Pickering emulsion stabilized by fish skin gelatin microgel and preparation method thereof

Abstract

The invention provides a Pickering emulsion stabilized by fish skin gelatin microgel and a preparation method thereof, and belongs to the technical field of emulsion preparation, and the preparation method comprises the following steps: A, preparing fish skin gelatin: extracting gelatin from fish skin by a salt extraction method, and performing vacuum freeze drying on a fish skin gelatin solution by a freeze dryer to obtain dried fish skin gelatin; b, preparing fish skin gelatin microgel: diluting the fish skin gelatin with deionized water until the protein concentration is 3%, and preparing the fish skin gelatin microgel by adopting an ultrasonic-microwave synergistic method; and C, preparing Pickering emulsion: mixing the fish skin gelatin microgel with soybean oil, and homogenizing to obtain the Pickering emulsion taking the fish skin gelatin microgel as the stabilizer. The fish skin gelatin microgel prepared through ultrasonic-microwave synergistic treatment is small in particle size, high in zeta potential absolute value, high in hydrophobicity and large in contact angle, can be adsorbed on a water-oil interface more easily, has the emulsifying property of 30% and the emulsifying stability of 33%, and can effectively stabilize Pickering emulsion.

Description

Technical Field

[0001] The present invention relates to the technical field of emulsion preparation, and particularly relates to a Pickering emulsion stabilized by fish skin gelatin microgels and a preparation method thereof. Background Art

[0002] Microgels are the particulate form of gels stabilized by covalent bonds and strong non-covalent interactions. They have the characteristics of small volume, large surface area, flexible structure, good colloidal stability, etc., and have a wide range of applications in the food industry. The deformability of microgels at the oil-water interface enables them to be used to stabilize emulsions, and microgel-stabilized high internal phase emulsions can be used for delivery systems and fat substitutes. Currently, microgels are prepared from whey protein, soy protein, egg white protein, and zein, usually by cross-linking proteins using heat or enzymatic treatment, followed by homogenization, and the process is complex. There are also methods such as spray drying and liquid anti-solvent precipitation, but they have disadvantages such as high production cost, insufficient particle size control, irregular microgel shape, and size dispersion.

[0003] Pickering emulsions are stabilized by solid particles and can be used for the encapsulation of active substances and fat substitutes. Most traditional emulsions use conventional small molecule surfactants as emulsifiers to stabilize the emulsion by electrostatic repulsion and reducing the interfacial tension. However, these surfactants are difficult to degrade and are harmful to health. Although protein microgels have great potential in stabilizing Pickering emulsions, the preparation methods are complex or the particle size is difficult to control. Therefore, it is of great significance to find a simple and inexpensive method for preparing microgels with controllable particle size as stabilizers for Pickering emulsions. Summary of the Invention

[0004] The purpose of the present invention is to provide a Pickering emulsion stabilized by fish skin gelatin microgels and a preparation method thereof to solve the existing technical problems.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A preparation method of a Pickering emulsion stabilized by fish skin gelatin microgels, comprising the following steps:

[0007] A. Prepare fish skin gelatin: After extracting gelatin from fish skin by the salt extraction method, vacuum freeze-dry the fish skin gelatin solution with a freeze dryer to obtain dry fish skin gelatin;

[0008] B. Prepare fish skin gelatin microgels: Dilute fish skin gelatin with deionized water to a protein concentration of 3%, and then prepare fish skin gelatin microgels by the ultrasonic-microwave synergistic method;

[0009] C. Prepare Pickering emulsion: Mix the fish skin gelatin microgels with soybean oil, and after homogenization, obtain a Pickering emulsion with fish skin gelatin microgels as the stabilizer.

[0010] Further, in step A, the specific process of extracting gelatin is as follows: Select 200 g of fresh fish skin, directly wash it with clear water, soak it in 500 mL of 1% NaOH solution for 12 h, then rinse it with distilled water, place it in 500 mL of 0.6% glacial acetic acid to swell for 3 h, and after washing, extract gelatin by the salting-out method.

[0011] Further, the fish skin is one of marine fish or freshwater fish.

[0012] Further, in step A, the specific process of freeze-drying is as follows: First, place the fish skin gelatin solution at -80 °C for 2 h, and then freeze-dry it with a freeze dryer for 12 - 24 h.

