A method for preparing an emulsion with fish skin gelatin and fucoidan as a composite carrier

Abstract

The application discloses a preparation method of an emulsion with fish skin gelatin and fucoidan as a composite carrier, and belongs to the technical field of emulsion preparation. The method comprises the following steps: S1, preparation of an aqueous phase: preparing a fish skin gelatin solution, and then adding fucoidan to obtain the aqueous phase; S2, preparation of an oil phase: adding a fat-soluble active substance to corn oil to make the fat-soluble active substance fully dissolved to obtain the oil phase; and S3, mixing the oil phase and the aqueous phase, high-speed dispersion and homogenization, so as to obtain the emulsion with the fish skin gelatin and the fucoidan as a composite carrier. In the application, the fish skin gelatin and the fucoidan compound are used as the aqueous phase, the corn oil is selected as the oil phase, and the fat-soluble active substance is used as the embedding material, and the embedding material can be encapsulated through the emulsion. The application is simple in operation, and the prepared emulsion has good storage stability and high ability of protecting the active substance.

Description

Technical Field

[0001] This invention belongs to the field of emulsion preparation technology, specifically relating to a method for preparing an emulsion using fish skin gelatin and fucoidan as a composite carrier. Background Technology

[0002] Plants contain many lipophilic active substances, such as carotene, vitamins, and polyphenols, which possess beneficial functional activities for human health, including anti-cancer, anti-inflammatory, blood sugar-lowering, blood lipid-lowering, and antioxidant properties. Benzyl isothiocyanate (BITC), a natural fat-soluble active substance also known as mustard oil, is a type of isothiocyanate compound (ITC) widely found in cruciferous vegetables. It has been proven to have anti-inflammatory, antibacterial, and antitumor effects, but its low water solubility, rapid degradation, and low bioavailability limit its widespread application. In food processing, encapsulation technology can mask irritating odors, improve stability, reduce volatility, and extend the duration of action. It can also allow for slow release of the encapsulated substance under more suitable conditions. Using an oil-in-water (O / W) emulsion delivery system to encapsulate fat-soluble active substances effectively protects them and improves their bioavailability. Therefore, using an emulsion delivery system to encapsulate BITC can effectively expand its application range.

[0003] Fish skin gelatin possesses excellent emulsifying properties. Structurally, it has numerous hydrophilic and hydrophobic sites, giving it both lipophilic and hydrophilic characteristics. When adsorbed onto the oil-water interface, fish skin gelatin reduces interfacial tension, thus exhibiting emulsifying properties. Compared to unstable emulsion systems with only protein added, the addition of polysaccharides as stabilizers improves the properties of protein emulsions. By altering the emulsion's rheological properties, it prevents droplet aggregation and achieves long-term stability. Proteins, acting as emulsifiers, and polysaccharides, acting as stabilizers, work in a complementary manner, forming a more stable composite emulsion delivery system. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for preparing an emulsion using fish skin gelatin and fucoidan as a composite carrier. Fish skin gelatin is used as an emulsifier, and fucoidan is used as a stabilizer to encapsulate fat-soluble active substances, thereby obtaining a stabilized emulsion that utilizes the intermolecular forces between fish skin gelatin and fucoidan to prepare a composite carrier and construct a structure capable of encapsulating fat-soluble active substances.

[0005] The first objective of this invention is to provide a method for preparing a stabilized emulsion using fish skin gelatin and fucoidan as a composite carrier, comprising the following steps:

[0006] S1. Preparation of the aqueous phase: Prepare a fish skin gelatin solution, then add fucoidan and mix to obtain the aqueous phase;

[0007] S2. Preparation of the oil phase: The fat-soluble active substance is dissolved in edible oil to obtain the oil phase;

[0008] S3. Mixing: The aqueous phase prepared in S1 and the oil phase prepared in S2 are mixed and dispersed at high speed, and then homogenized to obtain a stabilized emulsion with fish skin gelatin and fucoidan as composite carriers.

