Improved preparation and testing methods for salmon emulsion gels for dysphagia.

Abstract

This invention discloses a method for preparing and testing salmon emulsion gels that improve swallowing difficulties. It relates to the field of protein emulsion gel preparation and testing technology, addressing the problems of swallowing difficulties caused by protein-rich diets, insufficient intake of functional fatty acids in most elderly individuals, and the lack of a robust performance verification system for salmon emulsion gel products. At the molecular level, this invention transforms large-sized myofibril protein filament aggregates into nano-sized myofibril micelles. These nano-sized myofibril micelles dissociate in oil, linking and aggregating with other proteins. The molecular mechanism by which these micelles stabilize salmon emulsion gels and thicken their texture can be used to alter protein structure and self-assembly properties. Furthermore, it provides effective and comprehensive testing of salmon emulsion gel products, strongly demonstrating their effectiveness in improving swallowing difficulties.

Description

Technical Field

[0001] This invention relates to the field of protein emulsion gel preparation and testing technology, specifically to an improved method for preparing and testing salmon emulsion gels for patients with swallowing difficulties. Background Technology

[0002] Aging is a serious global problem because it is associated with many chronic diseases, such as dysphagia, osteoporosis, and frailty. Dysphagia is a clinical symptom characterized by difficulty swallowing, associated with weakening of the oral muscles, and is common in older adults. Population surveys estimate that nearly 20% of the population aged 50 and over suffer from dysphagia. Due to reduced food intake, older adults with dysphagia are more prone to chronic malnutrition and dehydration. Foods for dysphagia have often evolved from liquid to pureed forms to reduce the risk of choking. However, current dysphagia-specific foods often use large molecules extracted from raw foods, such as soy protein, whey protein, starch-rich foods, corn starch, gums, and plant polysaccharides. These formulations often lack bioactive compounds such as vitamins and minerals, and because the food lacks absorbency, it is difficult to stimulate appetite. Therefore, providing these individuals with well-designed, safe, delicious, and nutritionally rich, texture-modified foods is crucial.

[0003] Dysphagia is often associated with protein-energy malnutrition and frailty in older adults. A protein-rich diet helps promote muscle synthesis and maintain muscle function. Therefore, supplementing the diet with high-quality protein, such as fish protein, is a good nutritional intervention for older adults with dysphagia. Fish protein typically has a high digestible essential amino acid score (DIAAS), making it easier to digest and aiding in the absorption of essential amino acids. However, due to dysphagia, most older adults do not consume enough protein-rich diets compared to recommended intakes. Furthermore, polyunsaturated fatty acids (PUFAs), especially omega-3s, are recommended for older adults as they are beneficial for their cardiovascular health and immune system. However, most older adults do not consume enough of these functional fatty acids, and the performance verification system for salmon emulsion gel products is inadequate; therefore, existing needs are not met. To address this, we propose an improved method for the preparation and testing of salmon emulsion gels for dysphagia. Summary of the Invention

[0004] The purpose of this invention is to provide an improved method for preparing and testing salmon emulsion gels for those with swallowing difficulties, in order to address the problems mentioned in the background art, such as the difficulty in swallowing protein-rich diets compared to recommended intake, insufficient intake of functional fatty acids by most elderly people, and the lack of a robust performance verification system for salmon emulsion gel products.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing an improved salmon emulsion gel for swallowing difficulties, comprising the following steps:

[0006] Step 1: Mix fresh salmon meat and ice water in a 1:3 ratio in a blender and blend at high speed for 1 minute. Filter the mixture through a 60-mesh sieve.

[0007] Step 2: The obtained filtrate is pre-homogenized for 2 minutes using a disperser at a speed of 10,000 rpm;

[0008] Step 3: The dispersion is subjected to three cycles of high-pressure homogenization in the first step to obtain the initial protein suspension. The protein content in the suspension is then adjusted to 2.0 w / v%.

[0009] Step 4: Salmon oil extracted from salmon spine using cold micro-water extraction was added to a protein-based suspension to ensure that the oil volume fraction φ reached 0.5 and 0.6 respectively, thus obtaining two samples;

[0010] Step 5: Mix the sample using a disperser at 12,000 rpm for 2 minutes to obtain emulsion gel A;

[0011] Step 6: Perform a second high-pressure homogenization on emulsion gel A to obtain salmon emulsion gel.

[0012] Preferably, the temperature condition for the high-pressure homogenization treatment is 4°C, and the pressure condition for the high-pressure homogenization treatment is 50 MPa.

[0013] Preferably, salmon oil extracted from salmon spine comprises the following steps:

[0014] W1: The cleaned salmon spine is freeze-dried to reduce the moisture content, and the dried by-products are ground using a grinder.

[0015] W2: Rehydrate the ground sample with water until the oil volume fraction φ is adjusted to 75% of the volume. Stir tangentially with a top stirrer for 15 minutes, and then immediately centrifuge at 8000×g for 15 minutes at a temperature of 20℃.

[0016] W3: Obtain the upper layer of salmon oil and store it in a storage environment at -20℃.

[0017] An improved testing method for salmon emulsion gels for dysphagia includes the following steps:

[0018] S1: Salmon emulsion gels with different φ after the second high-pressure homogenization process are referred to as HPH0.5 and HPH0.6, respectively. Emulsion gel A without the second high-pressure homogenization process is referred to as Un-HPH0.5 and Un-HPH0.6, respectively. Each emulsion gel sample is stored in a 20 ml transparent glass vial and stored at 4°C.

[0019] S2: Color analysis was performed on all emulsion gel samples using a colorimeter, and microscopic observation and particle size analysis were conducted.

[0020] S3: Centrifugal stability tests were performed on all emulsion gel samples, and the measurements were taken using a dispersion analyzer;

[0021] S4: Differential scanning calorimetry was used to measure the formation and dissociation of ice crystals in salmon emulsion gels for all emulsion gel samples;

[0022] S5: IDSSI and rheological properties tests were performed on the salmon emulsion gel.

[0023] S6: Strain scanning test, viscosity shear test and frequency scanning test were performed on the salmon emulsion gel;

[0024] S7: Extract surface active proteins from salmon emulsion gel to obtain surface myofibrillar proteins, and analyze the morphology and interfacial rheological properties of the surface myofibrillar proteins.

[0025] S8: Repeat steps S1-S7 three times to calculate the mean and standard deviation of the data.

