Protein-polysaccharide composite microgel as well as preparation method and application thereof

Abstract

The invention relates to protein-polysaccharide composite microgel as well as a preparation method and application thereof, and belongs to the technical field of food science and functional materials. The composite microgel is prepared from soybean protein isolate and high-acyl gellan gum through phase separation and ultrasonic combined thermal induction processes. The composite microgel disclosed by the invention can be widely applied to semi-solid food as a fat substitute component, provides a novel material basis with an adjustable structure for developing healthy low-fat food, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of food science and functional materials technology, and relates to a protein-polysaccharide composite microgel, its preparation method and application. Specifically, it relates to a composite microgel prepared by using soy protein isolate and high acyl gellan gum as raw materials through phase separation, ultrasonic treatment and thermal induction process. The resulting microgel has nanoscale particle size, high absolute value of ζ-potential and suitable contact angle, and can be used as a fat substitute in low-fat foods to improve the structural stability and sensory characteristics of the food. Background Technology

[0002] In the food industry, with the increasing demand for healthy diets, the development of low-fat foods has become an important trend. Fat substitutes, as key components for reducing the saturated fatty acid content in food, have been widely used in sauces, dairy products, and meat products. Currently, common fat substitutes include protein-, polysaccharide-, or complex-based gel materials, which suffer from structural instability, rough texture, or poor lubricity. Protein-polysaccharide composite microgels, due to their excellent controllability and natural sources, have great potential in constructing fat substitutes. However, protein- or polysaccharide-based gels, as solid fat substitutes, typically exhibit enhanced texture properties, possessing high hardness and elasticity, but insufficient plasticity. Their simulation of a creamy, smooth texture also remains limited. Constructing food-grade microgels with good rheological properties, lubricity, and stability remains a challenge.

[0003] Protein-polysaccharide composite microgels show potential advantages in the field of fat substitution due to their natural origin and controllability. These microgels typically form a three-dimensional network structure through electrostatic bonding, hydrogen bonding, or hydrophobic interactions, providing a certain degree of elasticity and water retention. Soy protein isolate (SPI), as a high-purity plant protein with good nutritional value and digestibility, has been used to prepare microgels. For example, existing studies have used thermally induced methods to prepare SPI microgels, which exhibit high brittleness under nonlinear deformation conditions, but lack mechanical strength and are sensitive to changes in environmental pH and temperature, limiting their application as independent fat substitutes. To improve these shortcomings, researchers have attempted to introduce polysaccharide components, such as gellan gum. High-acyl gellan gum (HG) is an anionic polysaccharide that can form a soft gel network at low concentrations; when combined with proteins, it can enhance electrostatic repulsion and steric hindrance effects. Existing literature reports that phase separation occurs when whey protein is combined with gellan gum, resulting in reduced gel strength and weak deformability. Similarly, although the combination of SPI and low-acyl gellan gum improves functional properties, it still suffers from problems such as excessively high network rigidity and poor plastic deformation ability. Especially at low polysaccharide concentrations, the microgels tend to aggregate and have poor dispersibility, failing to effectively simulate the rheological behavior of semi-solid fats.

[0004] Secondly, controlling the particle size and distribution of microgels also presents some technical bottlenecks that need to be overcome. Only by controlling the particle size to the submicron level and ensuring uniform distribution can a grainy texture be avoided upon ingestion, and the aggregation of particles can reduce oral friction, thereby producing a rich, oily mouthfeel. It has been reported that the aggregation of fat microparticles in the 200–500 nm range in ice cream can give the product a smooth and delicate texture. Therefore, an ideal fat-substitute microgel should have a particle size of around several hundred nanometers. Currently, commonly used methods to achieve such small and uniform particle sizes include high-shear homogenization, ultrasonic treatment, or complex condensed phase separation. However, maintaining the stability of its internal network structure while reducing particle size remains a challenge.

[0005] Furthermore, existing preparation methods mostly rely on single thermal induction or enzymatic cross-linking processes, making it difficult to achieve synergistic control of microgel particle size, surface properties, and rheological characteristics. In the existing technology, there are no reports on addressing the insufficient plasticity of microgels under low polysaccharide dosage conditions, and structural-functional optimization analyses are also scarce. This limits the application of composite microgels in low-fat foods, such as margarine or mayonnaise, where it is difficult to provide a stable emulsification interface and plasticity, affecting the product's processing adaptability and storage stability.

[0006] Therefore, the main technical problem with existing protein-polysaccharide composite microgels is that at low polysaccharide concentrations, they have poor plastic deformation ability, insufficient lubricity and stability, and cannot meet the requirements as a substitute for semi-solid fats. Summary of the Invention

[0007] This invention aims to address the aforementioned bottlenecks by introducing a process combining phase separation, ultrasound, and thermal induction to construct composite microgels with small particle size, high potential, and suitable contact angle. This enables the adjustment of rheological properties and the improvement of lubricity, providing technical support for the structural optimization of low-fat foods.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A protein-polysaccharide composite microgel, wherein the composite microgel is prepared from soy protein isolate and high-acyl gellan gum through a synergistic process of phase separation, ultrasound and thermal induction.

[0010] The composite microgel has a particle size of 300-400 nm and an absolute value of ζ-potential greater than 30 mV; the water contact angle of the composite microgel is about 72° and the oil-water-solid three-phase contact angle is about 50°.

[0011] The preparation method of the protein-polysaccharide composite microgel includes the following steps:

[0012] (1) Dissolve soy protein isolate and high acyl gellan gum in 10-20 mmol / L PBS buffer (pH 6.8-7.2) and let stand overnight at 2-8°C;

[0013] (2) Add the acyl gellan gum solution to the soy protein isolate solution according to the final mass concentration of 3%-5% protein and 0.1%-0.4% polysaccharide, stir evenly, adjust the pH to 6.8-7.2, and let stand at room temperature for 30 minutes until a phase separation structure is formed;

[0014] (3) The phase separation system is ultrasonically treated with 200-400W for 10-20 minutes in pulse mode (on for 4 seconds, off for 2 seconds), and then heat-treated at 85-95℃ for 10-20 minutes. After cooling, the composite microgel dispersion is obtained.

