Formation and properties of β-conglycinin particle-Konjac Glucomannan co-stabilized Pickering emulsion: Dynamic adsorption behavior and complex interface microstructure

Formation and properties of β-conglycinin particle-Konjac Glucomannan co-stabilized Pickering emulsion: Dynamic adsorption behavior and complex interface microstructure | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 23 January 2025 V1 Latest version Share on Formation and properties of β-conglycinin particle-Konjac Glucomannan co-stabilized Pickering emulsion: Dynamic adsorption behavior and complex interface microstructure Authors : Yanan Guo , Tianfu Cheng , Shuo Zhang , Haotian Liu 0000-0001-9398-9062 , Ramnarain Ramakrishna , Lianzhou Jiang , Zhongjiang Wang , and Zengwang Guo 0000-0002-3996-7214 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173759969.99359333/v1 Published International Journal of Biological Macromolecules Version of record Peer review timeline 255 views 118 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The interaction between polysaccharides and proteins in co-adsorption played a critical role in determining interfacial properties and emulsion stability. This study focused on the regulation mechanism of different concentrations of polysaccharide on the dynamic adsorption behavior of protein particles at the subunit level, and how the interfacial membrane microstructure affected the stability of emulsion. The research demonstrated that Konjac Glucomannan (KGM) and β-conglycinin (7S) formed stable composite through non-covalent interactions such as hydrogen bonding and hydrophobic interactions. Additionally, low concentrations of KGM promoted the unfolding of 7S structures, enhancing the wettability of the 7S-KGM composite by modulating the hydrophilic-hydrophobic balance. The results from interfacial adsorption kinetics and Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) indicated that KGM could synergistically facilitate the initial diffusion, penetration, and rearrangement of 7S at the oil-water interface thus forming a dense and viscoelastic three-dimensional gel-like multilayer interfacial structure. Notably, high-density adsorption of the 7S-KGM composite at the interface significantly increased the thickness and the protein content of the interfacial layer, enhancing emulsion stability. Finally, The results of dissipative particle dynamics simulations further demonstrated that the synergistic adsorption of KGM enhanced the interfacial adsorption efficiency of 7S, thereby stabilizing the interface more effectively and reducing droplet size. These findings of experiments and simulations provided insights into the mechanisms by which polysaccharides enhanced protein emulsification performance at the subunit level, offering critical guidance for optimizing protein interfacial properties and enhancing emulsion stability. Formation and properties of β-conglycinin particle-Konjac Glucomannan co-stabilized Pickering emulsion: Dynamic adsorption behavior and complex interface microstructure Yanan Guo a,1 , Tianfu Cheng a,1 , Shuo Zhang a , Haotian Liu a , Ramnarain Ramakrishna b , Lianzhou Jiang a,b,c , Zhongjiang Wang a,c* , Zengwang Guo a** a College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang, 150030, China b Department of Plant Sciences, North Dakota State University, Fargo, ND 58108, USA c College of Food Science and Engineering, Hainan University, HaiKou, 570228, China * Corresponding author: College of Food Science, Northeast Agricultural University, Harbin, 150030, China. ** Corresponding author: College of Food Science, Northeast Agricultural University, Harbin, 150030, China. E-mail addresses: [email protected] (Y.N Guo), [email protected] (T.F Cheng), [email protected] (S Zhang), [email protected] (H.T Liu), [email protected] (Ramnarain Ramakrishna), [email protected] (L.Z Jiang), [email protected] (Z.J Wang), [email protected] (Z.W Guo) 1 These authors contributed equally to this work. Keywords: Konjac Glucomannan, β-conglycinin, Co-stabilized emulsion, Interfacial behavior, Interfacial microstructure, Dissipative particle dynamic Abstract: The interaction between polysaccharides and proteins in co-adsorption played a critical role in determining interfacial properties and emulsion stability. This study focused on the regulation mechanism of different concentrations of polysaccharide on the dynamic adsorption behavior of protein particles at the subunit level, and how the interfacial membrane microstructure affected the stability of emulsion. The research demonstrated that Konjac Glucomannan (KGM) and β-conglycinin (7S) formed stable composite through non-covalent interactions such as hydrogen bonding and hydrophobic interactions. Additionally, low concentrations of KGM promoted the unfolding of 7S structures, enhancing the wettability of the 7S-KGM composite by modulating the hydrophilic-hydrophobic balance. The results from interfacial adsorption kinetics and Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) indicated that KGM could synergistically facilitate the initial diffusion, penetration, and rearrangement of 7S at the oil-water interface thus forming a dense and viscoelastic three-dimensional gel-like multilayer interfacial structure. Notably, high-density adsorption of the 7S-KGM composite at the interface significantly increased the thickness and the protein content of the interfacial layer, enhancing emulsion stability. Finally, The results of dissipative particle dynamics simulations further demonstrated that the synergistic adsorption of KGM enhanced the interfacial adsorption efficiency of 7S, thereby stabilizing the interface more effectively and reducing droplet size. These findings of experiments and simulations provided insights into the mechanisms by which polysaccharides enhanced protein emulsification performance at the subunit level, offering critical guidance for optimizing protein interfacial properties and enhancing emulsion stability. 