil droplets, allowing them to easily migrate within the gel and break through the single-layer membrane, resulting in a decreased encapsulation rate.

[0070] In summary, the multilayer electrostatic assembly and sodium alginate-calcium chloride semi-interpenetrating network process in Example 3 significantly improved the encapsulation rate of fatty acids and amino acids in mussel oil by optimizing the encapsulation structure and gel carrier. This lays a key technological foundation for the efficacy stability and quality reliability of mussel oil-containing gel products, and has greater technical advantages and application value compared with the single-layer encapsulation and gelatin process in Comparative Example 3.

[0071] 3. Determination of release rate: The effect of using and not using an iontophoresis device on the release rate of mussel oil was investigated under simulated human body temperature and fluid flow conditions.

[0072] Iontophoresis device not used: Weigh 0.2g of test sample 3 and reference sample 3 separately and place them in dialysis bags, with each sample 3 in parallel. After sealing, place them in centrifuge tubes containing 20mL of phosphate buffer and put them in a constant temperature shaker at 37℃ and 100 r / min. At 0.5h, 1h, 2h, 4h, 6h, 8h, 12h and 24h respectively, 2mL of release medium was aspirated and an equal volume of fresh phosphate buffer was added. Weigh 0.2g of test sample 3 and reference sample 3 separately, with each sample 3 in parallel. Add 5 mL of methanol, sonicate, centrifuge at 3000 r / min for 5 min, and take the solution to obtain the encapsulated mussel oil.

[0073] The content of fatty acids and amino acids in the release medium and total mussel oil were determined according to the method for determining the content of fatty acids and amino acids in Experiment Example 1. The release rate was calculated by the ratio of mussel oil in the release medium to the mussel oil in the package. The results are shown in Table 3-3.

[0074] Table 3-3 Mussel oil release rate without iontophoresis

[0075] As shown in Table 3-3, at 0.5 h, the release rates of both components in Example 3 were 6.8%, while those in Comparative Example 3 were 20.0% and 19.3%, respectively. At 24 h, the release rate of Example 3 was 56%, while that of Comparative Example 3 was 97%. Example 3 exhibited a stable release process within 24 h, with a uniform increase in release rate at each time point, demonstrating long-lasting sustained-release characteristics. Comparative Example 3 showed a rapid release rate in the early stages, reaching over 67% at 6 h, but the release was nearly complete in the later stages, with no significant sustained-release effect. The chitosan-hyaluronic acid-chitosan multilayer encapsulation and sodium alginate-calcium chloride semi-interpenetrating network process in Example 3 effectively inhibited the release of active ingredients from mussel oil, while the single-layer chitosan encapsulation and gelatin gel process in Comparative Example 3, due to its weak encapsulation barrier and loose gel network, could not control the release of components.

[0076] Using an iontophoresis device: Insert the electrodes of the iontophoresis instrument into the centrifuge tube, weigh 0.2 g of test sample 3 and reference sample 3 into the dialysis bag, place each sample 3 in parallel, seal the bag and place it into a centrifuge tube containing 20 mL of phosphate buffer, and place it in a constant temperature shaker with an electric field of 0.5 mA / cm², 37 ℃ and 100 r / min. At 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h respectively, 2 mL of release medium was aspirated and an equal volume of fresh phosphate buffer was added. Weigh 0.2 g of test sample 3 and reference sample 3 separately, with each sample 3 in parallel. Add 5 mL of methanol, sonicate, centrifuge at 3000 r / min for 5 min, and take the solution to obtain the encapsulated mussel oil.

[0077] The content of fatty acids and amino acids in the release medium and total mussel oil were determined according to the method described in Experiment Example 1. The release rate was calculated by the ratio of mussel oil in the release medium to the mussel oil in the package. The results are shown in Table 3-4.