[0009] In one embodiment of the present invention, the mass concentration of the fish skin gelatin solution in S1 is 0.5~1.5%.

[0010] In one embodiment of the present invention, the mass ratio of fish skin gelatin to fucoidan in S1 is 8:1 to 1:1.

[0011] In one embodiment of the present invention, the edible oil in S2 is one or more of corn oil, soybean oil, sunflower seed oil, rapeseed oil, and olive oil, and is more preferably corn oil.

[0012] In one embodiment of the present invention, the fat-soluble active substance in S2 is one or more of fat-soluble vitamins, flavonoids, isothiocyanates, and carotene, and is more preferably benzyl isothiocyanate.

[0013] In one embodiment of the present invention, the concentration of the fat-soluble active substance in the oil phase of S2 is 1~10 mg / mL.

[0014] In one embodiment of the present invention, the concentration of the fat-soluble active substance in the oil phase of S2 is 5~6 mg / mL.

[0015] In one embodiment of the present invention, the volume ratio of oil phase to water phase in S3 is 1:5 to 1:15, and more preferably 1:8 to 1:12.

[0016] In one embodiment of the present invention, the parameters for high-speed dispersion in S3 are 8000~15000 r / min, and the high-speed dispersion time is 1~4 min.

[0017] In one embodiment of the present invention, the homogenization parameters in S3 are 8000~14000 psi, and the number of homogenization cycles is 4~8.

[0018] This invention provides a stabilized emulsion prepared by the method described above, using fish skin gelatin and fucoidan as a composite carrier.

[0019] The present invention also provides the application of the stabilized emulsion described above, which uses fish skin gelatin and fucoidan as a composite carrier, in the food industry.

[0020] The beneficial effects of this invention are:

[0021] (1) BITC, as a typical isothiocyanate compound, has significant antibacterial, anticancer, and anti-inflammatory activities in the field of biology, and has therefore become a research focus in recent years. However, due to the volatility, thermal instability, and acid-base instability of BITC, its bioavailability in practical applications is low, which to some extent restricts its further promotion and application. Encapsulating BITC in an oil-in-water emulsion delivery system is a method that can effectively reduce BITC loss, improve transportation efficiency, and enhance bioavailability. The fish skin gelatin selected in this invention has excellent functional properties, but the stability of emulsions constructed from a single protein is easily affected by the environment. Fucoidan, a stable and non-toxic natural sulfated polysaccharide derived from marine brown algae (such as kelp and seaweed), possesses higher water solubility and charge density. When bound to proteins, it exhibits superior electrostatic binding capacity, allowing it to alter emulsion rheological properties and prevent droplet aggregation and agglomeration for long-term stability. Furthermore, its strong hydrophilicity, good gelling properties, and excellent biocompatibility enable it to interact with proteins and influence their application characteristics, making it a valuable wall material raw material for enhancing protein emulsification. Current research on the stability of oil-in-water emulsions using protein-polysaccharide composite systems primarily focuses on the interaction between proteins and anionic polysaccharides such as gum arabic, pectin, and xanthan gum. Research on fucoidan is severely lacking, thus its application in stabilizing emulsion systems is still in its early stages, significantly limiting the role of fish skin gelatin-fucoidan composite systems in food processing. This paper mainly studies the interaction between fucoidan and fish skin gelatin, elucidates the formation and structural changes of the complex, and explores the synergistic effect of the fish skin gelatin-fucoidan complex system on emulsion stabilization based on this. It also studies the preparation of composite carriers and the construction of stable emulsions that can encapsulate fat-soluble active substances, providing new ideas for the construction of emulsion delivery systems.