1. Introduction Emulsion is commonly found in various practical food systems and it exhibit thermodynamic instability due to the high free energy of the system. Therefore, emulsifiers are required to facilitate formation and ensure long-term storage stability [1] . Although small molecular surfactants or inorganic particles in traditional emulsions can form compact interface structures, they have low stiffness and high mobility, and their fragile spatial structure makes them difficult to resist droplet deformation [2] . Therefore, during the storage of traditional emulsions, the intricate and dynamic behavior of unstable droplets can easily trigger instability phenomena [3] . Pickering emulsions, also known as particle-stabilized emulsions, utilize colloidal particles for stabilization [4] . These particles can attach to the interface via interactions between particles or between particles and the interface, leading to the formation of diverse interfacial microstructures. Additionally, due to the higher desorption energy of colloidal particles, the interface typically exhibits a greater dilational modulus, which helps enhance resistance to droplet coalescence under complex environmental (such as temperature, pH, and ionic strength) [5] . Therefore, the development of green, safe, and natural food-grade particulate stabilizers has become a critical focus in Pickering emulsion research. As food-grade colloidal particles, proteins possess natural amphiphilic properties, and are widely available, making them one of the most promising candidates for edible emulsifiers in Pickering emulsions. However, single proteins are not ideal stabilizers in practical applications, and their ability to stabilize interfaces is often limited. The polysaccharides can significantly enhance the rheological properties and stability of emulsions by increasing viscosity and providing steric hindrance [6, 7] . Besides, the non-covalent interactions between protein and polysaccharides contribute to enhancing emulsion stability. For example, Li et al. [8] used hydroxypropyl methylcellulose and xanthan gum to form complexes with SPI respectively, effectively improving SPI’s ability to stabilize oil-water interfaces and increasing the elastic behaviour of emulsions. Shi et al. [9] used three polysaccharides (sodium alginate, xanthan gum and arabic gum) to enhance the interaction of droplets in emulsion, thus increasing the viscoelasticity and stability of protein-based emulsion. Ren et al. [10] showed that the interaction between myosin and chitosan / carrageenan could make myosin more hydrophilic, which helped to form emulsion with more evenly distributed droplets. Therefore, polysaccharides can improve the interfacial performance of protein and enhance emulsion stability. However, current research predominantly focuses on the impact of polysaccharides on the rheological properties of protein-based emulsions [11, 12] . The mechanisms by which protein particles and polysaccharides reach the interface, and the detailed progress about interfacial cooperative or competitive adsorption behaviors during emulsion formation, remain largely unknown. Besides, the morphology of composite interfaces has not been thoroughly elucidated. Therefore, the complex dynamic interfacial behavior established by proteins and polysaccharides requires further exploration. Soy Protein Isolate (SPI) is commonly used as an emulsifier due to its low cost and high emulsifying performance. More importantly, SPI is primarily composed of two globulins: glycinin (11S) and β-conglycinin (7S). Compared to 11S, the 7S displays superior emulsifying activity, mainly due to its higher surface hydrophobicity, and easier unfolding at the oil-water interface [13] . Therefore, within SPI, 7S is considered the core component for expressing emulsifying functionality, which plays a crucial role in forming stable emulsions. The key to enhancing the performance of SPI-based emulsions is to enhance the emulsifying ability of 7S [14] . However, current research mostly focuses on the synergistic improvement mechanism of polysaccharides from the perspective of SPI, and the specific mechanism by which polysaccharides enhance SPI-based emulsifying characteristics at the subunit level has not been thoroughly explored. Considering the effect of electrostatic repulsion, mixing the SPI with negatively charged and polysaccharides with neutral charged in appropriate proportions might not lead to phase segregation [12] . Konjac Glucomannan (KGM), a naturally derived neutral polysaccharide, consists of D-glucose and D-mannose monomers and has substituent chains with an acetyl group content ranging around 10-15% [15] . Due to the presence of both hydroxyl and acetyl groups in KGM, the complexes of KGM and protein has been applied to successfully prepare Pickering emulsions in many reports [16-18] . However, there remains a lack of detailed understanding regarding how KGM precisely influence the structure of protein subunits and their dynamic interfacial behavior. In this context, this study utilized 7S and KGM at varying concentrations to co-stabilize Pickering emulsions. Initially, the structural characteristics and intermolecular interaction mechanisms of the binary composites were evaluated using Fourier Transform Infrared Spectroscopy, three-phase contact angle and so on. Subsequently, the complex adsorption behavior of the 7S-KGM composites at the oil-water interface was investigated through interfacial adsorption kinetics and Quartz crystal microbalance