[0026] Preferably, the colorimeter measurement parameters include luminance. Redness and yellowness The value;

[0027] The centrifugal stability test involved transferring the sample into a polycarbonate tube and collecting data at 20-second intervals in a centrifuge. The sample was centrifuged for 6000 seconds to destabilize it, and then the instability index and area percentage were calculated using LUMSEPviewV6.4 software.

[0028] Preferably, microscopic observation and particle size analysis include the following steps:

[0029] Step 1: Dilute the homogenized salmon emulsion gel in 1% SDS solution at a ratio of 1:10 to disperse the aggregated oil droplets. Dilute the non-homogenized emulsion in ultrapure water at a ratio of 1:5.

[0030] Step 2: Observe the microstructure of oil droplets in salmon emulsion gel using a fluorescence inverted microscope with a digital camera. The oil droplets are stained red with Nile red, then dropped onto a glass slide and covered with a coverslip.

[0031] Step 3: The particle size distribution of the fresh emulsion gel was determined by dynamic light scattering (DLS) in the range of 0.02 μm-2600 μm. The ultrasonic power was set to 35 W for 2 minutes, the stirring speed was set to 1200 rpm, and the measurement opacity was set to 5%-20%.

[0032] Preferably, the differential scanning calorimetry measurement includes the following steps:

[0033] Step 1: Place the fresh salmon emulsion gel into an aluminum crucible, introduce nitrogen gas at a flow rate of 20 mL / min. Cool the measured temperature from 20°C to -30°C at a cooling rate of 5°C / min;

[0034] Step 2: After equilibration for 5 minutes, the sample is heated from 30°C to 40°C at the same flow rate, and then cooled from 40°C to 20°C at a rate of 10°C / min.

[0035] Preferably, the rheological performance test is performed using a rheometer with a plate diameter of 40 mm and a gap distance of 1000 micrometers. The test sample is placed at ambient temperature for 1 hour to reach thermal equilibrium, and the test temperature is 25°C.

[0036] Strain scanning tests were performed to measure viscoelasticity in an oscillating amplitude mode within the linear viscoelastic region (LVR). The test temperature was 25℃, the frequency was 1Hz, and the strain range was 0.1% to 1000%.

[0037] Viscosity shear test involves placing the sample on a platform with a shear rate ranging from 0.1 to 100 s⁻¹, and evaluating the apparent viscosity by testing its response to different shear rates.

[0038] The frequency sweep test was conducted in the frequency range of 0.1 to 15 Hz to measure the energy storage modulus G' and loss modulus G”, at a temperature of 25 °C and a strain rate of 0.5%.

[0039] Preferably, the method for extracting surface-active proteins from salmon emulsion gel includes the following steps:

[0040] E1: Centrifuge 20g of fresh emulsion gel at 10,000×g for 30 minutes at 4°C to remove unabsorbed particles;

[0041] E2: After centrifugation, collect the top layer, mix the top layer with methanol at a weight-to-volume ratio of 1:2, and incubate at room temperature for 10 minutes;

[0042] E3: The mixture is then centrifuged at 4,700×g for 10 minutes and the top methanol phase is removed. This process is repeated three times.

[0043] E4: After washing with methanol, the precipitate was washed three times with n-hexane, and finally washed three times with ethanol. The collected precipitate was washed with 100mM phosphate buffer (pH 7.0) containing 100mM NaCl, 1mM MgCl2 and 1mM EDTA and centrifuged twice to obtain myofibrillar protein. The surface-active myofibrillar protein precipitate was dried by freeze-drying.

[0044] Preferably, the analysis of the morphology and interfacial rheological properties of surface myofibrillar proteins includes the following steps:

[0045] G1: The obtained surface myofibrillar proteins were measured by FTIR, XRD, SEM, AFM and TEM, and the particle size distribution was determined by DLS.

[0046] G2: Interfacial properties were measured using an optical surface analyzer. Medium-chain triglycerides (MCT) were used as the oil phase. Surfactant proteins were suspended in 10 mM PBS at a concentration of 500 μg / mL and filtered through a 0.45 μm filter to remove large protein aggregates.

[0047] G3: Protein concentration was quantified using the BCA Protein Assay Kit. A 15 mm² pendant droplet was created on the needle tip. The droplet was always equilibrated for 3 hours before any droplet area deformation was performed, and the oscillation amplitude was set to 10%. Amplitude scans were performed at a constant frequency of 0.05 Hz at 25 °C to obtain interfacial tension, dilatational elastic modulus Ed', and dilatational viscous modulus Ed".

[0048] Compared with the prior art, the beneficial effects of the present invention are:

[0049] This invention utilizes high-pressure homogenization (HPH) to transform raw salmon fillets and spinal cord oil into emulsion gels with high ω-3 oil content, specifically designed for patients with dysphagia. HPH improves the stability, swallowing safety, and rheological viscosity of emulsion gels containing 30-60 vol.% oil. Emulsion gels containing 50 and 60 vol.% oil exhibit excellent swallowing safety and rheological properties, and can be classified as level 4 according to the IDDSI framework. The enhanced emulsion stability and gel texture are due to the modification of myofibrillar proteins by HPH, including particle size, α-helical structure, aggregation, and interfacial properties.

[0050] At the molecular level, HPH transforms large myofibril protein filament aggregates into nanoscale myofibril micelles. These nanoscale myofibril micelles can dissociate and aggregate with other proteins in oil, revealing the molecular mechanism by which HPH stabilizes salmon emulsion gels and thickens their texture. High-pressure homogenization (HPH), as a non-thermal physical processing technique, can be used to alter the structure and self-assembly properties of proteins. When high-speed fluid passes through the narrow slit of the homogenization valve, it generates strong shear and impact forces. These forces disrupt and mix particles, cells, or colloids in the sample, making them smaller and more uniformly dispersed. Homogenization pressure causes proteins to unfold, aggregate, or form gels, effectively improving the storage stability and mouthfeel of liquids and purified foods. Furthermore, it provides effective and comprehensive testing for salmon emulsion gel products, strongly demonstrating the efficacy of salmon emulsion gel products in improving swallowing difficulties. Attached Figure Description

[0051] Figure 1 This is a flowchart illustrating the preparation process of the salmon emulsion gel of the present invention.

[0052] Figure 2 This is a graph showing the instability of the HPH of salmon emulsion gels with different oil contents according to the present invention.