[0015] (4) The obtained dispersion was centrifuged at 2-8℃ for 20-40 min under the condition of 8000-12000×g, and the precipitate was collected as the composite microgel; the soybean protein isolate-high acyl gellan gum composite microgel powder was obtained by freeze drying.

[0016] Furthermore, the application of the protein-polysaccharide composite microgel in low-fat foods.

[0017] Furthermore, the application of the protein-polysaccharide composite microgel in margarine.

[0018] Furthermore, the application of the protein-polysaccharide composite microgel in low-fat mayonnaise.

[0019] Furthermore, the application of the protein-polysaccharide composite microgel in plant-based meat sauce.

[0020] The beneficial effects of this invention are as follows:

[0021] (1) This invention uses soy protein isolate and high acyl gellan gum as raw materials, both of which are edible and natural, and conform to the trend of clean label and healthy consumption.

[0022] (2) It is prepared by a combined process of "phase separation-ultrasound-thermal induction". The process conditions are mild and the operation is controllable, which has good prospects for industrial application.

[0023] (3) The composite microgel of the present invention has the characteristics of small particle size, good dispersibility, strong amphiphilicity and good plasticity. It realizes the synergistic control of multiple parameters such as microgel particle size, surface properties and rheological behavior. It can be widely used in food systems such as low-fat sauces and margarine, and provides a theoretical basis and practical paradigm for future customized fat replacement systems with different food textures. Attached Figure Description

[0024] Figure 1 The microgel particle size (a), PDI (b), ζ-potential (c), and turbidity (d) are as described in the specific embodiments.

[0025] Figure 2The water contact angle (a), three-phase contact angle (b), and CLSM image (c) are shown in the specific embodiment.

[0026] Figure 3 The FTIR (a), fluorescence spectroscopy (b), UV-Vis analysis (c), and surface hydrophobicity (d) are described in the specific embodiments.

[0027] Figure 4 The small amplitude rheological properties described in the specific implementation are (a: frequency scanning, b: steady-state shear).

[0028] Figure 5 The strain scan (a) and loss tangent (b) are described in the specific implementation.

[0029] Figure 6 The elastic lissajous curve described in the specific implementation.

[0030] Figure 7 The viscous lissajous curve is described in the specific implementation method.

[0031] Figure 8 The particle size, potential, and turbidity of the Pickering emulsion described in the specific embodiments are given.

[0032] Figure 9 The water-holding capacity of the Pickering emulsion described in the specific embodiment.

[0033] Figure 10 The images show the morphology and optical microscope images of the Pickering emulsion described in the specific embodiments.

[0034] Figure 11 This is a CLSM diagram of the Pickering emulsion described in the specific embodiment.

[0035] Figure 12 The frequency scan (a), steady-state shear (b), strain scan (c), and loss tangent (d) of the Pickering emulsion described in the specific embodiment are shown. Detailed Implementation

[0036] To explain in detail the technical content, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments and accompanying drawings.

[0037] Example 1: Preparation of SPI-HG composite microgels

[0038] Preparation of 1SPI-HG composite microgels

[0039] SPI and HG were dispersed in 10 mM phosphate-buffered saline (PBS) and magnetically stirred for 2 h at room temperature until completely dissolved to prepare SPI (6% w / w) and HG (1.2% w / w) stock solutions, respectively. The pH was adjusted to 7.0 with 1.0 M and 0.1 M HCl, and stored at 4 °C.

[0040] The SPI and HG solutions were mixed to achieve a final protein concentration of 4% (w / w) and HG concentrations of 0%, 0.05%, 0.1%, 0.2%, and 0.4% (w / w). The treated mixture was then sonicated at 300W for 15 minutes using an ultrasonic cell disruptor (SCIENTZ-950E) with a probe (6mm diameter) inserted 1-2 cm deep. The pulse duration was 4 seconds followed by a 2-second pause, while maintaining the system temperature below 25°C using a circulating water bath. The mixture was then heated in a 90°C water bath for 15 minutes, cooled to room temperature in an ice bath, and allowed to stand at 4°C for 12 hours to obtain an SPI-HG microgel dispersion. The dispersion was centrifuged at 10,000 × g at 4°C for 30 minutes. The precipitate was collected as the composite microgel. A portion of the wet microgel was stored at 4°C for further characterization, while the other portion was freeze-dried at -50°C for 48 hours to obtain the SPI-HG composite microgel powder.

[0041] 2. Basic Physicochemical Characteristics

[0042] 2.1 Particle size and zeta potential measurement

[0043] The average particle size and zeta potential of the microgels were determined using a Mastersizer 3000 laser particle size analyzer (Malvern Instruments Ltd., Worchester, UK). The test temperature was 25℃, and the sample concentration was 0.1 mg / ml. Each sample was measured in triplicate, and the results were taken as the average ± standard deviation.

[0044] 2.2 Turbidity Measurement

[0045] The protein sample solution was diluted to a concentration of 0.5 mg / mL, and the absorbance of the sample was measured at 600 nm using a UV-1800 spectrophotometer. Simultaneously, an SPI solution of the same concentration was used as a control, and a deionized water sample was used as a blank control. Turbidity was expressed as a percentage of OD600 value relative to the SPI control.

[0046] 2.3 Contact Angle and Three-Phase Contact Angle

[0047] The contact angle was determined using an optical contact angle measurement device (OCA25) equipped with a high-speed camera. The freeze-dried SPI-HG sample was pressed into discs. For three-phase contact angle determination, the pressed sample was first soaked in soybean oil for 10 min. Then, a drop of deionized water (2 μL) was placed on the disc surface, the contact surface was imaged using the high-speed camera, and θ was calculated using the Young-Laplace equation.

[0048] 2.4 Laser Confocal Microscope (CLSM)

[0049] The microstructure of the samples was observed using a laser confocal microscope (LSM 880). Samples were treated with Nile blue (0.1 wt%, w / v, isopropanol) and 5-aminofluorescein (0.1 wt%, w / v, dimethyl sulfoxide) to stain proteins and polysaccharides, respectively. The samples were placed on glass slides, covered with coverslips, and observed using a 40× objective lens.