with dissipation monitoring. Further, an in-depth examination of the microstructure, interfacial layer morphology, microrheological properties, and physical stability of emulsions stabilized by different 7S-KGM composites was conducted to establish correlations between interfacial characteristics and stability. Finally, a mesoscopic model of 7S was constructed, and the dissipative particle dynamic method was employed to simulate the dynamic formation process of the interfacial layer stabilized by 7S-KGM composites. This work aims to deepen the understanding of polysaccharide-enhanced SPI emulsification mechanisms at the subunit level by revealing the complex interactions between 7S globulin and KGM and decoding the mechanism of interface co-adsorption. This study provided key guidance for optimizing the composition of Pickering emulsion and enhancing its stability. 2. Results and discussion 2.1 Structural characteristics of 7S particles with different KGM concentration 2.1.1 FTIR Through the analysis of FTIR spectroscopy changes, valuable information could be gained into the microenvironmental modifications, the formation of new functional groups, and the types of intermolecular forces involved during the interaction between 7S and KGM. This also helped to elucidate the effects of these interactions on the overall structure of the proteins [19] . The FTIR spectra of 7S at different KGM concentrations was shown in Figure 1A. It was clear that the 7S-KGM composite displayed no additional absorption peaks when compared to pure 7S, suggesting that the interaction between 7S and KGM did not result in the formation of new functional groups. As the concentration of KGM increased, the peak intensity of the 7S-KGM composite at 1025.39 cm -1 (C-O-H stretching) gradually increased. This phenomenon was due to the characteristic absorption peak of the pyranose ring in KGM [20] . The 7S characteristic peaks were distributed at 3400-3200 cm −1 (O-H stretching), 2956 cm −1 (C–H stretching), 1700-1600 cm −1 (amideⅠband, C=O stretching), 1600-1500 cm −1 (amide Ⅱ band, C-N stretching coupled with N-H blending mode) and 1070 cm −1 (C–O stretching). When introducing KGM, these characteristic peaks of 7S showed significant changes, indicating a strong interaction between 7S and KGM. Specifically, the changes in the intensity and shape of the characteristic peak of 7S at 2956 cm −1 indicated a change in the environment of the C-H bond, which might be due to the addition of KGM altering the protein conformation and forming new interactions with the 7S particle. In addition, for the broad absorption band of 7S at 3400-3200 cm⁻¹, the characteristic peak shifted to lower wavenumbers upon the addition of KGM. When the KGM concentration was 0.15%, the 7S-KGM exhibited the highest degree of redshift. This indicated the formation of hydrogen bonds between the hydroxyl groups of KGM and 7S, which led to changes in the hydrogen bond network. And the 7S-KGM 0.15 composite showed the strongest hydrogen bond. The absorption peak of 7S-KGM composite underwent a red shift at 1070 cm -1 . This might be because the hydrogen bonds formed between 7S and KGM can alter the electron distribution around the C-O bond, further affecting the stretching degree of the C-O group. The peak at approximately 1726.29 cm -1 (corresponding to C=O stretching vibration) usually represented the absorption peak of acetyl groups in KGM [21] . However, the absorption peak corresponding to the acetyl group in the 7S-KGM complex was nearly undetectable. Meanwhile, the intensity of the amide II peak of 7S-KGM composite increased to varying degrees. These findings together suggested the possibility of hydrophobic interactions occurring between the 7S and KGM acetyl groups. Therefore, KGM could interact with 7S particle through non covalent forces such as hydrogen bond and hydrophobic interaction. In addition, the amide I band (1500-1600 cm -1 ) and amide II band (1600-1700 cm -1 ) of 7S showed a red shift when bound to KGM, indicating that KGM induced the unfolding of the 7S structure and increased protein flexibility. To uncover more detailed structural information, the peakfit software was utilized to analyze and provide insights into the characteristics of the 7S’s secondary structure. The secondary structure of proteins determined their flexibility and stability, which was crucial for emulsifiers. Typically, α-helix within polypeptide chains were mainly used to construct the protein skeleton through hydrogen bonding. However, the densely packed structure of α-helix was not conducive to the conformational changes required for the functional characteristics of 7S, especially its emulsifying properties. In contrast, the random coil conformation was more flexible, aiding proteins in unfolding at interfaces and expressing their functional attributes [22] . Table 1 showed the proportions of secondary structures in different samples. Compared to 7S particles, the addition of KGM at varying concentrations resulted in a decrease followed by an increase in the relative content of α- helix, while β-sheet and random coil initially increased and then decreased. This trend suggested that the thermodynamic incompatibility between proteins and polysaccharides might induce varying degrees of unfolding and rearrangement of 7S structure [23] . Specifically, KGM, as a highly hydrated polysaccharide molecule with abundant hydroxyl groups, could form hydrogen bonds with water molecules, thereby weakening the internal hydrogen bond network of 7S and inducing structural unfolding [12] . This interventions not only altered the electronic density of 7S molecules but also facilitated the transformation of α-helix into β-sheet in the secondary structure, and increased the content of random coil, ultimately leading to a looser and more disordered conformation of 7S. However, when the concentration of KGM exceeded 0.15%, extensive cross-linking occured between KGM and 7S molecules, resulting in a dense configuration of the 7S-KGM composite. This led to a reorganization into a more structured and organized conformation, characterized by an increase in α-helix and a decrease in random coil. Han et al. [24] have similarly shown that an increase in α-helix content indicated strong non-covalent interactions between SPI and various polysaccharides, which was not conducive to the expression of its functional properties. Therefore, by adjusting the concentration of KGM, the structural characteristics of the 7S-KGM composite could be optimized to enhance the emulsifying effect. 