[0053] Figure 3 This is a comparison of the instability coefficient curves of the HPH of salmon emulsion gels with different oil contents according to the present invention.

[0054] Figure 4 This is a comparison of the interfacial motion rate curves of salmon emulsion gels with different oil contents and without HPH according to the present invention.

[0055] Figure 5 This is a comparison of the differential thermal scanning curves of the HPH of salmon emulsion gels with different oil contents according to the present invention.

[0056] Figure 6 The graph shows the instability curves of salmon emulsion gels with different oil contents according to the present invention after being analyzed by an HPH instability analyzer.

[0057] Figure 7 The graph shows a comparison of the instability coefficient curves of salmon emulsion gels with different oil contents according to the present invention after pH testing.

[0058] Figure 8 This is a comparison of the interfacial motion rate curves of salmon emulsion gels with different oil contents according to the present invention after passing through the pH.

[0059] Figure 9 This is a comparison of the differential thermal scanning curves of salmon emulsion gels with different oil contents according to the present invention after passing through the pH.

[0060] Figure 10These are microscopic images of the salmon emulsion gel of the present invention before and after HPH.

[0061] Figure 11 This is a graph showing the change in the number of bacterial colonies during the storage of the salmon emulsion gel of the present invention;

[0062] Figure 12 This is a comparison of the oscillation strain scanning curves of salmon emulsion gels with different oil contents and without HPH according to the present invention.

[0063] Figure 13 This is a comparison of the frequency scanning curves of the HPH of salmon emulsion gels with different oil contents according to the present invention.

[0064] Figure 14 This is a comparison of viscosity shear scan curves of salmon emulsion gels with different oil contents and without HPH according to the present invention.

[0065] Figure 15 This is a comparison of the oscillation-strain scanning curves of salmon emulsion gels with different oil contents according to the present invention after HPH.

[0066] Figure 16 This is a comparison of the frequency scanning curves of the HPH of salmon emulsion gels with different oil contents according to the present invention.

[0067] Figure 17 This is a comparison of the viscosity shear scan curves of salmon emulsion gels with different oil contents according to the present invention after HPH.

[0068] Figure 18 This is a comparison of the surface protein particle size distribution curves of Un-HPH0.6 and HPH0.6 of the present invention;

[0069] Figure 19 This is a comparison of the infrared spectral curves of Un-HPH0.6 and HPH0.6 of the present invention;

[0070] Figure 20 A comparison of the X-ray diffraction curves of Un-HPH0.6 and HPH0.6 of the present invention;

[0071] Figure 21 This is a SEM image of the surface proteins of the salmon emulsion gel of the present invention;

[0072] Figure 22 This is an AFM image of the surface proteins of the salmon emulsion gel of the present invention;

[0073] Figure 23 This is a TEM image of the surface proteins of the salmon emulsion gel of the present invention;

[0074] Figure 24 This is a schematic diagram illustrating the mechanism by which the present invention improves the stability and texture of salmon emulsion gel through HPH;

[0075] Figure 25 This is a comparison diagram of the interface expansion of Un-HPH0.6 and HPH0.6 in this invention;

[0076] Figure 26 This is a comparison diagram of the interfacial adsorption characteristics of Un-HPH0.6 and HPH0.6 of the present invention. Detailed Implementation

[0077] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0078] Please see Figures 1 to 6 One embodiment of the present invention provides an improved method for preparing salmon emulsion gel for swallowing difficulties, comprising the following steps:

[0079] Step 1: Mix fresh salmon meat and ice water in a 1:3 ratio in a blender and blend at high speed for 1 minute. Filter the mixture through a 60-mesh sieve.

[0080] Step 2: The obtained filtrate is pre-homogenized for 2 minutes using a disperser at a speed of 10,000 rpm;

[0081] Step 3: The dispersion undergoes a first-step high-pressure homogenization process. The temperature conditions for high-pressure homogenization are 4℃ and the pressure conditions are 50 MPa. This process is repeated three times to obtain the initial protein suspension. The protein content in the suspension is then adjusted to 2.0 w / v%.

[0082] Step 4: Salmon oil extracted from salmon spine using cold micro-water extraction was added to a protein-based suspension to ensure that the oil volume fraction φ reached 0.3, 0.4, 0.5, 0.6 and 0.7 respectively, and five samples were obtained.

[0083] Step 5: Mix the sample using a disperser at 12,000 rpm for 2 minutes to obtain emulsion gel A;

[0084] Step 6: Perform a second high-pressure homogenization on emulsion gel A. The temperature conditions for high-pressure homogenization are 4℃ and the pressure conditions are 50Mpa to obtain salmon emulsion gel.

[0085] Salmon oil extracted from salmon spines involves the following steps:

[0086] W1: The cleaned salmon spine is freeze-dried to reduce the moisture content, and the dried by-products are ground using a grinder.

[0087] W2: Rehydrate the ground sample with water until the oil volume fraction φ is adjusted to 75% of the volume. Stir tangentially with a top stirrer for 15 minutes, and then immediately centrifuge at 8000×g for 15 minutes at a temperature of 20℃.

[0088] W3: Obtain the upper layer of salmon oil and store it in a storage environment at -20℃.

[0089] The above-mentioned improved testing method for salmon emulsion gels that are difficult to swallow is characterized by comprising the following steps:

[0090] S1: The salmon emulsion gels with different φ after the second high-pressure homogenization process are abbreviated as HPH0.3, HPH0.4, HPH0.5, HPH0.6 and HPH0.7, respectively. The emulsion gel A without the second high-pressure homogenization process is abbreviated as Un-HPH0.3, Un-HPH0.4, Un-HPH0.5, Un-HPH0.6 and Un-HPH0.7, respectively. Each emulsion gel sample is stored in a 20 ml transparent glass vial and stored at 4°C.

[0091] S2: Perform color analysis on all emulsion and gel samples using a colorimeter. Colorimeter measurement parameters include brightness. Redness and yellowness The value was determined, and microscopic observation and particle size analysis were performed.

[0092] S3: Centrifugal stability tests were performed on all emulsion gel samples. The measurements were taken using a dispersion analyzer. The centrifugal stability test involved transferring the sample to a polycarbonate tube and collecting data at 20-second intervals in a centrifuge. The sample was centrifuged for 6000 seconds to make it unstable. The instability index and area percentage were then calculated using LUMSEPviewV6.4 software.