[0013] Further, in step B, the specific steps of the ultrasonic-microwave synergistic method are as follows: Take 50 mL of the diluted fish skin gelatin and add it to a three-necked flask, and treat it with a microwave ultrasonic combined extractor, with intermittent ultrasonic treatment, working for 1 s and stopping for 1 s.

[0014] Further, the ultrasonic-microwave treatment time is 1 - 7 min, the ultrasonic-microwave treatment temperature is 60 - 80 °C, and the ultrasonic-microwave treatment power is 200 - 600 W.

[0015] Further, in step C, the mass percentage of the fish skin gelatin microgel is 0.5 - 3 wt%, and the oil phase volume fraction of the soybean oil is 0.35 - 0.85.

[0016] Further, in step C, the specific process of homogenization is as follows: Homogenize with a high-speed disperser at 10000 rpm for 2 min.

[0017] A Pickering emulsion stabilized by fish skin gelatin microgel is prepared by a preparation method of a Pickering emulsion stabilized by fish skin gelatin microgel.

[0018] Due to the adoption of the above technical solutions, the present invention has the following beneficial effects:

[0019] 1. The present invention prepares fish skin gelatin microgel by the ultrasonic-microwave synergistic method. The microwave can quickly heat the protein, and the cavitation effect of ultrasonic waves can avoid excessive aggregation of proteins caused by microwaves. The combination of microwave and ultrasonic waves realizes the adjustment of the particle size of fish skin gelatin microgel, and quickly prepares fish skin gelatin protein microgel with good emulsifying properties.

[0020] 2. The microgel treated by the ultrasonic-microwave synergistic method in the present invention has a small particle size, a high absolute value of zeta potential, high hydrophobicity, a large contact angle, is more likely to adsorb on the water-oil interface, has high emulsifying properties, and shows great potential in the preparation of Pickering emulsions. Description of the Drawings

[0021] Figure 1 It is a diagram showing the effects of the US-MW treatment time (A), temperature (B), and US power (C) of the present invention on H0 and the contact angle;

[0022] Figure 2 It is a diagram showing the emulsifying properties of the raw material EW and the fish skin gelatin microgel sample of the present invention;

[0023] Figure 3 It is a diagram showing the intermolecular interaction forces of the fish skin gelatin microgel of the present invention;

[0024] Figure 4 It is a CLSM image of the Pickering emulsion with different concentrations of fish skin gelatin microgel of the present invention;

[0025] Figure 5 It is a CLSM image of the Pickering emulsion with different oil phase volume fractions of the present invention;

[0026] Figure 6 It is a diagram showing the apparent viscosity of the Pickering emulsion with different concentrations of fish skin gelatin microgel (A) and oil phase volume fractions (B) of the present invention;

[0027] Figure 7 It is the frequency sweep curve of the Pickering emulsion at different concentrations of fish skin gelatin microgel (A, B) and oil phase volume fractions (C, D) of the present invention;

[0028] Figure 8 It is the appearance and OHC value of the emulsion after centrifugation of the Pickering emulsion of the present invention;

[0029] Figure 9 It is the appearance and EI of the Pickering emulsion with different concentrations of fish skin gelatin microgel (A, B) and oil phase volume fractions (C, D) during 30 days of storage of the present invention;

[0030] Figure 10 It is the optical image of the emulsion with different concentrations of fish skin gelatin microgel of the present invention;

[0031] Figure 11 It is the optical image of the emulsion with different oil phase volume fractions of the present invention. Detailed Description of the Invention

[0032] To make the objectives, technical solutions and advantages of the present invention more clear and understandable, the following preferred embodiments are given with reference to the accompanying drawings for further detailed description of the present invention. However, it should be noted that many details listed in the specification are only for enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be implemented even without these specific details.

[0033] Comparative Example

[0034] A. Preparation of fish skin gelatin: Select 200 g of fresh fish skin, directly wash it with clean water, and soak it in 500 mL of 1% NaOH solution for 12 h; then rinse it with distilled water, and place it in 500 mL of 0.6% glacial acetic acid to swell for 3 h. After washing, extract gelatin by the salting-out method; finally, first place the fish skin gelatin solution at -80 °C for 2 h, and then use a freeze dryer (2KBTES-55, VirTis Co., Gardiner, NY, USA) to vacuum freeze-dry all the fish skin gelatin solution for 12 - 24 h to obtain dry fish skin gelatin;

[0035] B. Preparation of fish skin gelatin microgels: Take the fish skin gelatin microgels prepared by the water bath method (WB) as the control group. Dilute the fish skin gelatin to a protein concentration of 3% (W / V), add 50 mL of the diluted fish skin gelatin microgels to a beaker, and magnetically heat and stir in a 75 °C water bath for 5 min.