[0022] (2) The present invention found that compared with the simple fish skin gelatin solution, the contact angle of the fish skin gelatin-fucose polysaccharide complex first increased and then decreased with the increase of fucoidan concentration. The results show that when the ratio of fish skin gelatin to fucoidan is 2:1, the emulsion is more stable and the nanoparticles are more stable at the oil-water interface. Figures 1-6 During 14 days of storage, compared with simple fish skin gelatin emulsion, the appropriate addition of fucoidan significantly reduced the particle size of the composite emulsion. p <0.05) Figure 7 Furthermore, as the concentration of fucoidan increased, the particle size of the composite emulsion first decreased and then increased, and the zeta potential of the composite emulsion decreased. When the ratio of fish skin gelatin to fucoidan was 2:1, the particle size of the composite emulsion was the smallest, indicating that the composite emulsion at this ratio had better stability. Figure 7 , Figure 8Except for the fish skin gelatin-fucose polysaccharide ratio of 1:2, the fish skin gelatin-fucose polysaccharide composite emulsions with other ratios did not show obvious stratification during storage. Figures 9-13 Fluorescence microscopy revealed that on day 0 of storage, when the ratio of fish skin gelatin to fucoidan was 8:1 / 4:1 / 2:1 / 1:1, the oil droplets in the composite emulsion were relatively uniformly distributed, with no significant aggregation. Figures 14-19 On the 14th day of storage, the microstructure size of all compound emulsions with different ratios increased. Figures 20-25 After 14 days of storage, the fish skin gelatin-fucose polysaccharide composite emulsion exhibited the highest retention rate of lipid-soluble active substances (BITC) when the ratio of fish skin gelatin to fucoidan was 2:1. Figure 26 This indicates that under these conditions, the fish skin gelatin-fucose polysaccharide composite emulsion exhibits a better protective effect against lipid-soluble active substances (BITC).

[0023] (3) This invention provides a simple and convenient method for preparing fish skin gelatin-fucose polysaccharide composite emulsion without adjusting the pH of the aqueous phase. Attached Figure Description

[0024] Figure 1 This is a diagram showing the measurement of the contact angle in Comparative Example 1 of the present invention;

[0025] Figure 2 This is a diagram showing the measurement of the contact angle in Embodiment 1 of the present invention;

[0026] Figure 3 This is a diagram showing the measurement of the contact angle in Embodiment 2 of the present invention;

[0027] Figure 4 This is a diagram showing the measurement of the contact angle in Embodiment 3 of the present invention;

[0028] Figure 5 This is a diagram showing the measurement of the contact angle in Embodiment 4 of the present invention;

[0029] Figure 6 This is a diagram showing the measurement of the contact angle in Comparative Example 2 of the present invention;

[0030] Figure 7 This is a graph showing the particle size changes of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Examples 1-4 and Comparative Example 2, compared with the fish skin gelatin emulsion in Comparative Example 1, after being stored at room temperature for 0, 1, 4, 7, and 14 days.

[0031] Figure 8 This is a potential change graph of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Examples 1-4 and Comparative Example 2 of the present invention and the fish skin gelatin emulsion in Comparative Example 1 (simple fish skin gelatin) after being stored at room temperature for 0, 1, 4, 7 and 14 days.

[0032] Figure 9These are actual images of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Examples 1-4 and Comparative Example 2 of the present invention, and the fish skin gelatin emulsion in Comparative Example 1 (simple fish skin gelatin), stored at room temperature for 0 days. From left to right, they are Comparative Example 1, Example 1, Example 2, Example 3, Example 4, and Comparative Example 2.

[0033] Figure 10 These are actual images of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Examples 1-4 and Comparative Example 2 of the present invention, and the fish skin gelatin emulsion in Comparative Example 1 (simple fish skin gelatin), stored at room temperature for 1 day. From left to right, they are Comparative Example 1, Example 1, Example 2, Example 3, Example 4, and Comparative Example 2.