[0050] 3. Spectral characteristics

[0051] 3.1 Fourier Transform Infrared Spectroscopy (FTIR)

[0052] The lyophilized sample was mixed with KBr (1:100, w / w) and ground into a fine powder. The mixture was then formulated into tablets and measured using a Fourier transform infrared spectroscopy (FTIR) instrument (Nicolet iS10, Thermo Fisher, USA). The infrared spectrum was scanned in the range of 4000–500 cm⁻¹. -1 The resolution is 4cm. -1 And 32 scans, with background collected after each sample scan.

[0053] 3.2 Fluorescence spectrum

[0054] Fluorescence spectra were performed using an RF-6000 fluorescence spectrometer. All samples were diluted with 10 mmol / L PBS (pH 7.0) to obtain a protein concentration of 1 mg / mL. Emission spectra (300–450 nm) were recorded at an excitation wavelength of 290 nm with a scan rate of 600 nm / min.

[0055] 3.3 Ultraviolet Spectrum

[0056] UV spectroscopy was performed using an Evolution Pro UV spectrophotometer. All samples were diluted with 10 mmol / L PBS (pH 7.0) to obtain a protein concentration of 1 mg / ml. Absorbance was collected in the wavelength range of 220 nm–500 nm.

[0057] 3.4 Surface hydrophobicity

[0058] Surface hydrophobicity was determined using an RF-6000 fluorescence spectrometer. In short, SPI-HG lyophilized samples were dissolved in 10 mmol / L PBS (pH 7.0) to obtain a 1 mg / mL protein solution, which was then diluted to different concentrations (0.05, 0.1, 0.25, 0.5, and 1 mg / mL). Each diluted SPI solution (8 mL) was mixed with 40 μL of 8.0 mmol / L ANS solution (using ANS in PBS as a blank), and reacted in the dark for 15 min. The fluorescence intensity was then measured at an excitation wavelength of 390 nm and an emission wavelength of 470 nm (scanning speed 600 nm / min, slit width 5 nm). The fluorescence intensity showed a linear relationship with protein concentration, and the slope of this curve was the surface hydrophobicity index H0.

[0059] 4. Rheological properties

[0060] 4.1 Frequency Scan

[0061] Frequency sweep tests were performed using a TA rheometer (Discovery HR10) on a flat plate (40 mm in diameter, 1000 μm gap) at 25 °C, within the linear viscoelastic (LVR) region, with a strain of 0.1% and an angular frequency range between 0.1 and 100 rad / s.

[0062] 4.2 Steady-state shear

[0063] At 25°C, by measuring 1 second -1 up to 100s -1 The apparent viscosity at the shear rate was used to obtain the flow curve.

[0064] 4.3 Strain Scanning Test

[0065] The strain scanning test was performed at a constant frequency of 1 Hz, at a temperature of 25 ℃, with a strain range of 0.1%-1000%.

[0066] 4.4 Lissajous Curve

[0067] Stress-strain or stress-strain rate plots are used to characterize the changes in a specimen under a strain cycle. The entire curve of the plot forms a dense loop, called a Lissajous curve. Further analysis of the Lissajous plot shows that the area of ​​the Lissajous curve represents the energy consumption per unit volume per cycle; a larger area indicates more energy consumption.

[0068] 5 Results

[0069] 5.1 Effect of HG concentration on the properties of SPI-HG

[0070] like Figure 1As shown in Figure a, as the HG content increased from 0% to 0.2%, the average size of the microgels decreased significantly from 837.9 nm to 352.6 nm (Table 1). This is attributed to a phase separation mechanism, where the addition of HG slowed the growth of the SPI-enriched region before gelation, resulting in smaller particles. When the HG concentration was further increased to 0.4%, the particle size rose again to 860.1 nm, as excess unbound HG molecules induced depletion flocculation, leading to aggregation.

[0071] The polydispersity index (PDI) reflects the uniformity of particle size distribution: a PDI > 0.5 indicates a wide size distribution, while a PDI < 0.5 indicates relatively uniform particles. In this study, the PDI reached its minimum value of 0.49 ± 0.11 at 0.2% HG. Figure 1 b) indicates that the particle size is most uniform at this concentration.

[0072] Table 1. Particle size, PDI, ζ-potential, and turbidity of composite microgels at different HG concentrations.

[0073]

[0074] Note: All experimental results are expressed as mean ± standard deviation. Different lowercase letters in the same column indicate significant differences (p<0.05).

[0075] Figure 1 c shows the zeta potential of the SPI-HG microgel. The negative zeta potential indicates that electrostatic repulsion plays a dominant role in the system. As the HG concentration increases from 0% to 0.4%, the |ζ| value increases from 17.73 ± 0.60 mV to 37.70 ± 0.36 mV (Table 1), indicating enhanced electrostatic interaction. The larger absolute zeta potential implies stronger repulsive forces between particles; at high HG levels, strong electrostatic repulsion and thermodynamic incompatibility make the system more stable.

[0076] The turbidity of SPI-HG microgels is as follows Figure 1 As shown in Figure d, turbidity is an indicator of the degree of protein aggregation. A decrease in turbidity indicates dispersion of the composite microgel, while an increase in turbidity indicates aggregation of the complex. With increasing HG concentration, turbidity decreased from 22% to 5% (Table 1). This is because the physical force generated by ultrasonic treatment breaks down protein particles in the solution system, leading to greater dispersion of the composite microgel.

[0077] Therefore, at 0.2% HG, the microgels are smaller, more uniformly distributed, and carry a higher charge, making the system more stable. These changes in properties suggest that the interfacial properties and rheological behavior of the microgels will also be affected.

[0078] 5.2 Contact Angle and Laser Confocal Microscopy Analysis

[0079] Figure 2 The contact angle (θ) reflects surface wettability: θ < 90° indicates a hydrophilic surface, while θ > 90° indicates a hydrophobic surface. As the HG content increases to 0.2%, θ decreases from 104.15° to 72.67°, indicating a significant increase in hydrophilicity; the SPI-HG microgel with an HG content of 0.2% is the most hydrophilic and exhibits the best wettability.