[0036] Example

[0037] A method for preparing a Pickering emulsion stabilized by fish skin gelatin microgels, comprising the following steps:

[0038] A. Preparation of fish skin gelatin: Select 200 g of fresh fish skin, directly wash it with clean water, and soak it in 500 mL of 1% NaOH solution for 12 h; then rinse it with distilled water, and place it in 500 mL of 0.6% glacial acetic acid to swell for 3 h. After washing, extract gelatin by the salting-out method; finally, first place the fish skin gelatin solution at -80 °C for 2 h, and then use a freeze dryer (2KBTES-55, VirTis Co., Gardiner, NY, USA) to vacuum freeze-dry all the fish skin gelatin solution for 12 - 24 h to obtain dry fish skin gelatin;

[0039] B. Preparation of fish skin gelatin microgels: Prepare fish skin gelatin microgels by the ultrasonic-microwave synergistic method (US-MW). Dilute the fish skin gelatin with deionized water to a protein concentration of 3%, and take 50 mL of the diluted fish skin gelatin and add it to a three-necked flask. Treat it with a microwave-ultrasonic combined extractor (XH-300A+, Beijing Xianghu Science and Technology Development Co., Ltd., Beijing, China), and perform intermittent ultrasonic treatment (working for 1 s and stopping for 1 s); the ultrasonic-microwave treatment time is 1 min, 3 min, 5 min, 7 min, the ultrasonic-microwave treatment temperature is 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, and the ultrasonic-microwave treatment power is 200 W, 400 W, 500 W, 600 W.

[0040] C. Preparation of Pickering emulsion: Mix the fish skin gelatin microgel with soybean oil and homogenize it with a high-speed disperser at 10,000 rpm for 2 min to obtain a Pickering emulsion with the fish skin gelatin microgel as the stabilizer; the mass percentages of the fish skin gelatin microgel are 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, and the oil phase volume fractions of the soybean oil are 0.35, 0.45, 0.55, 0.65, 0.75, 0.85.

[0041] I. Test methods:

[0042] (I) Characteristics of fish skin gelatin microgel

[0043] 1. Determination of particle size and potential: Dilute the fish skin gelatin microgel with deionized water to 1 mg / mL, where the fish skin gelatin microgel concentration is the protein concentration, the same hereinafter. Use a particle size analyzer (NANO ZS90, Malvern Instruments, UK) to measure the average particle size, polydispersity index (PDI), and zeta potential of the fish skin gelatin microgel.

[0044] 2. Determination of surface hydrophobicity (H0): Dilute the fish skin gelatin microgel with PBS buffer (0.02 M, pH 7.4) to 0.05 - 0.2 mg / mL. Add 20 μL of 8 mM ANS solution (0.02 M PBS, pH 7.4) to 4 mL of the diluted solution and store it in the dark for 1 h. Measure the fluorescence intensity with a fluorescence spectrophotometer (F - 4500, Hitachi, Japan), with an excitation wavelength of 390 nm and an emission wavelength of 300 - 500 nm. Calculate the slope of the fluorescence peak intensity - protein concentration relationship curve with H0.

[0045] 3. Determination of contact angle: Take 100 μL of the fish skin gelatin microgel and drop it on a glass slide, dry it at 40 °C to form a fish skin gelatin microgel film. Use a video optical angle analyzer (Attention Theta, Biolin, Finland) to measure the contact angle of the fish skin gelatin microgel. Use OneAttension software to record the contraction angle.

[0046] 4. Determination of emulsification characteristics: Dilute the fish skin gelatin microgel with deionized water to 0.1% (V / V). Mix the diluted solution with soybean oil at a ratio of 3:1 (V / V), homogenize it with a high - speed disperser at 10,000 rpm for 1 min, and then add 50 μL of the emulsion to 5 mL of 1% w / v sodium dodecyl sulfate (SDS) aqueous solution. Measure the absorbance with a spectrophotometer (UV - 6100, Shanghai Jingke Industrial Co., Ltd., China) at a wavelength of 500 nm. Using the SDS solution as the blank solution, calculate the emulsifying capacity (EC) and emulsifying stability (ES) as follows:

[0047] EC(%) = A0 × 100% (Equation 1)

[0048] ES(%) = A 10 / A0 × 100% (Equation 2)

[0049] Where A0 is the absorbance measured for the first time, and A10 is the absorbance measured after 10 min.