[0034] Figure 11 These are actual images of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Examples 1-4 and Comparative Example 2 of the present invention, and the fish skin gelatin emulsion in Comparative Example 1 (simple fish skin gelatin), stored at room temperature for 4 days. From left to right, they are Comparative Example 1, Example 1, Example 2, Example 3, Example 4, and Comparative Example 2.

[0035] Figure 12 These are actual images of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Examples 1-4 and Comparative Example 2 of the present invention, and the fish skin gelatin emulsion in Comparative Example 1 (simple fish skin gelatin), stored at room temperature for 7 days. From left to right, they are Comparative Example 1, Example 1, Example 2, Example 3, Example 4, and Comparative Example 2.

[0036] Figure 13 These are actual images of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Examples 1-4 and Comparative Example 2 of the present invention, and the fish skin gelatin emulsion in Comparative Example 1 (simple fish skin gelatin), stored at room temperature for 14 days. From left to right, they are Comparative Example 1, Example 1, Example 2, Example 3, Example 4, and Comparative Example 2.

[0037] Figure 14 This is a fluorescence microscope image of the control group emulsion of pure fish skin gelatin prepared in Comparative Example 1 of this invention after being stored at room temperature for 0 days.

[0038] Figure 15 This is a fluorescence microscope image of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Example 1 of this invention after being stored at room temperature for 0 days;

[0039] Figure 16 This is a fluorescence microscope image of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Example 2 of this invention after being stored at room temperature for 0 days;

[0040] Figure 17 This is a fluorescence microscope image of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Example 3 of this invention after being stored at room temperature for 0 days;

[0041] Figure 18This is a fluorescence microscope image of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Example 4 of this invention after being stored at room temperature for 0 days;

[0042] Figure 19 This is a fluorescence microscope image of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Comparative Example 2 of this invention after being stored at room temperature for 0 days.

[0043] Figure 20 This is a fluorescence microscope image of the control group emulsion of pure fish skin gelatin prepared in Comparative Example 1 of this invention after being stored at room temperature for 14 days.

[0044] Figure 21 This is a fluorescence microscope image of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Example 1 of this invention after being stored at room temperature for 14 days;

[0045] Figure 22 This is a fluorescence microscope image of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Example 2 of this invention after being stored at room temperature for 14 days;

[0046] Figure 23 This is a fluorescence microscope image of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Example 3 of this invention after being stored at room temperature for 14 days;

[0047] Figure 24 This is a fluorescence microscope image of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Example 4 of this invention after being stored at room temperature for 14 days;

[0048] Figure 25 This is a fluorescence microscope image of the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Comparative Example 2 of this invention after being stored at room temperature for 14 days.

[0049] Figure 26 This is a diagram showing the encapsulation rate of BITC in the fish skin gelatin-fucose polysaccharide composite emulsion prepared in Examples 1-4 and Comparative Example 2 of the present invention, compared with that in the simple fish skin gelatin emulsion of Comparative Example 1. Detailed Implementation

[0050] Test method:

[0051] Determination of the contact angle of the composite system: Freeze-dried fish skin gelatin-fucose polysaccharide complex powder was pressed into tablets with a thickness of 2 mm and a diameter of 12 mm. These tablets were then soaked in corn oil, and the sample was measured using the sitting drop method. The syringe position and baseline position were adjusted, and the sample was analyzed using deionized water, with each drop being 2 μL. The specific analytical basis is that a contact angle of 90° is the critical value for amphiphilicity. Above the critical value, the oil-water interface tends to be hydrophobic, and below the critical value, it tends to be hydrophilic. The oil-in-water emulsion prepared at the critical value is the most stable.

[0052] Particle size and zeta potential determination of emulsions: The product emulsions prepared in Examples 1-4 and Comparative Example 2, and the control emulsion containing only fish skin gelatin, were stored for 0 days, 1 day, 4 days, 7 days, and 14 days, respectively. They were diluted 100 times with deionized water, and the average particle size and zeta potential of the sample emulsions were determined using a laser particle size analyzer. Specific experimental parameters were: number of runs: 10, test temperature: 25℃, equilibration time: 10 s. Different uppercase letters in the data represent significant differences in the same emulsion at different storage days, and different lowercase letters represent significant differences in different emulsions at the same storage days. p <0.05). Specific analysis and theoretical basis include:

[0053] (1) The smaller the average particle size of the emulsion, the more stable the system.