[0080] Figure 2 b. The three-phase contact angles of the SPI-HG microgels were measured. All samples exhibited three-phase contact angles (oil-water-solid) less than 90°, indicating that the microgel particles are mainly hydrophilic at the interface. As HG increased from 0% to 0.2%, the oil-water contact angle decreased from 75.72° to 50.65°, indicating enhanced hydrophilicity and improved adsorption performance of the particles at the oil-water interface. This increased hydrophilicity provides greater steric hindrance, preventing aggregation or flocculation, thereby improving the stability of the microgel. However, when HG reached 0.4%, the oil-water contact angle increased to 62.73°, possibly due to reduced microgel shrinkage caused by excessive HG, thus relatively increasing the exposure of the hydrophobic surface region.

[0081] Confocal microscope images ( Figure 2 c) The microgels exhibit bright yellow fluorescence (originating from the superimposed color development of protein and polysaccharide dyes). Compared to the absence of HG, higher HG content resulted in smaller and more uniformly distributed microgel particles in the SPI-enriched phase. The addition of HG may have enhanced the electrostatic repulsion effect and steric stabilization between particles. Confocal microscopy results confirmed that the microgels containing 0.2% HG had the smallest size, which is consistent with the particle size distribution data. The 0.2% HG sample exhibited the smallest and most uniform particle size, the highest zeta potential, and the lowest contact angle, indicating that the physical stability and amphiphilicity of the microgels were optimal at this concentration.

[0082] 5.3 The impact of HG on SPI structure

[0083] FTIR was used to assess the stretching of chemical bonds and conformational changes in the structure, such as... Figure 3 As shown in Figure a, all the spectra are almost identical, indicating that the incorporation of HG does not induce additional covalent bonds in the SPI-HG complex. (At 3300-3500 cm⁻¹) -1 The position represents the stretching and bending vibrations of the -OH group in amide A, 1500-1700 cm. -1 This is attributed to the stretching or bending of C=O and NH in amide I and amide II. When the HG concentration increases to 0.2%, the amide A band peak of the SPI-HG microgel shifts from 3369.53 cm⁻¹. -1 Offset to 3406.18cm -1Furthermore, the vibrational band broadens, indicating hydrogen bonding and electrostatic interactions between SPI and HG. The amide I band peak increases from 1636.77 cm⁻¹. -1 Moved to 1625.93cm -1 The peak value of the amide II band is from 1539.82 cm⁻¹. -1 Moved to 1531.43cm -1 This suggests that there may be hydrophobic interactions between SPI and HG.

[0084] Fluorescence spectra such as Figure 3 As shown in b, tryptophan, tyrosine, and phenylalanine residues in the protein exhibit intrinsic fluorescence, which can be used to detect conformational changes in the protein. With the addition of the anionic polysaccharide HG, the fluorescence intensity of SPI gradually decreased (except at 0.4% HG), indicating an interaction and complex formation between SPI and HG. Strong SPI-HG interaction led to fluorescence quenching; indeed, as HG increased from 0.05% to 0.2%, the protein fluorescence was gradually quenched. At 0.2% HG, quenching reached its maximum, indicating that HG bound to the most SPI sites, resulting in the burial of hydrophobic aromatic residues within the protein structure after heating. However, when HG was further increased to 0.4%, the fluorescence intensity increased again. This rebound may be due to the excess HG promoting the formation of SPI-rich regions, leading to protein aggregation and gelation (reducing quenching efficiency). Combining the FTIR and fluorescence results, it can be inferred that HG mainly binds to SPI through non-covalent interactions (hydrogen bonds, electrostatic attraction, etc.), without disrupting the chemical structure of the SPI backbone, but affecting the protein's spatial conformation and surface properties.

[0085] In the ultraviolet spectrum ( Figure 3 In c), the SPI-HG microgel exhibits characteristic absorption in the 250-300 nm range, attributed to the aromatic rings of tryptophan and tyrosine. Absorbance near 270 nm increases with increasing HG concentration, primarily due to π-π* transitions in the C=O groups of the peptide bonds. This indicates that SPI-HG interactions alter protein conformation by promoting intermolecular and intramolecular aggregation, exposing more aromatic residues (tryptophan, tyrosine). The presence of HG may lead to partial unfolding (chain extension) of the SPI, exposing hydrophobic groups and thus promoting stronger intermolecular hydrophobic interactions and aggregation.

[0086] Figure 3Figure d shows the surface hydrophobicity (H0) of the SPI-HG microgel, representing the relative abundance of hydrophobic groups on the protein surface (a key indicator of protein denaturation). The addition of HG reduces the surface hydrophobicity of SPI. With increasing HG concentration, H0 first decreases and then increases, reaching a minimum (5123.05) at 0.2% HG. This minimum H0 corresponds to the maximum surface wettability, consistent with the aforementioned contact angle wettability results. Tyrosine residues are typically buried in the hydrophobic core of proteins, exposed when SPI interacts with HG, and HG introduces new hydrophilic groups (hydroxyl, carboxyl groups), thereby reducing surface hydrophobicity.

[0087] These spectroscopic analyses consistently demonstrate that HG interacts with SPI through hydrogen bonding, electrostatic and hydrophobic interactions, leading to changes in the secondary / tertiary structure of SPI (e.g., buried hydrophobic groups are exposed at moderate HG levels, while they are reburied or aggregated at higher HG levels).

[0088] 5.4 Small Amplitude Oscillation Shear (SAOS)

[0089] Figure 4 The results of the frequency scan are presented. Storage modulus (G') and loss modulus (G”) increase with increasing angular frequency, indicating frequency-sensitive viscoelastic behavior. As HG increases from 0% to 0.2%, both G' and G” values ​​decrease, meaning the microgel becomes more liquid (less rigid and more easily deformable). Lower G' indicates a less rigid network structure (greater compliance), while lower G” implies less viscous dissipation, thus the material behaves closer to an ideal elastic solid. When HG is further increased to 0.4%, the modulus increases slightly compared to 0.2% (but remains below the value at 0% HG), likely due to some polysaccharide aggregation caused by exceeding the optimal HG ratio, partially restoring rigidity.

[0090] Figure 4 b shows the apparent viscosity of the SPI-HA microgels as a function of shear rate. All samples exhibited non-Newtonian (pseudoplastic) behavior: viscosity decreased with increasing shear rate (shear thinning). Higher HA content resulted in a decrease in overall viscosity. This suggests that the addition of gellan gum (especially at high contents) weakens the interactions and network formation in the SPI-HA system, leading to a mixed dispersion with a weaker molecular network.