2.1.2 Intrinsic fluorescence By examining changes in the intensity and position of fluorescence emission peaks in the 7S-KGM composite, we could gain insights into the structural and environmental polarity changes of the 7S molecule after binding to KGM, further elucidating the interaction mechanism between KGM and 7S [25] . The effect of adding different concentrations of KGM on the 7S fluorescence characteristics was shown in Figure 1B. The 7S protein exhibited a strong fluorescence peak at approximately 330 nm. With increasing KGM concentration, the fluorescence intensity of the 7S-KGM composite markedly decreased, and the maximum emission wavelength shifted towards longer wavelengths. This indicated that 7S and KGM could formed non covalent composites through hydrogen bonding, van der Waals forces, hydrophobic interactions, and so on, leading to fluorescence quenching [26] . In addition, polysaccharide chains might have a shielding effect, reducing the direct contact between chromophores and the external environment. Notably, the interaction between KGM and 7S caused a degree of unfolding in the 7S molecular structure. As a result, Tyr chromophores originally embedded within the protein became exposed to a more hydrophilic environment [27] , increasing the polarity of the surrounding solvent and leading to a red shift in the emission wavelength. 3.1.3 Surface hydrophobicity H 0 could reflect the distribution information of hydrophobic amino acid residues on protein surfaces, and its changes significantly affected the interfacial properties of proteins [28] . ANS was an anionic fluorescent probe used to study protein surface hydrophobicity and conformational changes, with its fluorescence intensity reflecting the surface hydrophobicity of 7S protein. As shown in Figure 1C, the peak intensity of the 7S fluorescence curve near 470 nm, which indicated the surface hydrophobicity of 7S, initially increased and then decreased with increasing KGM concentration, reaching a maximum at 0.15% KGM. KGM could induce the gradual unfolding of originally self-aggregated 7S molecules through steric hindrance, volume exclusion effects, and thermodynamic incompatibility with 7S [12] . This process loosened the tightly packed globular structure, exposing hydrophobic groups initially hidden within the globular 7S. However, when excess KGM (>0.15%) was added, a large number of KGM polysaccharide chains bound to the surface of 7S molecules via hydrophobic interactions [29] . This over-modification reduced the number of hydrophobic groups exposed on the surface of the 7S-KGM composite, leading to a decrease in surface hydrophobicity. It’s interesting that these findings were consistent with changes in β-sheet structure (Table 1). The hydrophobic interactions were a primary driving force for β-sheet formation in proteins [ 28] . When 7S molecules partially unfolded due to the presence of KGM, exposed hydrophobic groups promoted the generation of local β-sheet structures, increasing β-sheet content. However, after the surface hydrophobicity decreased due to excess KGM, this trend might reverse, inhibiting the formation of β-sheet structures. This phenomenon highlighted the delicate balance in the interaction between KGM and 7S, which could significantly influence structural changes in 7S. 3.1.4 Wettability The θ was crucial for evaluating their wettability and performance as emulsifiers. When θ was nearly 90°, it indicated that Pickering emulsions have formed stable nano composites with nearly equal hydrophilic and hydrophobic properties [30] . In MCT oil, the θ of KGM, 7S, and 7S-KGM composite particles were shown in Figure 1D. All θ of samples were less than 90°, indicating that 7S and KGM predominantly displayed hydrophilic characteristics. As a natural polymer, KGM possessed hydrophobic vinyl groups capable of free-radical polymerization and hydrophilic hydroxyl groups, exhibiting amphiphilicity [30] . As the concentration of KGM increased from 0% to 0.25%, the θ value first increased and then decreased, reaching a maximum of 88.64° at 0.15% KGM concentration. Combined with the results of FTIR and H 0 , the hydroxyl and acetyl groups in KGM could form different non-covalent bonds with 7S, which might expose hidden or sealed hydrophobic regions within the 7S structure, thus enhancing the complex’s wettability towards the oil phase [31] . At a KGM concentration of 0.15%, theθ of the 7S-KGM composite neared 90°, indicating almost perfect amphiphilic properties that enhanced particle adsorption ability. When the KGM concentration further increased beyond 0.25%, excess KGM promoted aggregation among 7S molecules, burying some hydrophobic groups while introducing additional hydrophilic hydroxyl groups, resulting in the system becoming more hydrophilic and the overall θ decreasing . Overall, an appropriate amount of KGM optimized the amphiphilic properties of the 7S-KGM composite, which contributed to the emulsification ability and anti oil droplet aggregation ability of the complex system. 