[0054] (2) The larger the absolute value of the emulsion potential, the more stable the system.

[0055] Observation of emulsion appearance: The product emulsions prepared in Examples 1-4 and Comparative Example 2, and the control group emulsion containing only fish skin gelatin, were stored for 0 days, 1 day, 4 days, 7 days, and 14 days, respectively, and their appearance was observed. The specific analysis was based on the following: the more stable the system, the less significant the change in the appearance of the emulsion.

[0056] Observation of oil droplet distribution in emulsions: Appropriate amounts of the product emulsions prepared in Examples 1-4 and Comparative Example 2, along with a control group emulsion containing only fish skin gelatin, were stored for 0 and 14 days. Fish skin gelatin and fucoidan, used as wall materials, were labeled with fluorescein isothiocyanate, and corn oil, used as the core material, was labeled with Nile Red. The dye and emulsion samples were mixed evenly at a ratio of 1:25. 5 μL of the stained sample was placed on a glass slide, and images of each sample were obtained using a fluorescence microscope. The specific analytical basis was that the more uniform and smaller the oil droplet distribution in the fluorescence microscope image, the more stable the system.

[0057] BITC Retention Rate Determination: Appropriate amounts of the product emulsions prepared in Examples 1-4 and Comparative Example 2, and the control group emulsion containing only fish skin gelatin, were stored for 0 days and 14 days. BITC in the emulsions was extracted with n-hexane and methanol. The BITC content was analyzed by high-performance liquid chromatography (HPLC). Based on the positive correlation between the concentration of BITC in the solution and the peak content, the retention rate of BITC in the emulsion was calculated: Retention rate (%) = Peak area of ​​BITC in the emulsion / Peak area of ​​BITC in the standard. Different uppercase letters represent significant differences in the same emulsion stored for different days, and different lowercase letters represent significant differences in the same emulsion stored for the same number of days. p <0.05). The specific analysis and theoretical basis are: the higher the encapsulation rate of the emulsion, the more stable the system.

[0058] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0059] Raw material source:

[0060] Fish skin gelatin and BITC were purchased from Sigma-Aldrich, USA; fucoidan was purchased from Shandong Jiejing Group; and the oil used in the experiment was Xiwang non-GMO corn germ oil purchased from Carrefour supermarket in Dalian.

[0061] Comparative Example 1 (Control Group consisting solely of fish skin gelatin)

[0062] S1. Prepare a 1% fish skin gelatin solution and stir it with a magnetic stirrer at room temperature for 1 hour to completely dissolve it.

[0063] S2. Preparation of the oil phase: Dissolve BITC in edible corn oil to prepare a BITC-corn oil solution with a mass concentration of 5 mg / mL;

[0064] S3. The oil phase prepared in S2 and the fish skin gelatin solution prepared in S1 were mixed at a volume ratio of 1:9 and dispersed at 10,000 r / min for 2 min. The mixture was then homogenized at 12,000 psi for 6 times to obtain a control group emulsion of pure fish skin gelatin.

[0065] Example 1

[0066] S1. Prepare a 1% (w / w) fish skin gelatin solution, stir with a magnetic stirrer at room temperature for 1 hour until completely dissolved, add fucoidan to the fish skin gelatin solution and mix to obtain an aqueous phase. The final (w / w) concentration of fucoidan is 0.125%.