[0091] Small strain tests identified 0.2% HG as the most flexible microgel, while higher concentrations (0.4%) partially reversed this softening due to network perturbations. It was crucial to evaluate this property under large deformations, which was addressed using LAOS analysis. LAOS results showed that the addition of HG reduced the strength of the microgel network, consistent with the aforementioned smaller, softer structure induced by HG. This improved network compliance suggests that the microgel may possess better plasticity under large deformations.

[0092] 5.5 Large Amplitude Oscillation Shear (LAOS)

[0093] 5.5.1 Strain Scan

[0094] Figure 5 A strain scan is presented. In the low strain region, G' and G” remain almost constant (linear viscoelastic behavior). Above approximately 100% strain, both moduli decrease significantly, indicating the onset of nonlinear behavior. As HG increases from 0% to 0.2%, the values ​​of G' and G” in the linear region are lower (consistent with the frequency scan results), and they decrease more rapidly with strain. After G” crosses over G', the gel exhibits liquid behavior at high strain. Loss tangent (tanδ) ( Figure 5 b) further illustrates this transition: when tanδ>1, viscous behavior dominates. tanδ exceeds 1 at approximately 40% strain (the gel point), indicating that beyond this strain, elasticity weakens, viscosity dominates, and gel yielding (plastic or permanent deformation) occurs.

[0095] 5.5.2 Lissajous Curve

[0096] Figure 6 The Lissajous diagram showing shear stress (σ) versus strain (γ) over a period is called the elastic Lissajous diagram. The closed region of the elastic Lissajous diagram is proportional to the dissipated energy and G”. When γ = 1%, the elastic Lissajous curve is almost a straight line, showing a perfect elastic response and low energy dissipation.

[0097] When γ = 20%, all elastic Lissajous plots show extremely narrow ellipses, and σ remains a straight line. The Lissajous plots at low HG concentrations are still very narrow, but as the HG concentration increases, the area of ​​the SPI-HG gel curve gradually increases, showing a significantly wider ellipse. This is because the addition of HG shortens the linear viscoelastic region, causing the microgel sample to enter the nonlinear viscoelastic region more quickly.

[0098] When γ = 100%, the area of ​​the ellipse in the microgel sample increases significantly, exhibiting a more rounded "rugby ball" shape, indicating that all samples have fully entered the nonlinear viscoelastic region. When γ = 350%, the area of ​​the curve continues to increase, exhibiting a larger "parallelogram" shape. σ also changes from a straight line to an "S" shape, meaning the slope of the stress curve gradually decreases, and the energy dissipation of the curve increases significantly.

[0099] As γ increases to 550%, the Lissajous curves begin to distort due to the gradual softening of the material. At a maximum strain of 750%, the loops become nearly rectangular, indicating that all samples have transitioned to near-plastic behavior and lost elastic recovery. The elastic Lissajous curves under large deformations are all elliptical, with the ellipse area increasing, indicating that increased strain leads to energy dissipation, and increased HG concentration also leads to increased dissipation of mechanical energy. After strain exceeds 20%, the slope of the stress curve gradually decreases with increasing strain. This hysteresis in stress change exhibits liquid-like characteristics. In summary, the addition of HG increases the degree of deformation of the elastic Lissajous curves at high strain but reduces the degree of shape distortion, indicating a weakening of the system's nonlinear behavior and a more compliant deformation.

[0100] Accordingly, from the viscous Lissajous curve ( Figure 7 A similar trend can be observed: at γ = 1% and 20%, the rings exhibit a flattened elliptical shape (“egg” shape). When γ = 100%, the rings shrink into an “olive” shape with a reduced area, indicating shear thinning due to partial network breakage. For strains exceeding 100%, the rings become distorted, exhibiting self-intersecting figure-eight shapes (similar to knotted candies), suggesting very rapid microstructure rearrangement (faster than the deformation timescale). As HG concentration increases, the viscous rings tend to straighten, implying a decrease in viscous stress response—consistent with liquid behavior and observations of elastic rings.

[0101] The above results indicate that 0.2% HG can significantly improve the flow-remodeling ability of composite microgels in high-shear processing while maintaining particle size uniformity and system stability, making it a semi-solid fat substitute with tunable performance.

[0102] Example 2: Application of composite microgels in plant-based emulsions

[0103] 1. Preparation of Pickering emulsion stabilized by composite microgel

[0104] The SPI solution and HG solution were mixed to achieve a final protein concentration of 4% (w / w) and a polysaccharide concentration of 0.2% (w / w). The mixture was then subjected to ultrasonic cell disruption using a titanium probe (6 mm in diameter) immersed to a depth of 1-2 cm below the liquid surface. Ultrasonic treatment was then performed for 15 min at output powers of 0, 100, 200, 300, and 400 W (pulse duration 4 s, off for 2 s), with ice used throughout the process to maintain the system temperature at 25°C. The mixture was then heated in a water bath at 90°C for 15 min. Afterward, the sample was cooled to room temperature in an ice bath and refrigerated overnight to obtain a microgel dispersion.

[0105] 12 mL of SPI-HG microgel dispersion was mixed with 3 mL of soybean oil and homogenized using a high-speed disperser at 12,000 r / min for 3 min to prepare a Pickering emulsion.

[0106] 2 Performance Characterization

[0107] 2.1 Particle size and zeta potential

[0108] The droplet size of different emulsions was measured at room temperature using a Baxter particle size analyzer (BT-9000ST, Liaoning, China). The spectral range of droplet size was 10%–20%, and the particle size distribution of the emulsions was recorded. All measurements were performed in triplicate, and the average value was reported.

[0109] The emulsion was diluted to 1 mg / ml at 25 °C using a Mastersizer 3000 (Malvern Instruments Ltd., Worchester, UK), and the zeta potential was measured by dynamic light scattering. All measurements were performed in triplicate, and the average value was reported.

[0110] 2.2 Turbidity

[0111] The concentration of a 1 mg / mL sample was measured at 400 nm using a UV spectrophotometer. The resulting optical density (OD) value was recorded and used to indicate turbidity. Three copies of each sample were measured.