[0093] S4: Differential scanning calorimetry was used to measure the formation and dissociation of ice crystals in salmon emulsion gels for all emulsion gel samples;

[0094] S5: IDSSI and rheological properties tests were performed on the salmon emulsion gel.

[0095] Rheological properties were measured using a rheometer with a plate diameter of 40 mm and a gap distance of 1000 μm. The test sample was placed at ambient temperature for 1 hour to reach thermal equilibrium, and the test temperature was 25 °C.

[0096] S6: Strain scanning, viscosity shear, and frequency scanning tests were performed on the salmon emulsion gel. The strain scanning test measured the viscoelasticity in an oscillation amplitude mode within the linear viscoelastic region (LVR). The test temperature was 25℃, the frequency was 1Hz, and the strain range was 0.1% to 1000%.

[0097] Viscosity shear test involves placing the sample on a platform with a shear rate ranging from 0.1 to 100 s⁻¹, and evaluating the apparent viscosity by testing its response to different shear rates.

[0098] Frequency scanning tests were conducted in the frequency range of 0.1 to 15 Hz to measure the storage modulus G' and loss modulus G", at a temperature of 25°C and a strain rate of 0.5%.

[0099] S7: Extraction of surface active proteins from salmon emulsion gel to obtain surface myofibrillar proteins. The extraction method for surface active proteins from salmon emulsion gel includes the following steps:

[0100] E1: Centrifuge 20g of fresh emulsion gel at 10,000×g for 30 minutes at 4°C to remove unabsorbed particles;

[0101] E2: After centrifugation, collect the top layer, mix the top layer with methanol at a weight-to-volume ratio of 1:2, and incubate at room temperature for 10 minutes;

[0102] E3: The mixture is then centrifuged at 4,700×g for 10 minutes and the top methanol phase is removed. This process is repeated three times.

[0103] E4: After washing with methanol, the precipitate was washed three times with n-hexane, and finally washed three times with ethanol. The collected precipitate was washed with 100mM phosphate buffer containing 100mM NaCl, 1mM MgCl2 and 1mM EDTA at pH 7.0 and centrifuged twice to obtain myofibrillar protein. The surface-active myofibrillar protein precipitate was dried by freeze-drying.

[0104] The morphological and interfacial rheological properties of surface myofibrillar proteins were analyzed, including the following steps:

[0105] G1: The obtained surface myofibrillar proteins were measured by FTIR, XRD, SEM, AFM and TEM, and the particle size distribution was determined by DLS.

[0106] G2: Interfacial properties were measured using an optical surface analyzer. Medium-chain triglycerides (MCT) were used as the oil phase. Surfactant proteins were suspended in 10 mM PBS at a concentration of 500 μg / mL and filtered through a 0.45 μm filter to remove large protein aggregates.

[0107] G3: Protein concentration was quantified using the BCA Protein Assay Kit. A 15 mm² pendant droplet was created on the needle tip. The droplet was always equilibrated for 3 hours before any droplet area deformation was performed, and the oscillation amplitude was set to 10%. Amplitude scanning was performed at a constant frequency of 0.05 Hz at 25 °C to obtain interfacial tension, dilatational elastic modulus Ed', and dilatational viscous modulus Ed".

[0108] S8: Repeat steps S1-S7 three times to calculate the mean and standard deviation of the data.

[0109] Microscopic observation and particle size analysis include the following steps:

[0110] Step 1: Dilute the homogenized salmon emulsion gel in 1% SDS solution at a ratio of 1:10 to disperse the aggregated oil droplets. Dilute the non-homogenized emulsion in ultrapure water at a ratio of 1:5.

[0111] Step 2: Observe the microstructure of oil droplets in salmon emulsion gel using a fluorescence inverted microscope with a digital camera. The oil droplets are stained red with Nile red, then dropped onto a glass slide and covered with a coverslip.

[0112] Step 3: The particle size distribution of the fresh emulsion gel was determined by dynamic light scattering (DLS) in the range of 0.02 μm-2600 μm. The ultrasonic power was set to 35 W for 2 minutes, the stirring speed was set to 1200 rpm, and the measurement opacity was set to 5%-20%.

[0113] Differential scanning calorimetry (DSC) measurement includes the following steps:

[0114] Step 1: Place the fresh salmon emulsion gel into an aluminum crucible, introduce nitrogen gas at a flow rate of 20 mL / min. Cool the measured temperature from 20°C to -30°C at a cooling rate of 5°C / min;

[0115] Step 2: After equilibration for 5 minutes, the sample is heated from 30°C to 40°C at the same flow rate, and then cooled from 40°C to 20°C at a rate of 10°C / min.

[0116] Water exudation was observed at the bottom of the salmon protein dispersion and during storage. No water exudation was observed when the oil phase volume fraction reached 60% and 70%. This high stability is likely due to the repulsive effect of the oil phase inhibiting protein aggregation, thus suppressing water exudation. High-pressure homogenization (HPH) further stabilized the emulsion gel, resisting water exudation. HPH can break down salmon micron-sized myofibril protein particles into smaller particles, thereby supporting the stability of water and oil. However, HPH emulsion gels containing 70% oil phase failed to form stable emulsion gels. Under such a high oil phase, the emulsion exhibited poor flowability when flowing through the homogenization valve, leading to emulsion disruption. Furthermore, the HPH sample remained in a condensed state during storage, exhibiting a creamy texture. Since salmon oil is light orange-red, color changes effectively reflect the degree of oil release in the sample, as shown in Tables 1A and 1B below on days 1 and 7 during storage (4°C).

[0117]

[0118]

[0119] The effects of HPH treatment on the surface color of the emulsion gel are shown in Tables 1A and 1B, respectively. On day 1, the HPH samples... The value was higher than that of the non-HPH sample, while and The value was low, and HPH treatment may have broken the oil droplets into smaller ones, thereby reducing red and yellow. After 7 days of storage, the surface color parameters (φ=0.3-0.6) of the HPH emulsion gel did not change significantly. Therefore, it can be concluded that 7 days of storage did not cause significant oil exudation in the HPH salmon emulsion gel.