[0067] S2. Dissolve BITC in edible corn oil to prepare a BITC-corn oil solution with a mass concentration of 5 mg / mL;

[0068] S3. The oil phase prepared in S2 is mixed with the aqueous phase prepared in S1 at a volume ratio of 1:9, and dispersed at high speed of 8000 r / min for 2 min; then homogenized under high pressure of 8000 psi 6 times to obtain an emulsion with fish skin gelatin and fucoidan composite as carrier.

[0069] Example 2

[0070] S1. Prepare a 1% (w / w) fish skin gelatin solution and stir it with a magnetic stirrer at room temperature for 1 hour until completely dissolved. Add fucoidan to the fish skin gelatin solution and mix to obtain an aqueous phase. The final (w / w) concentration of fucoidan is 0.25%.

[0071] S2. Dissolve BITC in edible corn oil to prepare a BITC-corn oil solution with a mass concentration of 5 mg / mL;

[0072] S3. The oil phase prepared in S2 is mixed with the aqueous phase prepared in S1 at a volume ratio of 1:9, and dispersed at high speed of 14000 r / min for 1 min; then homogenized at high pressure of 12000 psi 4 times to obtain an emulsion with fish skin gelatin and fucoidan composite as carrier.

[0073] Example 3

[0074] S1. Prepare a 1% (w / w) fish skin gelatin solution, stir with a magnetic stirrer at room temperature for 1 hour until completely dissolved, add fucoidan to the fish skin gelatin solution and mix to obtain an aqueous phase. The final (w / w) concentration of fucoidan is 0.5%.

[0075] S2. Dissolve BITC in edible corn oil to prepare a BITC-corn oil solution with a mass concentration of 5 mg / mL;

[0076] S3. Mix the oil phase prepared in S2 with the aqueous phase prepared in S1 at a volume ratio of 1:9, and disperse at 12000 r / min for 4 min.

[0077] The mixture was homogenized at 14,000 psi for 8 cycles to obtain an emulsion with a composite of fish skin gelatin and fucoidan as the carrier.

[0078] Example 4

[0079] S1. Prepare a 1% (w / w) fish skin gelatin solution, stir with a magnetic stirrer at room temperature for 1 hour until completely dissolved, add fucoidan to the fish skin gelatin solution and mix to obtain an aqueous phase, the final (w / w) concentration of fucoidan is 1%;

[0080] S2. Dissolve BITC in edible corn oil to prepare a BITC-corn oil solution with a mass concentration of 5 mg / mL;

[0081] S3. Mix the oil phase prepared in S2 with the aqueous phase prepared in S1 at a volume ratio of 1:9, and disperse at 13000 r / min for 1 min.

[0082] The mixture was homogenized five times under high pressure at 10,000 psi to obtain an emulsion with a composite of fish skin gelatin and fucoidan as the carrier.

[0083] Comparative Example 2

[0084] S1. Prepare a 1% (w / w) fish skin gelatin solution, stir with a magnetic stirrer at room temperature for 1 hour until completely dissolved, add fucoidan to the fish skin gelatin solution and mix to obtain an aqueous phase, the final (w / w) concentration of fucoidan is 2%;

[0085] S2. Dissolve BITC in edible corn oil to prepare a BITC-corn oil solution with a mass concentration of 5 mg / mL;

[0086] S3. The oil phase prepared in S2 is mixed with the fish skin gelatin solution prepared in S1 at a volume ratio of 1:9, and dispersed at high speed of 8000 r / min for 2 min; then homogenized under high pressure of 8000 psi 6 times to obtain an emulsion with fish skin gelatin and fucoidan as the carrier.