[0078] Table 3-4 Mussel oil release rate using iontophoresis device

[0079] As shown in Tables 3-4, the iontophoresis device significantly enhanced the release of Example 3 compared to Comparative Example 3. At 60 min, the release rates of fatty acids and amino acids in Example 3 reached 95.1%, while those in Comparative Example 3 were 58.0% and 58.1%, respectively, with the former's release efficiency being 1.6 times that of the latter. The release rate of Example 3 was significantly increased under the influence of an electric field, reaching over 30% at 10 min, over 86% at 30 min, and essentially complete release at 60 min. In contrast, the release rate of Comparative Example 3 was lower at all time points, below 60% at 60 min, indicating limited improvement in release rate. The multi-layered encapsulation and semi-interpenetrating network structure of Example 3 was easily disrupted under the influence of an electric field, thus accelerating the release of the active ingredient. The simple single-layered encapsulation structure of Comparative Example 3 showed less structural disruption under the influence of the electric field, resulting in a less significant release-enhancing effect.

[0080] In summary, the multi-layer encapsulation and semi-interpenetrating network process of Example 3 endows the product with long-lasting sustained-release capability, solving the problem of rapid ingredient loss under the single-layer encapsulation and gelatin gel process of Comparative Example 3. This makes it more suitable for gel-type masks that require long-term efficacy. The iontophoresis device can specifically enhance the release rate of Example 3, enabling flexible switching between long-lasting sustained release and rapid release. Comparative Example 3, due to process limitations, cannot effectively control release through this method, further demonstrating the technical advantages of the process in Example 3. Whether using or not using the iontophoresis device, Example 3 exhibits superior release characteristics, meeting both long-lasting needs in conventional scenarios and addressing special scenarios requiring rapid onset of action, thus supporting the diversified application of mussel oil-containing gel products.

[0081] Experiment Example 4 This experimental example verifies the shelf life of Example 3 and Comparative Example 4 by measuring appearance, active ingredient content, and microorganisms. The specific steps include: Both test sample 3 and reference sample 4 were stored at 25°C, 60%RH, and protected from light.

[0082] 1. Appearance measurement: At the 0th, 3rd, 6th, 9th, 12th and 13th months of storage, the presence of stratification, precipitation and discoloration of the samples was recorded by visual observation. The test results are shown in Table 4-1.

[0083] Table 4-1 Results of Appearance Measurement

[0084] Note: "-" indicates spoilage; stop observation. Layering, sedimentation, and discoloration indicate spoilage.

[0085] As shown in Table 4-1, reference sample 4 exhibited obvious stratification in the third month, marking the earliest point of appearance deterioration. The stratification continued into the sixth month without precipitation, but it already met the criteria for deterioration, indicating extremely poor system stability and inability to withstand long-term storage. Test sample 3 showed no stratification or precipitation for the first 12 months, maintaining a completely stable appearance, a stark contrast to reference sample 4. Only in the 13th month did it show slight stratification without precipitation, indicating very mild deterioration, demonstrating that its system effectively delayed stratification and its stability was far superior to reference sample 4. Reference sample 4 initially appeared pale yellow, deepening to a deep yellow in the sixth month, a significant color change reflecting potential oxidation and degradation reactions within the sample, further proving its poor stability. Test sample 3 maintained stable color from month 0 to month 13, indicating that its system effectively protected active ingredients such as mussel oil, preventing oxidation and degradation reactions that cause color changes, demonstrating significantly better color stability than reference sample 4.

[0086] In summary, test sample 3 uses a chitosan-hyaluronic acid-chitosan multilayer encapsulation of mussel oil and a sodium alginate-calcium chloride cross-linked semi-interpenetrating network structure. This structure can form a stable gel system, reduce the contact between mussel oil and the external environment, and delay stratification and component deterioration. In contrast, reference sample 4 is simply a mixture of glycerol and xanthan gum without a stable encapsulation or cross-linking structure. The system is easily affected by storage conditions and quickly shows stratification and color changes.

[0087] 2. Determination of active ingredient content: At storage months 0, 3, 6, 9, 12, 13, and 14, the contents of fatty acids and amino acids in each storage period were determined according to the method described in Experimental Example 1. The retention rates of fatty acids and amino acids were calculated by comparing the mussel oil obtained after storage with the mussel oil obtained in month 0.