[0120] For the centrifugal stability of salmon emulsion gels, proteins tend to aggregate during storage, leading to water exudation, while oil droplets tend to coalesce, resulting in oil droplet separation. Therefore, an accelerated stability analyzer (LUMiSizer) was used to investigate the potential storage stability of salmon emulsion gels. Centrifugal emulsification leads to oil droplet coalescence and more severe oil droplet separation, as well as water exudation, such as... Figures 2-9 As shown, during centrifugation (1000×g), the overall transmittance spectra of non-high pressure homogenized (Un-HPH) and high pressure homogenized (HPH) emulsion gels were obtained. Due to the instability of oil droplets, the emulsion gel with the highest oil content φ=0.7 showed obvious oil droplet separation, which is consistent with their appearance. The non-high pressure treated emulsion 0.3-0.6 had a slight oil droplet separation layer at the top, while the HPH 0.3-0.6 emulsion had no oil droplet separation, indicating that the oil phase was more stable. However, both had a water layer at the bottom from 110 mm to 130 mm. Therefore, the water seepage during centrifugation can indicate the instability index.

[0121] Compared with Un-HPH0.3-0.6, the instability index of HPH0.3-0.6 was significantly reduced. The increase in emulsion stability can be attributed to the modification of protein structure and interfacial properties by high-pressure shear, thereby improving oil / water adsorption capacity. In addition, the instability index decreased as the φ value increased from 0.3 to 0.6, indicating that the emulsion gel with a higher oil volume fraction can stabilize more water. The instability index decreased with increasing φ value. Overall, HPH0.5 and HPH0.6 emulsions exhibited excellent emulsion stability, with HPH0.6 showing the highest stability.

[0122] The thermal stability of the salmon emulsion gels was determined by differential scanning calorimetry (DSC). The crystallization temperature (Tc) and melting point (Tm) of all emulsion gels were -17 to -21 °C and 5 to 8 °C, respectively. The Tc and phase transition enthalpy (ΔH) of the HPH0.3-0.6 emulsion gel were lower than those of the un-high pressure treated (Un-HPH0.3-0.6) emulsion gel, indicating that it has higher thermal stability.

[0123] For the microstructure and particle size of salmon emulsion gels, enhanced stability largely depends on the oil droplet size, such as... Figure 10 As shown, microscopic observation of the emulsion oil droplets revealed that HPH significantly reduced the size of most oil droplets, from over 20 micrometers to nearly 2 micrometers, consistent with the DLS particle size distribution results. The mechanical forces generated by high-speed shearing and cavitation on the emulsion gel led to the breakup of large oil droplets. In the Un-HPH0.3-0.6 emulsion gel, oil droplets were dispersed individually. However, after high-pressure homogenization, even when dispersed in 1% SDS to reduce aggregation, the oil droplets in the HPH0.3-0.6 emulsion gel aggregated into large flocculent particles. High-pressure homogenization can alter protein structure, such as protein unfolding and exposure of hydrophobic groups, reducing protein particle size, increasing surface hydrophobicity, promoting their electrostatic interactions, and enhancing emulsifying and lubricating properties. The terminal regions of surface myofibrillar proteins extend into the aqueous phase, potentially promoting a stable gel structure through steric hindrance interactions. Therefore, high-pressure homogenization effectively reduced the droplet size of salmon emulsion gels and promoted droplet aggregation, which positively impacts their storage stability.

[0124] IDDSI provides a globally recognized standard and testing method for evaluating texture-modified foods for patients with dysphagia. The IDDSI test includes the fork drop test, spoon tilt test, and fork pressure test, categorized into eight levels (0-7) from liquid beverages to semi-solid foods. Salmon emulsion gels were evaluated using the IDDSI framework. The fork drop test assessed liquid-form foods (levels 3 and 4), while the spoon tilt test assessed the food's cohesiveness and viscosity (levels 4 and 5). All un-HPH 0.3-0.6 emulsion gels showed liquid consistency but failed to meet the standards of the level 3 and 4 fork drop and spoon tilt tests because they dripped rapidly from the fork and stuck to the spoon. In contrast, all HPH 0.3-0.6 emulsion gels were in a highly viscous and purified state, exhibiting higher cohesiveness than un-hyperpressed 0.3-0.6 emulsion gels. However, HPH 0.3 and HPH 0.4 were in liquid form and could not completely slide off the spoon, failing the level 4 spoon tilt test. As φ increases from 0.3 to 0.6, viscosity increases. HPH0.5 and HPH0.6 exhibit excellent stacking ability on a spoon, sliding slowly when tilted without any splashing. They also stack on a fork; in the fork crush test, HPH0.5 and HPH0.6 are easily crushed under slight pressure, leaving a clear pattern on the fork surface (level 4). Overall, the results indicate that HPH0.5 and HPH0.6 can be classified as level 4, meaning they are non-sticky, generally edible with a spoon, and require no chewing. Therefore, they are ideal for those with dysphagia due to their ease of swallowing.

[0125] For the viscoelasticity test of salmon emulsion gel, the emulsion gel exhibits a series of thickening and viscoelastic behaviors when subjected to shear force. Rheological properties are widely recognized as being used to quantify the oral performance and swallowability of emulsion gel. Before testing the viscosity, it is necessary to evaluate the hardness of the salmon emulsion gel. Oscillatory shear rheology (strain and frequency scan) was performed before shearing to obtain the storage modulus (G') and loss modulus (G").

[0126] In the strain sweep test, the G' of all samples was higher than G'', indicating that the salmon emulsion gels exhibited elasticity-dominated behavior regardless of whether they were homogenized or not. Generally, at the same φ value, the G' and G'' of the HPH0.3-0.6 emulsion gels were one order of magnitude higher than those of the non-HPH0.3-0.6 emulsion gels. In addition, G' and G'' gradually increased with the increase in the oil volume fraction, that is, the mechanical strength of the emulsion gels was improved. The higher G' and G'' of HPH0.3-0.6 might imply higher viscosity and hardness. When the strain reached the LVR, G' and G'' began to decrease non-linearly, indicating that the network structure was damaged. Compared with the HPH0.3-0.6 emulsion gels, the non-HPH0.3-0.6 samples had a shorter viscoelastic region (LVR), indicating their brittle texture. During the oral processing and swallowing of the non-HPH0.3-0.6 emulsion gels, disordered and heterogeneous structures might occur, which would have a negative impact on bolus adhesion during the passage of the bolus through the pharynx, thus increasing the swallowing risk for dysphagia patients.