2.2 Interface characteristics of 7S particles with different KGM concentration 2.2.1 Dynamic surface tension The variation of dynamic surface tension over time directly reflected the interfacial adsorption efficiency of 7S-KGM composite materials, which could further indicated the impact of KGM concentration on interfacial interactions, molecular dynamics, and structural changes [32] . The interfacial tension of 7S-KGM composite was shown in Figure 2A. Within the first 100 s, all samples exhibited a rapid decrease in interfacial tension. This was attributed to 7S being amphiphilic molecules that could spontaneously and quickly migrate from the aqueous phase to the interface, effectively reducing the system’s Gibbs free energy and enhancing interfacial stability [33] . As the adsorption process continued, the interfacial tension gradually tended to equilibrium, indicating that amphiphilic molecules at the interface reached saturation along with an increase in the electrostatic energy barrier associated with adsorption. Furthermore, as the KGM concentration increased, the interfacial tension initially decreased and then increased, with the 7S-KGM 0.15 composite showing the lowest interfacial tension. Combining results from FTIR (Figure 1A), surface hydrophobicity (Figure 1C), and contact angle (Figure 1D), it was observed that the interaction between KGM and 7S promoted the unfolding of the 7S molecular structure, exposing more hydrophobic groups. This improved the protein’s hydrophilic-hydrophobic balance and made its wettability closer to neutrality. At this point, the 7S-KGM composite exhibited higher surface activity, reducing kinetic barriers to adsorption and achieving high diffusion flux of 7S-KGM composite to the oil-water interface [34] . However, when the concentration of KGM was too high, a large number of high-molecular-weight KGM chains rapidly adsorbed and entangled on the oil droplets due to their inherent amphiphilicity. This might result in competitive adsorption with 7S particles at the interface, thereby disrupting the effective formation and stability of the oil/water interface [32] . Moreover, excess KGM might induce protein aggregation, thereby burying hydrophobic groups and reducing the adsorption efficiency at the interface, which could ultimately lead to an increase in surface tension. Nonetheless, the dynamic adsprption processes of the interfacial film needed further research and deeper exploration. 2.2.2 Adsorption kinetics at the oil-water interface Generally, the adsorption of surfactants onto an interface entails three steps such as diffusion, permeation, and reorganization [35] . As shown in Figure 2B, during the initial interval from 0 to 100 s, a rapid increase in the π was noted, a phase referred to as the rapid increase period. This phase was characterized by diffusion-driven transport mechanisms. Consequently, the slope of the curve plotting π against t 1/2 was used to determine the diffusion rate (K diff ). As illustrated in Figure 2C, the plot of time versus ln[(π7200-πt)/(π7200-π0)] exhibited two distinct linear segments. The development of the interfacial structure was elucidated by interpreting the first segment’s slope as the permeation rate (K p ), and the second segment’s slope as the reorganization rate (K R ) [36] . The K diff , K p , and K r were detailed in Table 2. It was clear that the values of K diff , K p , and K p firstly increased and then decreased when increasing the KGM concentration. The 7S-KGM 0.15 composite showed the highest K diff , K p , and K p. During the diffusion phase, KGM with low concentrations induced 7S particles to become a more disordered and hydrophobic conformation, enhancing their affinity for the oil phase. This facilitated the diffusion of 7S-KGM composite from the bulk to the interface. Simultaneously, the adsorption of non-ionic polysaccharide KGM at the interface might alter the charge distribution at the interface [37] . The adjustment of this charge distribution increased the electrostatic attraction of the interface to 7S particles, accelerating their diffusion to the interface, thereby increasing K diff . Moreover, the thermodynamically incompatible repulsive effect between 7S and KGM could promote more 7S particles to diffuse from the continuous phase to the interface, which was known as the interfacial concentration effect [38] . However, high concentrations of KGM could introduce steric hindrance and increase system viscosity. This formed a mobility resistance for 7S particles, restricting their diffusion at the oil-water interface. Thus, K diff significantly decreased. In the penetration phase, low concentrations of KGM increased the K P of 7S-KGM composite, indicating a positive effect of KGM in promoting the penetration of 7S. Generally, when protein particles migrated to the oil-water interface, their surface hydrophilic/hydrophobic sites would adjust to overcome energy barriers and anchor at the interface [39] . The presence of low concentrations of KGM adjusted the hydrophilic-hydrophobic balance of 7S particles, enhancing hydrophobic interactions between 7S and the interface, reducing the energy barrier for 7S to anchor at the interface. This meant that KGM made 7S to penetrate into the interface more easily and remain stable. Furthermore, KGM at the interface could attract more 7S in the local phase to underwent lateral permeation and accumulation at the interface through non-covalent interaction forces, synergistically improving the K P of 7S-KGM composite. However, as the concentration of KGM further increased, K P eventually decreased to -2.94×10 4 s -1 . This decrease was due to the dense packing and