[0112] 2.3 Water Holding Capacity (WHC)

[0113] Weigh 3g of the emulsion gel and place it in a 10ml centrifuge tube for centrifugation (6000g, 10 minutes, 4℃). Remove the water discharged during centrifugation.

[0114] WHC = m2 / m1 × 100%

[0115] Where m2(g) is the weight after centrifugation to remove water; m1(g) is the initial weight.

[0116] 2.4 Microstructure

[0117] The morphology of the emulsion droplets was observed using an optical microscope equipped with a 40× objective lens. Measurements were taken immediately after emulsion preparation to avoid the influence of oil droplet polymerization.

[0118] A confocal laser scanning microscope (CLSM) was used to observe oil droplets in the emulsion (63× oil immersion). The excitation wavelengths for oil (green) and protein (red) were 488 nm and 633 nm, respectively.

[0119] 2.5 Rheological Measurement

[0120] Shear rate scan test: apparent viscosity in the range of 0.1-100 s⁻¹ -1 As a function of shear rate.

[0121] Frequency sweep test: The sample was subjected to a frequency sweep test at a constant strain of 0.1% in the linear viscoelastic region (LVR) to record G' and G” as a function of the sweep frequency from 0.1 to 100 Hz.

[0122] Strain scanning test: At a constant frequency of 1 Hz, record G' and G” as a function of 0.1-1000% scan strain.

[0123] 3 Results

[0124] 3.1 Particle size, zeta potential, and turbidity

[0125] Figure 8 As shown in the particle size distribution diagram, at an ultrasonic power of 300W, the distribution is concentrated in the small to medium particle size range, with the average particle size reaching a minimum of 8.498μm. In the control group (without ultrasonication), the particle size is mostly concentrated around 219μm, indicating a larger particle size. Increasing the ultrasonic power from 100W to 300W clearly shows a shift in particle size distribution to the left, i.e., towards smaller particle sizes. This is because low-power ultrasound is beneficial for reducing the size of emulsion droplets, resulting in a more uniform droplet distribution. However, when the ultrasonic power continues to increase, from 300W to 600W, the particle size actually increases to 20.340μm. This may be because excessively high ultrasonic power leads to enhanced cavitation, and the resulting shock waves and turbulent currents alter the molecular structure of the emulsion droplet surface, causing excessive droplet breakup and aggregation, thus widening the droplet size distribution.

[0126] Figure 8The change in emulsion potential also confirms the results regarding particle size. Ultrasonic power affects the viscosity, dielectric constant, and other properties of the emulsion, causing changes in the emulsion potential. Low-power ultrasound (300W) increases the specific surface area of ​​the droplets, resulting in a relative increase in the number of charges per unit area, and the absolute value of the emulsion potential increases to 41.567mV. When the ultrasonic power exceeds 300W, the absolute value of the emulsion potential begins to decrease, reaching a minimum of 38.267mV at an ultrasonic power of 600W. Excessively high ultrasonic power widens the droplet size distribution of the emulsion, and the potential distribution also becomes uneven.

[0127] An appropriate increase in ultrasonic power will reduce the turbidity of the emulsion, such as... Figure 8 As shown in Figure c, the turbidity of the untreated emulsion was relatively high. When the ultrasonic power increased from 100W to 300W, the turbidity decreased significantly because the cavitation and mechanical effects of ultrasound broke down larger emulsion droplets, dispersing them into finer particles. The particle size results also confirmed this; the reduction in droplet size altered the light scattering efficiency, decreasing light scattering and absorption, thus reducing emulsion turbidity. When the ultrasonic power continued to increase to 600W, the emulsion turbidity began to rise. This is because excessive ultrasonic power generates excessive cavitation, leading to localized overheating and droplet aggregation in the emulsion system. Droplet aggregation increases the particle size, altering the dispersion state of the emulsion particles. It may also disrupt the stability of the emulsion, exposing more particles to light, increasing light scattering and absorption, and consequently increasing emulsion turbidity.

[0128] 3.2 Water Holding Capacity (WHC)

[0129] Figure 9 The water-holding capacity of emulsions is affected by different ultrasonic powers. Low ultrasonic power (100W) has little effect on the water-holding capacity of emulsions. At this level, the ultrasonic energy is low and has little effect on the internal structure and properties of the emulsion. The water in the emulsion is mainly retained in the system through physical interactions between emulsion particles, and the water-holding capacity is relatively stable.

[0130] Moderate ultrasonic power (300W) improved the water-holding capacity of the emulsion, increasing it from 82.333% (0W) to 90.333% (300W). This is because moderate ultrasonic power generates cavitation and mechanical agitation, reducing the droplet size and making the distribution more uniform, thus increasing the specific surface area of ​​the emulsion. This facilitates the interaction between emulsion particles and water, allowing more water to be adsorbed or encapsulated around the emulsion particles, thereby improving the water-holding capacity. Simultaneously, the ultrasonic effect may also cause moderate denaturation of components such as proteins in the emulsion, exposing more hydrophilic groups, further enhancing the adsorption capacity for water and improving water retention.

[0131] High ultrasonic power (500W) reduced the water-holding capacity of the emulsion from 90.333% (300W) to 88.000% (500W). Excessive ultrasonic power subjectes the emulsion system to excessive mechanical and thermal effects. On one hand, it may lead to excessive fragmentation and aggregation of emulsion particles, disrupting the stable structure of the emulsion and releasing previously encapsulated water, thus reducing water-holding capacity. On the other hand, the heat generated by excessive ultrasonic power may cause water evaporation from the emulsion, also leading to a decrease in water-holding capacity. Furthermore, high ultrasonic power may also cause excessive denaturation of components such as proteins, severely disrupting their spatial structure and reducing the activity of hydrophilic groups, thereby weakening their ability to adsorb water and affecting the water-holding capacity of the emulsion.

[0132] When the ultrasonic power was further increased to 600W, the water-holding capacity of the emulsion began to increase again, showing no significant difference compared to 300W. This may be because the proteins and other components in the emulsion form a three-dimensional structure that can bind water under high-power ultrasound, which is beneficial for water retention. When both methods achieve the same water-holding capacity, using 300W ultrasonic power can reduce production costs, decrease energy consumption, and is more energy-efficient and environmentally friendly. Furthermore, the emulsion particle size and turbidity at 600W ultrasonic power are larger than those at 300W, and other adverse changes may occur in the emulsion under high ultrasonic power, such as excessive protein denaturation, affecting emulsion quality.