[0087] Results analysis:

[0088] This invention, by simultaneously encapsulating BITC with a composite emulsion of fish skin gelatin and fucoidan at different concentrations, found that:

[0089] Contact angle results showed that fish skin gelatin alone was highly hydrophilic with a small contact angle (61.75 ± 1.87°). With the gradual increase of fucoidan content, when the fish skin gelatin to fucoidan ratio was 2:1 (Example 3), the contact angle increased to a maximum of 83.85 ± 0.21°. This indicates that the change in surface wettability increased the ability of the fish skin gelatin-fucoidan composite particles to adsorb onto the oil-water interface. At this point, the particles could quickly adsorb onto the oil-water interface, forming a thicker barrier that prevented emulsion aggregation. The hydrophobicity of the emulsion system gradually increased, becoming amphiphilic. However, with further increases in fucoidan (fish skin gelatin:fucoidan = 1:1 / 1:2) (Example 4, Comparative Example 2), the contact angle decreased instead. The reason for this might be due to the enhanced hydrophobic interaction between the excess polysaccharide and the protein. Figures 1-6 ).

[0090] With the addition of fucoidan, the particle size of the emulsion initially decreased and then increased. The particle size of the fish skin gelatin emulsion was smaller than that of the fish skin gelatin-fucoidan composite emulsion. This is because the anionic polysaccharide and cationic protein molecules formed large molecules through electrostatic interactions. When fucoidan was added to a certain amount (fish skin gelatin:fucoidan = 2:1) (Example 3), the emulsion particle size reached its minimum, at which point the emulsion stability was highest, and the two molecules reached their most stable composite aggregate during this process. When the fucoidan content continued to increase to its maximum proportion (fish skin gelatin:fucoidan = 1:2) (Comparative Example 2), there was an excess of fucoidan. The fish skin gelatin molecules in the system were completely bound by the fucoidan, and the strong electrostatic repulsion led to the formation of an electrically neutral complex, which further inhibited the protein-polysaccharide molecule interaction. Therefore, at this ratio, the emulsion particle size was the largest, and the composite system was the least stable. After 14 days of storage, compared with freshly prepared compound emulsions, the average particle size of fish skin gelatin-fucose composite emulsions with different compounding ratios was significantly increased. Figure 7 ).

[0091] Compared to emulsions containing only fish skin gelatin, the addition of fucoidan caused the zeta potential of the composite system to change from positive to negative. This change is because fucoidan is an anionic polysaccharide, and the positive charge carried by the fish skin gelatin molecules is gradually neutralized by fucoidan, thereby enhancing the overall electronegativity of the system. This observation indicates that the interaction between fish skin gelatin and fucoidan leads to a change in the surface charge characteristics of fucoidan and the exposure of more negatively charged groups on its surface. However, with prolonged storage time, the absolute value of the zeta potential of fish skin gelatin-fucoidan composite emulsions with different ratios showed a decreasing trend. When the emulsion ratio was 2:1 (Example 3), the zeta potential value fluctuated relatively little during the storage period, ranging from -53.47 ± 0.42 mV to -51.57 ± 0.35 mV. Figure 8 ).

[0092] The emulsions were milky white and uniform in texture immediately after preparation, with no adverse conditions. Observation of the emulsion appearance during storage revealed that on days 1, 4, 7, and 14 after preparation, except for the sample with a fish skin gelatin-fucose ratio of 1:2 (Comparative Example 2), the other samples showed no flocculation or precipitation, and remained stable. Figures 9-13 ).

[0093] Fluorescence inverted microscope images showed that the trend of particle size variation with fucoidan concentration observed under the fluorescence inverted microscope was the same as that observed in the particle size determination experiment of the emulsion. Figures 14-19 ), except for the emulsion with a compounding ratio of 1:2 (Comparative Example 2) Figure 19 In addition to the above, no obvious oil droplet aggregation was observed in the other emulsion groups. This indicates that the continuous aqueous phase of the emulsion system was dispersed around the oil phase, and the oil droplets were uniformly and stably distributed within the aqueous phase. The fish skin gelatin-fucose composite emulsion had a mixing ratio of 2:1 (Example 3). Figure 17 The system contained no large oil droplets, and the distribution of oil droplets was more uniform. The fish skin gelatin-fucose polysaccharide complex overlapped with the oil droplets, forming a good oil-in-water emulsion delivery system, exhibiting the best performance. With increasing storage days, the microstructure size of all compound sample groups increased, a result consistent with the trend of emulsion particle size variation. Figures 20-25 ).