[0050] 5. Determination of intermolecular forces: Fish skin gelatin microgels were diluted to 10 mg / mL with deionized water, urea (6 M), SDS (0.5% w / v), dithiothreitol DTT (30 mM), and NaCl (1 M), respectively, and then centrifuged at 10,000 rpm for 15 min. The solubility of the protein in the supernatant was determined by the biuret spectrophotometric method.

[0051] (II) Characterization of Pickering emulsions

[0052] 1. Microstructure of Pickering emulsions

[0053] (1) Optical microscope

[0054] The microstructure of the emulsion was observed using an optical microscope (DMIRB, Leica, Germany). 50 μL of the emulsion was placed on a glass slide and observed with a 40× objective lens.

[0055] (2) Confocal laser scanning microscope (CLSM)

[0056] A confocal laser scanning microscope (FV3000, Olympus Corporation, Japan) was used. 20 μL of Nile red (0.1% w / v) and Nile blue (0.1% w / v) were added to 1 mL of the emulsion to label the oil and protein, and observations were made at excitation wavelengths of 488 nm and 633 nm, respectively.

[0057] 2. Droplet size

[0058] The Pickering emulsion was diluted with deionized water, and then its droplet size was measured using a laser particle size analyzer (Microtrac S3500, Microtrac Instrument Corporation, USA). The refractive indices of the particles and the dispersion medium were 1.476 and 1.333, respectively.

[0059] 3. Emulsification index (EI)

[0060] The degree of phase separation of the emulsion was evaluated by measuring the total height and the height of the cream layer of the emulsion. The calculation method of EI is as follows.

[0061] EI = H0 / H × 100% (Equation 3)

[0062] Among them, H0 is the height of the cream layer and H is the height of the emulsion.

[0063] 4. Rheological analysis

[0064] The apparent viscosity, storage modulus (G′), and loss modulus (G″) of the emulsion were measured using a rheometer (HAAKE MARS60, Germany). The test gap was set to 1 mm and the temperature was 25 °C. The apparent viscosity was measured at a shear rate of 0.1 - 100 s-1. Dynamic scans were performed at a frequency of 0.1 Hz to 10 Hz at 1% strain to determine the storage modulus (G′) and loss modulus (G″).

[0065] 5. Centrifugation and storage stability

[0066] Regarding centrifugal stability, the emulsion was centrifuged at 6000 rpm for 2 min, the appearance of the emulsion was observed, and its oil-holding capacity (OHC) was measured. The calculation method is as follows.

[0067] OHC = m1 / m0 × 100% (Equation 4)

[0068] In the formula, m0 is the mass of the emulsion and m1 is the mass of the emulsion after centrifugation.

[0069] Regarding storage stability, sodium azide (0.02% w / v) was added to the fish skin gelatin microgel solution as an antibacterial agent to prepare Pickering emulsion. The Pickering emulsion was stored at 4 °C for 0, 15, and 30 days. The appearance of the emulsion was observed, and EI, droplet size, and zeta potential were measured to evaluate storage stability.

[0070] (III) Statistical analysis

[0071] Each measurement was repeated three times. ANOVA was performed using SPSS 25.0 software. The differences between groups were statistically significant (p < 0.05).

[0072] II. Result analysis

[0073] (I) Effects of US-MW treatment on fish skin gelatin microgel

[0074] 1. Particle size and zeta potential

[0075] At a US power of 500 W and 75 °C, the effect of treatment time on fish skin gelatin microgel was measured. As can be seen from Table 1, as the time extended, the particle size of fish skin gelatin microgel gradually decreased from 1 min to 5 min, and then increased to 285.27 ± 12.93 nm when extended to 7 minutes. With the passage of time, the decrease in PDI indicated that the size of fish skin gelatin microgel particles became more uniform, and the treatment time had no significant effect on the zeta potential.

[0076] Table 1 Effects of US-MW treatment time on particle size, PDI and zeta potential of fish skin gelatin microgels

[0077]

[0078] Note: Different letters indicate differences in fish skin gelatin microgels (p < 0.05).