gel-like network that high concentrations of KGM formed, establishing a physical barrier at the interface which restricted the mobility and infiltration of 7S particles. Through contrast, it was found that K R was higher than K P for all samples, suggesting that the formation of the interface was more influenced by the rearrangement of protein rather than by their penetration. K R was primarily determined by the conformational flexibility of proteins [40] . Combining FTIR results, KGM induced an increase in random coil content in the secondary structure of 7S through non-covalent interactions. This enhanced the conformational flexibility of 7S, promoting the rearrangement and deformation of 7S-KGM composite at the interface. However, under high concentrations of KGM, the flexibility of 7S-KGM composite decreased, easily restricting their rearrangement ability and proper positioning at the oil-water interface, ultimately leading to a decrease in K R . Generally, higher diffusion, penetration, and rearrangement rates were beneficial for the formation of a stable interfacial layer. However, the information obtained from interface adsorption kinetics was limited. In order to further analyze the interfacial adsorption behavior and obtain real-time and highly sensitive kinetic data, QCM-D technology was subsequently used to explore the kinetic characteristics of interfacial adsorption and its impact on the formation of interfacial films. 2.2.3 QCM-D As a piezoelectric material, the quartz crystal could transform the mass change on the sensor surface into electrical signal. Therefore, by recording the changes in dissipation (D) and frequency (f) using QCM-D technology, the differences in the interfacial adsorption behavior of 7S-KGM composite formed at different KGM concentrations could be reflected [41] . In brief, the Δf was closely related to the adsorption/deposition masses of 7S-KGM composite at the oil-water interface, while the ΔD was closely related to the alterations in the viscoelastic properties of the films [42] . In this work，the gold-coated sensors were hydrophobically modified by 1-hexadecanethiol to simulate the triglyceride-water interface [43] . Figure 3A and 3B illustrated the equilibrium-adsorption-cleaning process. Initially, to achieve stable dissipation and frequency values, the gold-coated sensor was flushed with buffer solution to establish a baseline. Subsequently, different 7S-KGM composite were flowed through the piping system onto the sensor surface. Due to the adsorption and interaction of the 7S-KGM composite on the sensor surface, the Δf and ΔD values shifted over time, eventually reaching equilibrium. This indicated that all samples continuously adsorbed and rearranged on the gold-coated sensor, causing rapid energy dissipation on the quartz crystal and forming a soft and thick interfacial film [44] . At the end of the adsorption phase, cleaning the sensor surface revealed a gradual decrease in ΔD and an increase in Δf. This was mainly due to the buffer solution removing loosely bound 7S-KGM molecules. Further analysis found that the ΔD1 for 7S alone was less than 1×10 -6 /10 Hz, while all 7S-KGM composites had ΔD1 greater than 1×10 -6 /10 Hz. This suggested that 7S molecules alone could only form rigid interfacial films, which were not conducive to stabilizing emulsions. The 7S-KGM composites could more tightly combine and arrange at the oil-water interface, forming dense, hydro-swollen viscoelastic soft films [45] . After increasing KGM concentration, the values of Δf1 and ΔD1 first increased and then decreased, reaching maximum values at a KGM concentration of 0.15%. This indicated that at lower concentrations, KGM optimized the adsorption kinetics of 7S-KGM composite, enabling them to rapidly absorb and orderly arrange on the interface. This enhanced interfacial intermolecular interactions such as hydrogen bonding, hydrophobic interaction and van der Waals forces, thereby increasing the mass and viscoelasticity of the interfacial film. However, as the KGM concentration increased further, the molecular distribution at the interface transitioned from sparse to dense. Eventually, due to excessive KGM molecules competing for limited adsorption sites, the interface underwent restructuring. This led to a looser structure of the adsorbed layer, with reduced mass and viscoelastic properties. Both 7S and 7S-KGM 0.05 samples exhibited lower Δf2 and ΔD2, attributed to low loading and firm adsorption of molecules at the interface. As the KGM concentration increased further, the values of Δf2 and ΔD2 became larger, especially for 7S-KGM 0.15 , which had the largest Δf2 and ΔD2. This was because the 7S-KGM composites in the first layer could firmly adsorb through anchoring of hydrophobic groups. The outer layer consisted of multilayer films driven by the interaction between KGM, 7S, and 7S-KGM composites through hydrogen bonding, van der Waals forces, and hydrophobic interactions. These outer layer could fill the gaps of the first layer to promote the formation of multilayer interfacial films and long-term emulsion stability [46] . However, because of the low desorption energy of the outer layer, this film lacked stability and could be readily removed. Upon reaching a new equilibrium following desorptio, 7S-KGM 0.15 composites still had the largest absolute values of ΔF and ΔD, indicating that the 7S-KGM 0.15 composite had strong interfacial activity and could undergo multilayer adsorption through stronger molecular interactions, producing thicker and more viscoelastic interfacial film. The slope of ΔD-Δf plots could provide information on the adsorption and desorption kinetics and conformational changes of the adsorbed film, such as Figure 3C. The slopes of the ΔD-Δf