[0094] Compared to emulsions containing only fish skin gelatin, the BITC encapsulation rate of emulsion delivery systems constructed with an appropriate proportion of fucoidan was significantly increased. Analysis showed that, compared to hydrophilic fish skin gelatin, increasing the fucoidan concentration improved the emulsifying properties of the fish skin gelatin-fucose complex, reduced the interfacial tension between oil and water, and significantly improved the encapsulation ability of the emulsion. The highest BITC encapsulation rate was observed when the ratio of fish skin gelatin to fucoidan was 2:1 (Example 3). However, further increasing the polysaccharide concentration to fish skin gelatin:fucose = 1:1 and 1:2 resulted in a continuous decrease in BITC retention. This phenomenon is presumably due to the enhanced hydrophobic interaction between excessive polysaccharide and proteins. After 14 days of storage, the BITC encapsulation rate of all emulsion samples decreased to varying degrees. When the ratio of fish skin gelatin to fucoidan was 2:1, the BITC retention rate decreased from 91.08 ± 1.86% to 88.05 ± 2.26%. Compared with emulsions with other ratios, the BITC retention rate showed less change during storage, and the retention rate was higher after 14 days of storage. Figure 26 ).

[0095] The above results indicate that the emulsion contact angle of the 2:1 ratio is 83.85 ± 0.21°, closest to 90°, suggesting that the fish skin gelatin-fucose complex at this ratio has the best interfacial stability. During the 14-day storage period, the 2:1 fish skin gelatin to fucoidan ratio group (Example 3) had the smallest particle size and relatively small ranges of variation in Zeta potential and particle size. The potential variation range was -53.47 ± 0.42 mV ~ -51.57 ± 0.35 mV, and the particle size variation range was 311.40 ± 6.13 nm ~ 367.37 ± 4.67 nm. After 14 days of storage, the BITC encapsulation rate of the fish skin gelatin:fucose = 2:1 sample (Example 3) decreased from 91.08 ± 1.86% to 88.05 ± 2.26%. It had the highest encapsulation rate and the smallest change, indicating that the fish skin gelatin-fucose composite emulsion at this ratio can reduce BITC loss more effectively than the fish skin gelatin-fucose composite emulsion, thus protecting the fat-soluble active substances and effectively preventing their degradation.

[0096] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing an emulsion using fish skin gelatin and fucoidan as a composite carrier, characterized in that, Includes the following steps: S1. Preparation of the aqueous phase: Prepare a fish skin gelatin solution, then add fucoidan and mix to obtain the aqueous phase; the mass concentration of the fish skin gelatin solution is 0.5~1.5%; the mass ratio of fish skin gelatin to fucoidan is 2:1; S2. Preparation of the oil phase: The fat-soluble active substance benzyl isothiocyanate is dissolved in edible oil to obtain the oil phase; the concentration of the fat-soluble active substance in the oil phase is 1~10 mg / mL; S3. Mixing: The aqueous phase prepared in S1 and the oil phase prepared in S2 are mixed and dispersed at high speed, and then homogenized to obtain a stabilized emulsion with fish skin gelatin and fucoidan as a composite carrier; the volume ratio of the oil phase to the aqueous phase is 1:8 to 1:

12.

2. The method for preparing an emulsion using fish skin gelatin and fucoidan as a composite carrier according to claim 1, characterized in that, The edible oil in S2 is one or more of the following: corn oil, soybean oil, sunflower seed oil, rapeseed oil, and olive oil.

3. An emulsion prepared by the method of claim 1 or 2, comprising fish skin gelatin and fucoidan as a composite carrier.

4. The application of the emulsion with fish skin gelatin and fucoidan as a composite carrier as described in claim 3 in the food industry.

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