[0127] In the frequency sweep test, as Figure 12-17 shown, the G' and G'' of the salmon emulsion gels with frequency. Since G'>G'', the emulsion gels were elastic in the range of 0.1-15 Hz. Both G' and G'' increased with the increase in the oil content, which might be due to the exposure of hydrophobic groups on the surface of salmon proteins, resulting in an increased degree of interaction between salmon proteins and oils. In addition, at the same φ value (0.3-0.6), the G' (600-5000 Pa) of the HPH0.3-0.6 emulsion gels was almost 6-8 times that of the Un-HPH0.3-0.6 emulsion gels (100-600 Pa). The salmon emulsion gels changed from liquid state to mud state after high-pressure homogenization. The possible reason was that high-pressure homogenization changed the structure of salmon proteins, improved the interfacial properties of proteins, and enhanced the emulsifying ability.

[0128] The value of tanδ (G" / G') can be used to characterize the liquid or gel state of the emulsion gels. tanδ>1 indicates a dilute solution, while 0.1<tanδ<1 indicates a weak gel. Food boluses with tanδ values in the range of 0.1-1 are easier to swallow for dysphagia patients. The tanδ values of the emulsion gels of HPH0.3, HPH0.4, HPH0.5, and HPH0.6 were in the range of 0.1-0.3, indicating that they were weak emulsion gels and were safe for dysphagia patients from the perspective of rheological properties.

[0129] During swallowing, shear is considered the main deformation force applied to the bolus. According to the American National Dysphagia Diet Guidelines, shear is recommended as the standard for evaluating the swallowing thickness of dysphagia patients. The shear rate is highly correlated with the pharyngeal force applied to the food bolus. Therefore, shear viscosity was measured in the range of 0.1 to 100 s⁻¹ to adequately assess the swallowability of the salmon emulsion gel. The viscosity of the sample decreased with increasing shear rate, indicating that these emulsion gels exhibit non-Newtonian shear thinning behavior, which is necessary for safe swallowing.

[0130] Thickening fluids (grades 1-4) have a strong viscosity-dependent therapeutic effect on oropharyngeal patients with severe dysphagia, increasing the safe swallowing rate from 63% at 0.1 Pa × sec to 95% at 1.6 Pa × sec. (shearing at time), all Un-HPH0.3-0.6 emulsion gels in The viscosity at the shear rate is between 0.14 and 0.58 Pa × s, indicating that these foods may have an unsafe swallowing rate of over 20-40%. HPH0.4, HPH0.5, and HPH0.6 have viscosities of 2.24, 7.65, and 8.65 Pa × s, respectively, indicating a high safe swallowing rate of over 95%. Grade 4 commercial thickeners typically exhibit low shear viscosity, ranging from 0.8 to 2.7 Pa × s. Recently, Grade 4 dysphagia diets prepared using various gel mixtures (1-2%) have exhibited higher shear viscosities at 50 s⁻¹, ranging from 10-20 Pa × s, making them ideal for the production of 3D-printed dysphagia foods.

[0131] Based on the above investigation, it is clear that HPH0.6 contains more ω-3 salmon oil and exhibits excellent emulsification stability, IDDSI texture, and rheological properties. Therefore, we further evaluated the relationship between the storage time of HPH0.6 at 4°C and the cumulative colony count, such as... Figure 11 As shown, without the addition of any preservatives, the cumulative colony counts of HPH0.6 on days 7, 10, and 14 were 5.6 × 10³ CFU / g, and According to Chinese standard (GB10136-2015), the cumulative bacterial count of edible aquatic animal products should reach [a certain threshold]. This is the acceptable limit, meaning that HPH 0.6 can be consumed within 10 days.

[0132] like Figure 18-20As shown, high-pressure homogenization significantly improved the physical stability of salmon emulsion gel and altered its texture and rheological properties due to changes in myofibrillar protein structure. To investigate this phenomenon, surface myofibrillar proteins were extracted and analyzed from the control group (a suspension based on salmon protein), the unhomogenized 0.6 group, and the homogenized 0.6 group. After high-pressure homogenization, the particle size decreased. The myofibrillar proteins in the control group showed a bimodal size distribution, with two main peaks of approximately 90 μm and 1200 μm, respectively. The samples treated with high-pressure homogenization (HPH0) showed a decrease in particle size. The main peak of Un-HPH0.6 is concentrated at about 90 micrometers, while the main peak of Un-HPH0.6 without high-pressure homogenization is concentrated at about 90 micrometers. This indicates that high-pressure homogenization reduces the particle size and aggregation of surfactant proteins. According to FTIR and XRD patterns, high-pressure homogenization treatment reduces α-helices, XRD 2θ decreases by 9°, FTIR absorption peaks between 1650-1660 decrease, and XRD intensity decreases, which also indicates a decrease in the crystallinity of protein particles. This is because high-pressure homogenization treatment reduces the degree of myofibril filamentary structure.

[0133] like Figure 21 As shown, the protein aggregates in the control group and Un-HPH0.6 samples exhibited fibrous and rod-like shapes, consistent with the filamentous structure of fish myofibrillar proteins. Between cross-linking, the surface of the protein fibrillary aggregates was rough with large gaps. HPH significantly altered the morphology of myofibrillar proteins. The protein aggregates of HPH0.6 exhibited a more granular shape with small voids between the aggregates, while the rod-like fibrillary structure disappeared. The gaps between HPH0.6 myofibrillar proteins were smaller, more uniform, and denser at the nanoscale, which is a marker of the protein's transformation from large fibrillary aggregates to small micelle aggregates.

[0134] like Figure 22 and Figure 23 As shown, atomic force microscopy (AFM) was used to detect the surface roughness and aggregation of myofibril proteins at the nanoscale. The control protein exhibited large aggregation peaks, characterized by high root mean square roughness (Rq; 10.8 nm), average roughness (Ra; 7.22 nm), and a large surface height value (47.4 nm). After dispersion with oil, the protein Ra and surface height decreased to 2.64 nm and 13.3 nm, respectively. This reduction is attributed to the hydrophobic forces from the oil, which inhibit the aggregation of myofibril filaments. The surface roughness of the HPH0.6 protein decreased to Ra = 1.59 nm in a more compact form, exhibiting a more uniform small peak. This indicates that the salmon myofibril filaments are broken down into shorter and thinner aggregates.