[0088] Table 4-2 Results of determination of active ingredient content

[0089] Note: "-" indicates that the product has deteriorated and testing should be stopped; a retention rate of less than 50% of the active ingredient content is considered as deterioration.

[0090] As shown in Table 4-2, the retention rates of fatty acids and amino acids in test sample 3 showed a slow downward trend with prolonged storage time. At 3 months, the retention rates of both fatty acids and amino acids were 98.3%; at 14 months, they were 78.2% and 78.3%, respectively, remaining at a high level, indicating that their active ingredients could be stably retained for a relatively long period. The retention rate of the active ingredients in reference sample 4 decreased significantly faster than that of test sample 3. At 3 months, the retention rates of fatty acids and amino acids were 85.2% and 85.6%, respectively; at 9 months, they had decreased to 58.1% and 58.0%, respectively, approaching the 50% deterioration threshold; at 12 months, they further decreased to 45.8% and 45.3%, below 50%, meeting the deterioration criteria.

[0091] In summary, Example 3, compared to Comparative Example 4, can more effectively retain the active ingredients, has better stability, and can maintain the content of active ingredients in the product for a longer period of time.

[0092] 3. Microbial assay: Samples were taken out for microbial analysis at 0, 3, 6, 9, 12, 13, and 14 months of storage.

[0093] Sample pretreatment: Take 10g of each of Example 3 and Comparative Example 4, place them in a sterile homogenizing bag, add 100mL of sterile 0.9% sodium chloride solution, seal and place in a sterile homogenizer, homogenize at 9000r / min for 2min to prepare a sample homogenate, and use no sample as a blank control.

[0094] Determination of total bacterial count: Dissolve 10g peptone, 3g beef extract, 5g sodium chloride and 15g agar in 1000 mL of distilled water, adjust the pH to 7.2, autoclave at 121℃ for 15 min, and cool to 50℃ to obtain nutrient agar medium. Transfer 1 mL of the sample homogenate into a sterile petri dish, pour in 15 mL of nutrient agar medium, and after the medium solidifies, incubate upside down at 36℃ for 48 h. Calculate the total bacterial count based on the product of the plate colony count and the dilution factor. The results are shown in Table 4-3.

[0095] Determination of total mold and yeast colony count: Dissolve 5 g peptone, 20 g glucose, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 15 g agar, and 0.033 g Bengal red in 950 mL of distilled water, adjust the pH to 5.6, autoclave at 121 °C for 15 min, cool to 50 °C, and add 50 mL of 1 mg / mL chloramphenicol solution to obtain Bengal red agar medium. Transfer 1 mL of sample homogenate into a sterile petri dish, pour in 15 mL of Bengal red agar medium, and incubate at 28°C upside down for 72 h after the medium solidifies. Calculate the total number of mold and yeast colonies based on the product of the number of colonies on the plate and the dilution factor. The results are shown in Table 4-3.

[0096] Table 4-3 Results of Microbial Detection

[0097] Note: "-" indicates spoilage, stop testing; a total bacterial count above 30 CFU / g is considered spoilage.

[0098] As shown in Table 4-3, for test sample 3, the total bacterial count and total mold and yeast count were both less than 10 CFU / g during the 0-12 month period, indicating a low level. At 13 months, the total bacterial count and total mold and yeast count were both 20 CFU / g. At 14 months, the total bacterial count was 30 CFU / g, and the total mold and yeast count was 20 CFU / g. Although there was an increase within 13 months, none of these values ​​exceeded the 30 CFU / g deterioration threshold, indicating a low risk of microbial contamination and good stability over a relatively long period. For control sample 4, at 3 months, the total bacterial count reached 40 CFU / g, and the total mold and yeast count was 30 CFU / g, both exceeding or approaching the 30 CFU / g deterioration threshold, indicating poor microbial stability and susceptibility to microbial contamination and deterioration.

[0099] In summary, Example 3 demonstrates superior microbial stability compared to Comparative Example 4, maintaining a lower microbial count for a longer period and delaying spoilage.