curves has K1, K2, K3, and K4, where K1 and K2 represented the adsorption process, and K3 and K4 represented the desorption process. Typically, a lower slope value indicates a stiffer and thinner layer, while a higher slope value suggests a more viscoelastic and thicker layer [47] . All slopes of different samples were shown in Table 3. K2 was much smaller than K1 for all samples. This indicated that initially, the 7S-KGM composite rapidly formed a loosely bound and flexible film. As more emulsifier was continuously added, additional molecules adsorbed onto this layer, and water molecules were gradually expelled. This process led to a structure that became increasingly compact and rigid over time. Thus, the interfacial film underwent a transformation from a hydrated, softer state to a more compact and viscoelastic configuration [47] . This was attributed to the removal of coupled water and multilayer adsorption behavior between 7S-KGM molecules. Additionally, K4 was much smaller than K3. This was due to the interfacial film becoming denser and harder as molecules continuously desorb from the adsorption film [46] . Furthermore, with increasing KGM concentration, K1, K2, K3, and K4 first increased and then decreased, with the 7S-KGM 0.15 composite having larger values for K1, K2, K3, and K4. This indicated that adding an appropriate concentration of KGM polysaccharide chains was more favorable for the adsorption and reorganization of the interfacial film, resulting in a denser, thicker, and more elastic multilayer interfacial film. Simultaneously, the outer viscoelastic film formed by the 7S-KGM composite at the interface provided significant steric hindrance. This strong spatial barrier effectively prevented droplet flocculation and coalescence, ensuring emulsion stability. Moreover, QCM-D could provide information regarding the thickness, elastic modulus, mass, and viscosity of the adsorbed film [48] . As shown in Figure 3D and 3E, with increasing KGM concentration, the thickness, elastic modulus, mass, and viscosity of the adsorbed layer first increased and then decreased. Firstly, the θ of the 7S-KGM composite was relatively large, reflecting a high degree of particle amphiphilicity. Secondly, the cooperative adsorption of KGM at the oil-water interface might lead to in-plane aggregation of 7S particles, thereby increasing the thickness and mass of the adsorbed film. Furthermore, the more effective lateral rearrangement and attractive longitudinal interactions of 7S-KGM composite at the interface could promote the formation of a more viscoelastic, ordered 3D gel-like interfacial structure [45] . Consequently, the 7S-KGM composite tended to extend both laterally and longitudinally, forming denser and more ordered adsorption films. This not only increased the thickness of the adsorbed film but also enhanced its structural integrity, thereby improving the viscoelasticity of the interfacial film. When the KGM concentration was too high, excessive KGM long chains rapidly adsorbed and entangled on the limited gold-coated sensor surface, hindering the effective adsorption of 7S particles. This reduced the thickness, elasticity, and mass of the adsorbed layer. Meanwhile, the structure of the adsorbed layer became looser, with decreased internal friction, leading to a drop in viscosity. Therefore, the dynamic behavior of 7S-KGM composite at the interface directly affected the properties of the interfacial film, and thus determined the long-term stability of Pickering emulsion. 2.3 Emulsion characteristics of 7S particles with different KGM concentration 2.3.1 EAI and ESI EAI and ESI were fundamental indicators for assessing the emulsifying properties of proteins. EAI reflected the ability of proteins to bind at the oil-water interface [49] . Compared to 7S, the EAI of 7S-KGM emulsions significantly increased, with the EAI of the 7S-KGM 0.15 emulsion increasing by 46.52 m²/g (Figure 3F). The 7S-KGM composite could disperse more effectively at the oil-water interface, thereby reducing interfacial energy and ultimately enhancing the EAI [50] . ESI reflected the capability of proteins to maintain stability at the oil-water interface [49] . With the increase in KGM concentration, the ESI of samples increased from a minimum of 58.81 min (7S emulsion) to a maximum of 95.32 min (7S-KGM 0.15 emulsion), indicating that the addition of KGM could improve the emulsifying stability of 7S. As KGM was added, 7S, KGM, and the 7S-KGM composite could physically adsorb and intertwine longitudinally on the oil surface, forming a multilayer interface. The outer layer, loosely adsorbed, provided additional mechanical strength and elasticity, effectively preventing droplet coalescence. Alavi and Chen [51] also proposed that complex interactions between proteins and pectin contribute to the formation of a longitudinal network during emulsification, ultimately enhancing ESI. Moreover, the high viscosity of KGM could dampen emulsions’ Brownian motion, thereby decreasing droplet collisions and enhancing ESI [15] . This further demonstrated that KGM could promote the adsorption and retention of 7S at the oil-water interface, resulting in better dispersibility and emulsifiability. However, when the KGM content exceeded 0.15%, the EAI and ESI of the 7S-KGM mixture gradually decreased. This might be due to competitive adsorption among complex particles in the interfacial layer and the high viscosity of the solution system leading to depletion flocculation between emulsion droplets. These results were consistent with the results of interfacial adsorption kinetics (Figure 2) and QCM-D (Figure 3), indicating that the interfacial behavior of 7S-KGM composites and the microstructure of the interfacial layer had a direct impact on their emulsifying properties. 