[0079] At 500 W and a treatment time of 5 min, the effects of temperature on fish skin gelatin microgels are shown in Table 2. The particle size of fish skin gelatin microgels was 161.27 ± 9.98 nm at 70 °C, 233.23 ± 6.49 nm at 75 °C, and 413.30 ± 20.72 nm at 80 °C, indicating that increasing temperature led to the unfolding of the protein structure and an increase in particle size. However, the differences in PDI were not significant, indicating that the degree of uniformity in the particle size distribution of fish skin gelatin microgels was similar. The absolute value of the zeta potential increased with increasing temperature, indicating that the stability of fish skin gelatin microgels could be enhanced under high-temperature conditions.

[0080] Table 2 Effects of temperature on particle size, PDI and zeta potential of fish skin gelatin microgels

[0081]

[0082]

[0083] Note: Different letters indicate differences in fish skin gelatin microgels (p < 0.05).

[0084] Table 3 shows the effects of US power on fish skin gelatin microgels. The particle size and PDI of fish skin gelatin microgels without US treatment were significantly higher than those of fish skin gelatin microgels treated with US, indicating that without US treatment, large protein aggregates with uneven sizes were formed in fish skin gelatin microgels. As the US power increased, the particle size of fish skin gelatin microgels gradually decreased, and the absolute value of the zeta potential increased, indicating that high-power US reduced protein aggregation, resulting in smaller particle sizes and larger surface areas, causing fish skin gelatin microgels to carry more negative charges on their surfaces.

[0085] Table 3 Effects of US power on particle size, PDI and zeta potential of fish skin gelatin microgels

[0086]

[0087] Note: Different letters indicate differences in fish skin gelatin microgels (p < 0.05).

[0088] 2. Hydrophobicity (H0) and contact angle

[0089] As Figure 1As shown in (A), the highest H0 value of US-MW treatment occurred at 5 min. The increase in H0 from 1 min to 5 min indicated that US-MW treatment caused the unfolding of the protein structure, exposing hydrophobic groups. The contact angles of all fish skin gelatin microgels were less than 90°, indicating that they were all hydrophilic. The maximum shrinkage angle occurred at 5 min ( Figure 1 (A)), indicating the lowest hydrophilicity, which was consistent with the result of H0. According to Figure 1 (A), both H0 and the contact angle increased with increasing temperature, indicating that heat denaturation caused by high temperature led to the exposure of more hydrophobic groups. Figure 1 (B), H0 and the contact angle increased with increasing temperature, indicating that heat denaturation caused by high temperature led to the exposure of more hydrophobic groups.

[0090] According to Figure 1 (C), the H0 of fish skin gelatin microgels without US treatment was significantly lower than that of other fish skin gelatin microgels. With the increase of US power, the content of H0 increased, and the contact angle increased from 56.49° to 76.33°, indicating an increase in hydrophobicity. According to the results of particle size, zeta potential, H0, and contact angle, the fish skin gelatin microgels prepared by US-MW treatment at 75 °C and 500 W US power for 5 min had a small particle size, a high absolute value of zeta potential, the highest H0 and contact angle, and a better adsorption effect on the water-oil interface.

[0091] 3. Emulsifying properties

[0092] The particle size, H0, and contact angle of fish skin gelatin microgels prepared by the WB method (water bath method) were as follows: large particle size, small H0, and small contact angle, which were not conducive to the stability of the emulsion. The emulsifying properties of raw materials EW and fish skin gelatin microgel samples were as shown in Figure 2 . The raw materials EC and ES were lower than those of the heat-treated samples. The fish skin gelatin microgels treated by US-MW had the highest EC and ES, showing significant differences from both MW and WB, indicating that US-MW significantly reduced the formation of large protein aggregates, making the particle size of fish skin gelatin microgels smaller and the emulsifying performance better.

[0093] 4. Intermolecular forces

[0094] In the present invention, urea, SDS, DTT, and NaCl disrupted hydrogen bonds, hydrophobic interactions, disulfide bonds, and electrostatic interactions in fish skin gelatin microgels, respectively. When the intermolecular forces maintaining the microgel structure were disrupted, the solubility of the protein increased. As shown in Figure 3 , after different treatments (MW, US-MW, WB) of fish skin gelatin microgels, the protein solubility increased significantly after adding urea and SDS, while there was no significant change in protein solubility after adding DTT and NaCl, indicating that the main intermolecular forces maintaining the fish skin gelatin microgel structure were hydrogen bonds and hydrophobic interactions. In addition, the protein solubility of US-MW fish skin gelatin microgels was significantly higher than that of other fish skin gelatin microgels ( Figure 3 Figure 3), indicating that heating and US treatment unfold the protein structure, expose amino acid residues and hydrophobic groups, and enhance hydrogen bonding and hydrophobic interactions.