[0100] The above description is only used to illustrate the technical solution of the present invention and is not intended to limit it. Equal modifications and variations made by those skilled in the art to the technical solution of the present invention, as long as they do not depart from the overall concept of the present invention, shall still fall within the scope of the present invention.

Claims

1. A method for preparing mussel oil, characterized in that: The preparation method of the mussel oil specifically includes the following steps: S001, add magnetic complex enzyme to mussel homogenate, adjust pH, enzymatically hydrolyze, separate with a magnet, collect the solution to obtain mussel enzymatic hydrolysate; S002, an extractant was added to the mussel enzymatic hydrolysate for extraction, and the extracted organic phases were combined; S003, nitrogen gas is introduced, rotary evaporation is performed, and rosemary extract is added to obtain crude mussel oil; S004: After heating the crude mussel oil, add ultrapure water and stir. After standing and separating the layers, collect the upper layer, add sodium hydroxide solution to neutralize it, and wash with ultrapure water until neutral. S005, add activated clay, stir, filter, vacuum distill, filter again to obtain refined mussel oil; S006, refined mussel oil and ethanolamine are added to a three-necked flask, nitrogen gas is introduced and heated, and the mixture is stirred until dissolved. Cyclohexylcarbodiimide and 4-dimethylaminopyridine are added. After the reaction, ethyl acetate is added for extraction. The organic phases are combined, washed and distilled under reduced pressure to obtain aminated mussel oil, denoted as mussel oil.

2. The method for preparing mussel oil according to claim 1, characterized in that: Mussel oil is encapsulated in a multilayer membrane of hyaluronic acid and chitosan, utilizing the electrostatic interaction between the two to achieve layer-by-layer assembly. This process balances the encapsulation efficiency of oil-soluble components with the release performance during iontophoresis, resulting in a gel mask containing mussel oil. The specific steps include: S101, mix mussel oil with Tween-80, dilute with phosphate buffer, and then emulsify by ultrasonication to obtain mussel oil emulsion; S102, slowly add an equal volume of chitosan solution and mix, stir, centrifuge, discard the supernatant, and wash twice with phosphate buffer. S103, add an equal volume of hyaluronic acid solution, stir, centrifuge, discard the supernatant, and wash twice with phosphate buffer. S104, slowly add an equal volume of chitosan solution and mix, stir, centrifuge, discard the supernatant, wash twice with phosphate buffer to obtain chitosan-hyaluronic acid-chitosan-encapsulated mussel oil, which is recorded as a multi-layer encapsulated material; S105, add calcium chloride solution, stir, add an equal volume of sodium alginate solution, add calcium chloride solution dropwise for cross-linking, ultrafilter, sterilize and filter to obtain a gel mask containing mussel oil.

3. The method for preparing mussel oil according to claim 1, characterized in that: The mussel homogenate solution is obtained by pretreating mussels through washing, desalination, and homogenization steps, specifically including the following steps: S201. The fresh mussel meat is rinsed, drained, soaked in sodium chloride solution, and then rinsed again to obtain clean mussel meat. S202, clean mussel meat is added to a phosphate buffer solution containing antioxidants and homogenized to obtain a mussel homogenate.

4. The method for preparing mussel oil according to claim 1, characterized in that: The magnetic composite enzyme is prepared by co-precipitation of magnetic iron oxide nanoparticles, followed by silanization and glutaraldehyde activation. Neutral protease and lipase are then immobilized on the surface of a magnetic carrier by covalent bonds. The composite enzyme is composed of neutral protease and lipase in a mass ratio of 2:

1.

5. The method for preparing mussel oil according to claim 1, characterized in that: The extractant is composed of n-hexane and ethanol containing antioxidants in a volume ratio of 3:

1. The antioxidants are composed of 0.1% vitamin E and 0.05% citric acid.

6. The method for preparing mussel oil according to claim 3, characterized in that: In step S202, phosphate buffer containing antioxidants is added at a material-to-liquid ratio of 1:

2. The antioxidants are composed of ethylenediaminetetraacetic acid (EDTA) and tea polyphenols, and the concentrations of both EDTA and tea polyphenols are 0.05%.

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