[0135] Transmission electron microscopy was also used to observe surface myofibrillar proteins in the control group, non-HPH0.6, and HPH0.6. Proteins from the control group formed large protein aggregates exceeding 6 μm, while proteins from non-HPH0.6 exhibited filamentous aggregates approximately 3 μm in length. Myosin and actin filaments (or sarcomeres) were typically approximately 2.5 μm in length. In contrast, proteins treated with high pressure (HPH0.6) formed smaller aggregates at both the micrometer (approximately 1 μm) and nanometer scales. These observations are consistent with atomic force microscopy results, indicating that high-pressure homogenization reduces the size of protein aggregates, changing their shape from filamentous to a more loosely aggregated state. High-pressure homogenization with oil can break down myofibrillar fibrillary aggregates, disintegrating them into subprotein species. These smaller units can then aggregate in an aqueous phase, forming different forms depending on their exposed hydrophobicity. The cavitation and shear forces generated by high-pressure homogenization likely promote hydrophobic adsorption of oil molecules, disrupting electrostatic and hydrophobic interactions between protein molecules; therefore, high-pressure homogenization effectively reduces protein aggregation and size.

[0136] like Figure 25 and 26 As shown, the interfacial properties of surface proteins are mainly determined by nanoparticles, leading to the aggregation and interfacial characteristics of these proteins. Micro-sized protein aggregates were removed by filtration through a 0.45-micron filter. The nano-sized protein particles were observed using transmission electron microscopy (TEM). The nano-sized particles in the control group and those without HPH0.6 exhibited a chain-like, coiled, helical fibrillary structure. The filtrate showed aggregated fibrils with chain lengths exceeding 2 microns and chain widths less than 0.45 microns. The HPH0.6 nano-sized particles, approximately 400 nanometers in diameter, were spherical and uniformly distributed, and are likely protein micelles.

[0137] The interfacial properties of the control group and the non-HPH0.6 myofibril nanofilaments were compared with those of the HPH0.6 myofibril micelles. The interfacial tension of the myofibril nanofilament micelles was lower than that of the control group and the non-HPH0.6 myofibril nanofilaments. This indicates that more myofibril nanofilament micelles adsorb at the oil / water interface. The expanded elastic modulus (Ed') was higher than the viscous modulus. The difference is an order of magnitude, indicating that the elastic component dominates the expansion response. The elastic modulus of the HPH0.6 nanomyofibrillar micelles is higher than that of the control group and the non-HPH0.6 myofibrillar protofilaments, implying higher interfacial stiffness and a stronger interface. This is due to the smaller size of the myofibrillar micelles, which also exposes hydrophobic regions. Furthermore, the myofibrillar micelles may dissociate at the interface. Therefore, the oil droplets of HPH0.6 are more stable at the interface than those of Un-HPH0.6.

[0138] Based on the results and viewpoints of the above sections, such as Figure 24As shown, a schematic diagram of the mechanism by which HPH improves stability and texture is presented. The myofibrillar protein of salmon is mainly myosin, with a molecular weight of about 200 kilodaltons.

[0139] Fish muscle myosin molecules consist of a long α-helical tail and two pear-shaped heads. The tail is 155 nm long and 2 nm in diameter, while the head axis is approximately 20 nm long and 6 nm wide, exhibiting a rotation radius of 80–90 nm. Therefore, the diameter of a single myosin molecule is likely 160–180 nm. Since the nanoparticles in HPH0.6 are nearly spherical with a diameter of approximately 400 nm, we propose assembling myosin molecules into two length-scale nanomicelles. HPH treatment reduces the α-helix and exposes the protein's internal hydrophobic regions, which, upon exposure to water, are likely to aggregate into spherical shapes due to hydrophobic-driven interactions. In non-HPH0.6, more than half of the oil droplets are dispersed in a large size of 20 micrometers, while most of the oil droplets in HPH0.6 are much smaller (<4 micrometers). Notably, the surfactant myofibrillar protein should be an order of magnitude smaller than the oil droplets, which suggests that the surfactant protein in non-HPH0.6 and HPH0.6 should be smaller than 2 micrometers and 400 nanometers, respectively, which is consistent with the size of the fibrils and micelles observed by transmission electron microscopy.

[0140] In non-HPH0.6 emulsion gels, individual myofibrillar protein molecules, each about 160-180 nanometers long, aggregate to form filament units of about 2.5 micrometers. These filament units may further aggregate to form large protofibrillary aggregates. The oil droplets stabilized by the protofibrillary structure have poor stability, and the cohesive interaction between protein protofibrils is too low to form a thick structure.

[0141] From a molecular perspective, high-pressure homogenization disrupts the myofibrillar aggregate structure, reduces α-helices, and exposes the inner hydrophobic regions of myofibrillar proteins. These proteins assemble into nanoscale micelle structures (approximately 400 nm) under the aggregation of hydrophobic heads to prevent contact between the hydrophobic heads and water. Protein micelles may dissociate at the oil interface. The surface proteins in the oil droplets of HPH0.6 are likely myofibrillar protein monomers, with their hydrophobic heads attached to the oil interface and their long tails extending into the aqueous phase. Therefore, the oil droplets of HPH0.6 are much more stable. Compared to HPH0.3-0.4, the HPH0.6 system is more compact, with a denser arrangement of oil droplets. Because HPH partially unfolds the protein tails, the tails in the aqueous phase are likely to promote droplet aggregation through electrostatic forces and steric hindrance interactions between the droplets. This contributes to the formation of a gel-like structure and increases the cohesive rheology and viscosity of the emulsion gel.

Claims

1. An improved method for preparing salmon emulsion gel for swallowing difficulties, characterized in that, Includes the following steps: Step 1: Mix fresh salmon meat and ice water in a 1:3 ratio in a blender and blend at high speed for 1 minute. Filter the mixture through a 60-mesh sieve. Step 2: The obtained filtrate is pre-homogenized for 2 minutes using a disperser at a speed of 10,000 rpm; Step 3: The dispersion is subjected to three cycles of high-pressure homogenization in Step 1. The temperature conditions for high-pressure homogenization are 4℃ and the pressure conditions are 50 MPa, resulting in an initial protein suspension. The protein content in the suspension is then adjusted to 2.0 w / v%. Step 4: Add salmon oil extracted from salmon spine using cold micro-water extraction to a protein-based suspension to ensure that the oil volume fraction φ reaches 0.5 and 0.6 respectively, obtaining two samples. The salmon oil extracted from salmon spine includes the following steps: W1: Freeze-dry the cleaned salmon spine to reduce the moisture content, and grind the dried by-product using a grinder; W2: Rehydrate the ground sample with water until the oil volume fraction φ is adjusted to 75% of the volume, stir tangentially with a top stirrer for 15 minutes, and then immediately centrifuge at 8000×g for 15 minutes at 20°C. W3: Obtain the upper layer of salmon oil and store it in a storage environment at -20℃; Step 5: Mix the sample using a disperser at 12,000 rpm for 2 minutes to obtain emulsion gel A; Step 6: Perform a second high-pressure homogenization on emulsion gel A to obtain salmon emulsion gel.