[0095] (II) Pickering emulsions stabilized by fish skin gelatin microgels

[0096] 1. CLSM of Pickering emulsions

[0097] The appearance and microstructure of Pickering emulsions with different fish skin gelatin microgel concentrations are as Figure 4 shown. In the CLSM images, the oil is shown in red and the fish skin gelatin microgels are shown in green. As can be seen from Figure 4 , the minimum fish skin gelatin microgel concentration required to prepare a stable emulsion is 1.5 wt% (φ = 0.75). At lower fish skin gelatin microgel concentrations, the emulsion is fluid and cannot form a stable HIPE structure. At fish skin gelatin microgel concentrations of 0.5 wt% and 1.0 wt%, not enough fish skin gelatin microgels are adsorbed at the oil-water interface to stabilize the emulsion, so the droplets are larger and the uncoated oil phase makes the emulsion fluid, resulting in poor stability. When the fish skin gelatin microgel concentration exceeds 1.5 wt%, stable HIPEs are formed and the fish skin gelatin microgels are stably adsorbed on the surface of the oil droplets. The increase in the fish skin gelatin microgel concentration leads to closer packing and smaller size of the oil droplets, improving the stability of the emulsion.

[0098] As Figure 5 shown, emulsions with low oil phase volume fractions consist of a cream phase and a serum phase. When are 0.75 and 0.85 respectively, only a single emulsified phase HIPE is formed. At a certain fish skin gelatin microgel concentration, as increases, the particle size of the emulsion gradually increases, indicating that the fish skin gelatin microgel concentration and the oil phase volume fraction have a great influence on the stability of the emulsion and are of great significance for preparing a stable emulsion.

[0099] 2. Rheological properties of Pickering emulsions

[0100] (1) Apparent viscosity

[0101] As the shear rate increases, the apparent viscosity of all samples decreases ( Figure 6 ), and the apparent viscosity gradually increases with the increase in the fish skin gelatin microgel concentration ( Figure 6 (A)), indicating that the fish skin gelatin microgels contribute to improving the strength of the emulsion and forming a more stable emulsion. As shown in Figure Figure 6(As shown in (B), when φ < 0.65, phase separation occurred in the emulsion, which was not suitable for rheological measurement; with the increase of the oil phase volume fraction (φ > 0.65), the apparent viscosity increased, indicating that the high oil phase volume also improved the emulsion strength.)

[0102] (2) Dynamic rheological properties

[0103] From Figure 7 (A, B), it can be seen that for emulsions with different concentrations of fish skin gelatin microgels, both G′ and G″ showed a parallel increasing trend with the increase of shear frequency, and G′ was always higher than G″, indicating that the emulsion exhibited elastic gel properties. In addition, from Figure 7 (A, B), it can be seen that at a fixed oil phase volume fraction, with the increase of the fish skin gelatin microgel concentration, G′ and G″ gradually increased, and the higher the G′ and G″, the higher the viscoelasticity of the emulsion. This is consistent with Figure 6 (A), indicating that a high concentration of fish skin gelatin microgels is beneficial to the formation of stable emulsions. The G′ and G″ of emulsions with a fish skin gelatin microgel concentration greater than 1.5 wt% were significantly higher than those of emulsions with a lower fish skin gelatin microgel concentration, indicating that the increase in the fish skin gelatin microgel concentration can enhance the strength of the emulsion. This is consistent with the appearance results, that is, stable HIPE is formed when the fish skin gelatin microgel concentration is greater than 1.5 wt%, while too low a fish skin gelatin microgel is not sufficient to stabilize the high volume fraction of the oil phase, resulting in unstable emulsions.)

[0104] As Figure 7 (C, D) shows, the G′ and G″ of the emulsion increased with the increase of the oil phase volume (φ > 0.65). In the range of 0.1 - 10 Hz, the G′ value of the emulsion was higher than G″, indicating that the emulsion was mainly elastic. The G′ and G″ (φ = 0.65) of the emulsion were significantly lower than those of the emulsions (φ = 0.75, 0.85), while there was no significant difference between the emulsions (φ = 0.75, 0.85), which was consistent with the results of the CLSM images of the emulsions( Figure 5 ).)