[0133] 3.3 Microstructure

[0134] like Figure 10 As shown, the oil droplets in the non-ultrasonic group were large and unevenly distributed, exhibiting poor stability. In the 100W group, the oil droplets became smaller, although some large particles remained; the distribution became slightly more uniform, cavitation began to appear, and the emulsification effect improved. In the 200W group, the oil droplet size further decreased; the distribution was significantly more uniform; showing a better emulsification effect. In the 300W group, the particle size became finer and the distribution narrower; there were almost no large oil droplets, and the emulsion structure was delicate; reaching a relatively optimal emulsification state. In the 400W group, some aggregation tendency began to appear, and ultrasonic cavitation may have slightly exceeded the optimal range. In the 500W group, some areas showed aggregation boundaries; indicating that excessive ultrasonic energy may lead to oil droplet re-aggregation or damage to the emulsion structure. In the 600W group, oil droplet aggregation intensified, forming clumps or rupture; high power led to emulsion structure damage and decreased stability.

[0135] Laser confocal microscopy of emulsions, such as Figure 11As shown, in the 0W group, the red and green fluorescent patches are large and dispersed; the green oil droplets are large in diameter and unevenly distributed; the red-green boundary in the synthesis diagram is blurred, indicating poor emulsification; there are obvious aggregation and stratification phenomena. In the 100W-200W group, the oil droplet size decreases and the number increases; red fluorescence surrounds some green oil droplets, indicating that the emulsifier gradually covers the surface of the oil droplets; a clear interface is formed in the synthesis diagram; the distribution becomes more uniform, and the cavitation effect begins to appear. In the 300W group, the oil droplets become smaller and the distribution is extremely uniform; in the synthesis diagram, the green droplets are completely wrapped by the red film, forming a typical "core-shell structure"; the red and green are distinct, and the structure is stable; the optimal emulsification range: the emulsion structure is dense, the oil droplets are small, and the emulsifier is well coated. In the 400W-600W group, the oil droplet size increases slightly, and some show a tendency to aggregate; the red fluorescence becomes blurred or broken, indicating that the interface structure may be damaged; the oil droplet boundary in the synthesis diagram is unclear, and there is an overlap phenomenon; excessive sonication may cause oil droplet breakage or emulsifier desorption, and the emulsion stability decreases.

[0136] 3.4 Rheological Analysis

[0137] Frequency scan results as follows Figure 12 As shown in a, G′>G″ holds true across the entire frequency range, indicating that the sample exhibits elastic dominance. As ultrasonic power increases, both G′ and G″ increase overall, suggesting that ultrasound enhances the degree of the "cross-linked network" of the microstructure and improves viscoelastic properties. 0-300W: In the structural enhancement stage, ultrasonic cavitation makes the oil droplets finer and more evenly distributed; at the same time, it promotes the rapid adsorption of proteins or emulsifiers on the surface of the oil droplets, forming a dense interface; the microparticle or network structure is enhanced, and G′ increases accordingly, resulting in enhanced elasticity. 400-500W: Structural disruption stage. Excessive cavitation leads to the depolymerization of emulsifier proteins and localized peeling of the interfacial stabilizing layer. Excessive collisions, aggregation, and even structural breakage may occur between oil droplets. The microstructure is partially destroyed, G′ and G″ decrease, and energy storage capacity weakens. 600W: Structural reorganization or particle re-aggregation stage. Secondary structural reorganization occurs, and high power induces protein recombination, forming new aggregate states. Large particles aggregate to form new networks: broken oil droplets aggregate to form coarse clumps or weak gel structures, which enhance G′ at higher frequencies. Strong ultrasound induces some proteins to aggregate into three-dimensional networks, increasing modulus at low or medium frequencies. However, these structures are usually coarse, low-stability "pseudo-network" structures, not the stable network of the previously fine emulsion. Ultrasound power from 0 to 300W helps network construction, 400-500W leads to structural disruption, while 600W may cause secondary networks due to aggregation or recombination, resulting in a "rebound" in modulus.

[0138] Steady-state shear results are as follows Figure 12As shown in b, all samples exhibit typical shear-thinning behavior: the higher the shear rate, the lower the viscosity, indicating that the sample has a certain structural network that is gradually destroyed during shearing, resulting in increased fluidity. The 0W sample has the lowest viscosity, a loose structure, and large oil droplets. The 300W sample has higher viscosity: the oil droplet size decreases, the specific surface area increases, protein adsorption is more complete, and the interface is more compact. This indicates that the emulsion network structure is most complete and the oil droplets are the finest, resisting flow. When the ultrasonic power is 400W-500W, the viscosity decreases significantly, the oil droplets coalesce, the number of particles decreases, and the overall specific surface area decreases. Network breakage and particle aggregation cause a decrease in internal fluid resistance, manifested as a decrease in apparent viscosity. At 600W, the viscosity begins to increase again, possibly due to the increased temperature of the overheated reaction. Oil droplet aggregation forms a flocculent large-particle network, and the hydrophobic aggregation between proteins is enhanced, resulting in a "weak gel structure." High-energy input induces more non-covalent aggregation behaviors (such as hydrogen bonding and hydrophobic interactions). Although not a high-quality network, these large-scale structures can still provide flow resistance, causing a slight increase in viscosity. The emulsion viscosity was optimal at medium power (300W) and then decreased due to structural disruption, but slightly increased at higher power (600W) due to the inefficient network structure formed by particle aggregation.