2. A method for testing improved salmon emulsion gels for dysphagia according to claim 1, characterized in that, Includes the following steps: S1: Salmon emulsion gels with different φ after the second high-pressure homogenization process are referred to as HPH0.5 and HPH0.6, respectively. Emulsion gel A without the second high-pressure homogenization process is referred to as Un-HPH0.5 and Un-HPH0.6, respectively. Each emulsion gel sample is stored in a 20 ml transparent glass vial and stored at 4°C. S2: Color analysis was performed on all emulsion gel samples using a colorimeter, and microscopic observation and particle size analysis were conducted. S3: Centrifugal stability tests were performed on all emulsion gel samples, and the measurements were taken using a dispersion analyzer; S4: Differential scanning calorimetry was used to measure the formation and dissociation of ice crystals in salmon emulsion gels for all emulsion gel samples; S5: IDSSI and rheological properties tests were performed on the salmon emulsion gel. S6: Strain scanning test, viscosity shear test and frequency scanning test were performed on the salmon emulsion gel; S7: Extract surface active proteins from salmon emulsion gel to obtain surface myofibrillar proteins, and analyze the morphology and interfacial rheological properties of the surface myofibrillar proteins. S8: Repeat steps S1-S7 three times to calculate the mean and standard deviation of the data.

3. The method for testing improved salmon emulsion gels for dysphagia according to claim 2, characterized in that: The colorimeter measures parameters including luminance. Redness and yellowness The value; The centrifugal stability test involved transferring the sample into a polycarbonate tube and collecting data at 20-second intervals in a centrifuge. The sample was centrifuged for 6000 seconds to destabilize it, and then the instability index and area percentage were calculated using LUMSEPviewV6.4 software.

4. The method for testing improved salmon emulsion gels for dysphagia according to claim 2, characterized in that: Microscopic observation and particle size analysis include the following steps: Step 1: Dilute the homogenized salmon emulsion gel in 1% SDS solution at a ratio of 1:10 to disperse the aggregated oil droplets. Dilute the non-homogenized emulsion in ultrapure water at a ratio of 1:

5. Step 2: Observe the microstructure of oil droplets in salmon emulsion gel using a fluorescence inverted microscope with a digital camera. The oil droplets are stained red with Nile red, then dropped onto a glass slide and covered with a coverslip. Step 3: The particle size distribution of the fresh emulsion gel was determined by dynamic light scattering (DLS) in the range of 0.02 μm-2600 μm. The ultrasonic power was set to 35 W for 2 minutes, the stirring speed was set to 1200 rpm, and the measurement opacity was set to 5%-20%.

5. The method for testing improved salmon emulsion gels for dysphagia according to claim 2, characterized in that: The differential scanning calorimetry measurement includes the following steps: Step 1: Place the fresh salmon emulsion gel into an aluminum crucible, introduce nitrogen gas at a flow rate of 20 mL / min, and cool the measured temperature from 20°C to -30°C at a cooling rate of 5°C / min. Step 2: After equilibration for 5 minutes, the sample is heated from 30°C to 40°C at the same flow rate, and then cooled from 40°C to 20°C at a rate of 10°C / min.

6. The method for testing improved salmon emulsion gels for dysphagia according to claim 2, characterized in that: The rheological properties were measured using a rheometer with a plate diameter of 40 mm and a gap distance of 1000 μm. The test sample was placed at ambient temperature for 1 hour to reach thermal equilibrium, and the test temperature was 25°C. Strain scanning tests were performed to measure viscoelasticity in an oscillating amplitude mode within the linear viscoelastic region (LVR). The test temperature was 25℃, the frequency was 1Hz, and the strain range was 0.1% to 1000%. Viscosity shear test involves placing the sample on a platform with a shear rate ranging from 0.1 to 100 s⁻¹, and evaluating the apparent viscosity by testing its response to different shear rates. The frequency sweep test was conducted in the frequency range of 0.1 to 15 Hz to measure the energy storage modulus G' and loss modulus G”, at a temperature of 25 °C and a strain rate of 0.5%.

7. The method for testing improved salmon emulsion gels for dysphagia according to claim 2, characterized in that: The extraction method for surfactant proteins from salmon emulsion gel includes the following steps: E1: Centrifuge 20g of fresh emulsion gel at 10,000×g for 30 minutes at 4°C to remove unabsorbed particles; E2: After centrifugation, collect the top layer, mix the top layer with methanol at a weight-to-volume ratio of 1:2, and incubate at room temperature for 10 minutes; E3: The mixture is then centrifuged at 4,700×g for 10 minutes and the top methanol phase is removed. This process is repeated three times. E4: After washing with methanol, the precipitate was washed three times with n-hexane, and finally washed three times with ethanol. The collected precipitate was washed with 100mM phosphate buffer (pH 7.0) containing 100mM NaCl, 1mM MgCl2 and 1mM EDTA and centrifuged twice to obtain myofibrillar protein. The surface-active myofibrillar protein precipitate was dried by freeze-drying.

8. The method for testing improved salmon emulsion gels for dysphagia according to claim 7, characterized in that: The analysis of the morphology and interfacial rheological properties of surface myofibrillar proteins includes the following steps: G1: The obtained surface myofibrillar proteins were measured by FTIR, XRD, SEM, AFM and TEM, and the particle size distribution was determined by DLS. G2: Interfacial properties were measured using an optical surface analyzer. Medium-chain triglycerides (MCT) were used as the oil phase. Surfactant proteins were suspended in 10 mM PBS at a concentration of 500 μg / mL and filtered through a 0.45 μm filter to remove large protein aggregates. G3: Protein concentration was quantified using the BCA Protein Assay Kit. A 15 mm² pendant droplet was created on the needle tip. The droplet was always equilibrated for 3 hours before any droplet area deformation was performed. The oscillation amplitude was set to 10%. An amplitude scan was performed at a constant frequency of 0.05 Hz at 25 °C to obtain interfacial tension, expansion modulus Ed', and expansion viscous modulus Ed".

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