[0105] 3. Centrifugal stability

[0106] As Figure 8 shown, when the fish skin gelatin microgel concentration was 0.5 wt%, obvious phase separation occurred after centrifugation, a large amount of oil was precipitated, and the OHC was the lowest, indicating that the emulsion was unstable. When the fish skin gelatin microgel concentration was 1.0 wt%, a small amount of oil was precipitated. The remaining emulsion phase and water phase were stable, no oil phase precipitation occurred, and the centrifugal stability was good with a high OHC value. The higher the fish skin gelatin microgel concentration, the higher the viscosity and viscoelasticity of the emulsion, the more stable the structure, and the better the anti-coalescence or demulsification effect.)

[0107] 4. Storage stability

[0108] (1) Emulsification index (EI)

[0109] The EI method was used to evaluate the phase separation of Pickering emulsions during storage. In Figure 9 (A, B), when the concentration of fish skin gelatin microgel was less than 1 wt%, phase separation occurred in the emulsion during storage, and the EI was significantly lower than that of the emulsion with a high fish skin gelatin microgel concentration. The emulsion with a high fish skin gelatin microgel concentration showed good stability when the EI was about 100%, indicating that the high viscosity of the emulsion and the close arrangement of the droplets restricted the movement of the droplets.

[0110] As Figure 9 (C, D) shows, the emulsion with a low oil volume fraction undergoes aqueous phase separation in a short time. As the storage time prolongs, the height of the aqueous phase increases and the EI decreases. When , the excess aqueous phase separates from the emulsion, and the height of the water layer gradually increases under the action of gravity. HIPEs remain stable during storage, without precipitation of the oil phase and the aqueous phase, and the EI is 100%. Compared with the oil volume fraction of other long-term stable emulsions usually being less than 0.75, fish skin gelatin microgel shows a stronger ability to stabilize a large amount of oil phase

[0111] (2) Emulsion droplet size

[0112] The average particle size (D 4，3 ) of the emulsion droplets at different fish skin gelatin microgel concentrations is shown in Table 4. When the concentration of fish skin gelatin microgel is less than 1.5%, the D4,3 of the emulsion droplets decreases with the increase of the concentration. When the concentration further increases, D 4，3 no longer changes. This is consistent with the optical image ( Figure 10 ). The D 4，3 of the emulsion with a low fish skin gelatin microgel concentration gradually increases with the prolongation of the storage time, indicating the aggregation of oil droplets. While the D 4，3 of the emulsion with a high fish skin gelatin microgel concentration changes insignificantly and has good stability, indicating that a small particle size is beneficial for the emulsion to maintain stability (see Figure 11 ).

[0113] The D 4，3 of the emulsion droplets with different oil volume fractions (φ) is shown in Table 5. When φ < 0.75, D 4，3 increases with the increase of the φ value, while D 4，3 remains unchanged when HIPEs are formed; when φ > 0.75, the D 4，3 of the emulsion does not increase significantly.

[0114] Table 4 D4,3 of Pickering emulsions with different fish skin gelatin microgel concentrations during 30-day storage

[0115]

[0116] Content of Pickering emulsions with different oil phase volume fractions in Table 5D4, 3

[0117]

[0118]

[0119] The present invention uses a simple "one-step" method of ultrasonic-microwave synergistic method (US-MW) to prepare fish skin gelatin microgels and uses them to stabilize Pickering emulsions. The fish skin gelatin microgels prepared by the US-MW method have small particle size, high absolute value of zeta potential, high hydrophobicity, large contact angle, are easy to adsorb at the water-oil interface, have high emulsifying property, and have great application potential in Pickering emulsions. In addition, the effects of fish skin gelatin microgel concentration and oil phase volume fraction on Pickering emulsions were also studied. High fish skin gelatin microgel concentration (>1.5%) is beneficial to the formation of stable emulsions. When the fish skin gelatin microgel concentration is 3.0 wt%, the fish skin gelatin microgel has a stronger ability to stabilize high volume oil phase fractions These stable Pickering emulsions have high apparent viscosity, elastic gel properties, and small particle size, thus preventing droplet aggregation and flocculation, and have high stability. The present invention provides a method for rapidly preparing fish skin gelatin microgels with good emulsifying properties, and the fish skin gelatin microgels can be used as stabilizers for Pickering emulsions and further applied to active substance delivery and fat substitution.