[0139] Figure 12 c shows the strain response of the emulsion. At 0W (untreated group), G′ and G″ values ​​are lowest, and the LVR region is very narrow, indicating a loose emulsion structure that is easily disrupted by slight strain. G′ falls below G″ very early, exhibiting typical liquid behavior. In the 100-200W treated group, G′ is significantly increased, the LVR region expands, and G′ is significantly higher than G″, indicating an elastic-dominated structure. At 200W, the yield point shifts to the right, indicating structural enhancement and improved resistance to deformation. At 300W (optimal power region), G′ reaches its peak and is significantly higher than G″, with the widest LVR region and the most stable structure; the yield point (crossover point) is at a higher strain, indicating that the sample can resist greater deformation stress. In the 400W treated group, G′ remains high, but a downward trend begins, and the LVR region narrows slightly. Ultrasonic treatment may have begun to damage some of the network structure, leading to a decrease in resistance to deformation. In the 500-600W treatment group, G′ decreased while G″ increased slightly; the G′ / G″ crossover point occurred earlier, and LVR shortened, indicating further structural damage, the formation of a weak network or flocculated structure, and decreased shear resistance; although the viscosity appeared high, stability and elasticity weakened. With increasing ultrasonic power, the linear viscoelastic region (LVR) of the emulsion significantly expanded to 300W, and G′ exceeded G″ over a wide strain range, indicating the formation of a strongly elastic network. Yield strain also increased, reflecting enhanced structural integrity. However, above 400W, G′ began to decrease, LVR shrank, and the crossover point between G′ and G″ shifted to the left, suggesting partial structural damage and reduced mechanical stability due to overtreatment.

[0140] Loss tangent Figure 12 As shown in Figure d, when the strain is less than 100%, G′ is significantly higher than G″ when the ultrasonic power is 100W-200W, and tanδ<1, indicating that the emulsion begins to exhibit weak gel characteristics, with enhanced elasticity, a more stable structure, and improved anti-flow properties. At 300W, G′ is at its maximum, G″ is at its minimum, and tanδ is at its lowest, indicating the strongest elasticity dominance and the densest network structure, representing the strongest and most stable state of the emulsion network. At 400W, G′ begins to decrease, G″ increases slightly, and tanδ increases. This indicates that the emulsion structure begins to break down, elasticity decreases, and viscosity increases; excessive ultrasonic power begins to affect the protein stabilizing layer. At 500-600W, G′ further decreases, G″ continues to increase, and tanδ further increases, indicating weakened elasticity and a looser structure in the system, possibly with oil droplet aggregation and structural disorder.

[0141] The evolution of tanδ during strain scanning further supports the structural transformation of the emulsion. At 300 W, tanδ reaches a minimum, confirming a dominant elastic, well-structured network characterized by a weakly gel-like system. In contrast, higher ultrasonic power (400–600 W) leads to a gradual increase in tanδ, indicating a shift in viscous-dominant behavior due to partial disruption or droplet coalescence. These results are consistent with observed trends in G′, G″, and yield strain.

[0142] The above results demonstrate that the Pickering emulsion prepared using this microgel system can form a fine-particle-size, uniformly structured, well-adsorbed, and stable emulsion system under appropriate ultrasonic power treatment (e.g., 300W). This is mainly attributed to the enhanced directional adsorption and compact arrangement of the protein microgels at the oil-water interface through ultrasonic cavitation, resulting in a dense and elastic interfacial film, thereby significantly improving emulsifying activity and stability. This study provides theoretical support and a technical pathway for constructing a highly stable Pickering emulsion system based on protein microgels. The prepared Pickering emulsion can be widely used in the food industry as a substitute for margarine.

[0143] Example 3: Application of composite microgels in low-fat mayonnaise

[0144] Using SPI-HG composite microgel as the main emulsifying structure agent, 30g of composite microgel, 60g of soybean oil, 5g of white vinegar, 3g of white sugar, and 1g of salt were premixed and then homogenized using a homogenizer (10000rpm, 1min) to prepare low-fat mayonnaise. The product has a moderate consistency, stable structure, good spreadability, and a fat content of approximately 50% of traditional mayonnaise, making it suitable as a healthy alternative.

[0145] Example 4: Application of composite microgels in plant-based meat sauce

[0146] SPI-HG microgels were mixed with puffed vegetable protein particles, seasoning liquid, and vegetable oil in a specific ratio to simulate the fat distribution in animal-based meat sauces. Sensory and textural analyses of the resulting meat sauce sample showed good spreadability and flavor encapsulation, while significantly reducing saturated fat content, making it suitable for vegetarians / low-fat diets.

[0147] It should be noted that although the above embodiments have been described herein, this does not limit the scope of patent protection of the present invention. Therefore, any changes and modifications made to the embodiments described herein based on the innovative concept of the present invention, or equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, directly or indirectly applying the above technical solutions to other related technical fields, are all included within the scope of patent protection of the present invention.

Claims

1. A protein-polysaccharide composite microgel, characterized in that, The composite microgel is prepared from soy protein isolate and high-acyl gellan gum through phase separation, ultrasonication combined with thermal induction process.

2. The protein-polysaccharide composite microgel according to claim 1, characterized in that, The composite microgel has a particle size of 300-400 nm, an absolute value of ζ-potential of 30-40 mV, a water contact angle of 70-75°, and an oil-water-solid three-phase contact angle of 48-52°.

3. A method for preparing the protein-polysaccharide composite microgel as described in claim 1 or 2, characterized in that, The preparation method includes the following steps: Soy protein isolate and high-acyl gellan gum were dissolved separately in 10-20 mmol / L PBS buffer (pH 6.8-7.2) and incubated overnight at 2-8°C. Add the acyl gellan gum solution to the soy protein isolate solution at a final mass concentration of 3%-5% protein and 0.1%-0.4% polysaccharide, stir evenly, adjust the pH to 6.8-7.2, and let stand at room temperature for 30 minutes until a phase separation structure is formed; The phase-separated system was ultrasonically treated at 200-400W for 10-20 minutes in pulse mode, and then heat-treated at 85-95℃ for 10-20 minutes. After cooling to room temperature, the composite microgel dispersion was obtained. The resulting dispersion was centrifuged at 2-8℃ for 20-40 min at 8000-12000×g, and the precipitate was collected as the composite microgel; the soybean protein isolate-high acyl gellan gum composite microgel powder was obtained by freeze drying.

4. The application of a protein-polysaccharide composite microgel as described in any one of claims 1-3 in low-fat foods.

5. The application of the protein-polysaccharide composite microgel according to claim 4 in the preparation of low-fat foods, characterized in that, The low-fat foods mentioned include margarine, low-fat mayonnaise, or plant-based meat sauce.

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