[0120] ​In the present invention, fish skin gelatin microgels were prepared by ultrasonic-microwave synergistic treatment (US-MW), and the ability of the gels to stabilize emulsions was evaluated. The cavitation effect of ultrasound (US) can avoid the excessive aggregation of proteins caused by microwave (MW), and achieve the regulation of the particle size of fish skin gelatin microgels. Compared with the fish skin gelatin microgels prepared by MW and water bath treatment, the microgels treated by US-MW have a small particle size (233.23 ± 6.49 nm), a high absolute value of zeta potential (-25.27 ± 0.74 mV), a high hydrophobicity (505.87 ± 5.22), a large contact angle (78.92°), are more likely to adsorb on the water-oil interface, have a high emulsifying property, and show great potential in the preparation of Pickering emulsions. Emulsions with a fish skin gelatin microgel concentration > 1.5% have good stability and show the ability to stably disperse a high volume fraction of the oil phase (φ = 0.85). Confocal laser scanning microscopy images show that fish skin gelatin microgels adsorb on the surface of oil droplets, forming a physical barrier for stable emulsions. Rheological results indicate that the emulsions (fish skin gelatin microgel concentration > 1.5% and φ > 0.75) have a high apparent viscosity and modulus. This study provides a basis for the simple preparation of fish skin gelatin protein microgels with high emulsifying ability, and fish skin gelatin microgels can effectively stabilize Pickering emulsions and be further applied to the delivery of active substances and fat substitution.

[0121] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a Pickering emulsion stabilized by fish skin gelatin microgel, characterized in that: The following steps are involved: A, preparing fish skin gelatin: after fish skin is extracted gelatin by salt extraction, vacuum freeze drying fish skin gelatin solution is carried out with freeze dryer to obtain dry fish skin gelatin; B. Preparation of fish skin gelatin microgel: After diluting the fish skin gelatin to a protein concentration of 3% with deionized water, an ultrasound-microwave synergistic method was used to prepare the fish skin gelatin microgel; C. Preparation of Pickering emulsion: fish skin gelatin microgel and soybean oil are mixed and homogenized to obtain a Pickering emulsion with fish skin gelatin microgel as a stabilizer.

2. The method for preparing a Pickering emulsion stabilized by fish skin gelatin microgel according to claim 1, characterized in that: In step A, the specific process of extracting gelatin is as follows: 200g of fresh fish skin is selected and directly washed with clean water, then immersed in 500mL 1% NaOH solution for 12h, then rinsed with distilled water, and placed in 500mL 0.6% glacial acetic acid for expansion for 3h, and then washed and extracted with salt extraction method.

3. The method for preparing a Pickering emulsion stabilized by fish skin gelatin microgel according to claim 2, characterized in that: The fish skin is made of either marine fish or freshwater fish.

4. The method for preparing a Pickering emulsion stabilized by fish skin gelatin microgel according to claim 1, characterized in that: In step A, the specific process of freeze drying is: firstly, the fish skin gelatin solution is placed at -80°C for 2 hours, and then freeze dried by a freeze dryer for 12-24 hours.

5. The method for preparing a Pickering emulsion stabilized by fish skin gelatin microgel according to claim 1, characterized in that: In step B, the specific steps of the ultrasound-microwave synergistic method are: taking 50 mL of diluted fish skin gelatin and adding it into a three-can bottle, treating it with a microwave-ultrasonic combined extractor, and performing ultrasonic intermittent treatment, working for 1 second and stopping for 1 second.

6. The method for preparing a Pickering emulsion stabilized by fish skin gelatin microgel according to claim 5, characterized in that: The ultrasonic-microwave treatment time is 1-7 minutes, the ultrasonic-microwave treatment temperature is 60-80° C., and the ultrasonic-microwave treatment power is 200-600W.

7. The method for preparing a Pickering emulsion stabilized by fish skin gelatin microgel according to claim 1, characterized in that: In step C, the mass percentage of the fish skin gelatin microgel is 0.5-3wt%, and the oil phase volume fraction of the soybean oil is 0.35-0.

85.

8. The method for preparing a Pickering emulsion stabilized by fish skin gelatin microgel according to claim 1, characterized in that: In step C, the specific process of homogenization is: homogenizing with a high-speed disperser at 10000 rpm for 2 minutes.

9. A Pickering emulsion stabilized by fish skin gelatin microgel, characterized in that: The method for preparing a Pickering emulsion stabilized by fish skin gelatin microgel according to any one of claims 1 to 8.

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