2.3.2 CLSM Typically, an ideal emulsion was characterized by oil droplets with uniform distribution and small volume [52] . By analyzing images obtained from CLSM (Figure 4A and 4B), we gained more information into the emulsion droplets. In this visualization, oil droplets were stained red with fluorescent dye, while 7S protein appeared green. To clearly demonstrate the changes in droplet size distribution among different samples, we selected representative droplets for size analysis at the same magnification and plotted the data distribution graph (Figure 4C). The results showed that when stabilized solely by 7S, the oil droplets exhibited a spherical structure encased by 7S particles, with a relatively large average diameter and non-uniform distribution, approximately 24.79±5.70 μm. However, as the concentration of KGM increased, the droplet sizes gradually decreased, and their distribution became more uniform. Notably, the emulsions stabilized by the 7S-KGM 0.15 composite displayed the smallest and most uniform droplet size distribution, with an average diameter of only 8.95±1.68 μm. This indicated that the synergistic adsorption of KGM played a crucial role in emulsion stability. The 7S-KGM composite could co-adsorb and accumulate at the oil-water interface, forming a tightly packed layer with strong steric hindrance, thereby maintaining effective particle distribution at the interface. Moreover, this complex not only acted as a surfactant to reduce interfacial tension but also functioned as a structural enhancer that increases system viscosity, effectively reducing droplet mobility and tendency to aggregate, thus promoting the formation of smaller and more uniform droplets [52] . When the concentration of KGM was too high, it led to noticeable aggregation and deformation of droplets, causing the size distribution to become uneven again, with an average diameter increasing to 16.80±6.17 μm. Besides, The fluorescence intensity of the interface layer decreased and the localized green fluorescence patches in the continuous phase increased, suggesting an increase in 7S particle aggregates in the aqueous phase. This situation could be ascribed to high concentrations of KGM causing a more rigid conformation in 7S particles (Table1), which hindered their adsorption at the interface. As a result, the formation of a stable interface layer was compromised, leading to a decrease in green fluorescence intensity at the interface. Simultaneously, excessive KGM chains competed with 7S for interfacial contact area, pushing some 7S particles outside the interfacial layer [52] , leading to the gradual formation of large protein aggregates. In summary, the addition of KGM significantly affected the 7S’s spatial distribution at the interface, thereby altering the morphology and stability of emulsion droplets. 2.3.3 In situ characterization of interfacial particle To more intuitively elucidate the mechanism by which the 7S-KGM composite stabilized emulsions, SEM was used to characterize the interfacial organization and distribution of complex particles [53] . For this purpose, a styrene-water emulsion was prepared as a substitute for the MCT oil-water emulsion and subsequently polymerized into solid beads. SEM images of the resulting polystyrene beads are shown in Figure 4D, 4E. 7S particles were embedded within the outer layer of the polystyrene, forming a monolayer interfacial film. The interfacial structure was unstable due to the lack of particle interaction, leading to droplet aggregation. Upon the addition of KGM, there was a notable transformation in the morphology of the interfacial layer. The 7S-KGM composite could uniformly cover the droplet surface, with the robust polysaccharide network of KGM linking individual particles, thereby enhancing the cross-linking degree between interfacial particles. In addition, KGM had extended bridges between adjacent droplets, enhancing the dispersion between oil droplets through steric hindrance effect. Consequently, particles adsorbed by the 7S-KGM composite gradually accumulated on the interface, forming a multilayer interfacial film that enriched and stabilized the interfacial structure. When the concentration of KGM was 0.15%, the distribution of interfacial particles became more uniform, and droplet size significantly decreased. This might be due to the fact that at this concentration, KGM more easily induced conformational changes in 7S particles and exposed hydrophobic groups, promoting interfacial interactions and the formation of a stronger viscoelastic interfacial layer [54] , consistent with QCM-D results. However, when the concentration of KGM exceeded 0.15%, excessive accumulation and aggregation of KGM long chains occured within the interfacial structure, hindering the adsorption behavior of 7S particles at the interface. Ultimately, the heterogeneous and deformed interfacial layers were formed, causing droplet aggregation and cross-linking. Therefore, the 7S-KGM composite significantly influenced emulsion stability by modulating the composition and spatial distribution of interfacial active particles. 2.3.4 Interface layer thickness and AP By measuring the layer thickness and the AP, the interfacial layer morphology and integrity of different samples were further quantitatively investigated. Figure 4F and 4G respectively showed the layer thickness and the AP on the interface of emulsion prepared by different samples. As the concentration of KGM increased, the thickness layer showed a trend of first increasing and then decreasing. This was consistent with the interface layer thickness results fitted in QCM-D. The content of interface proteins also showed the same trend. When only 7S particles were present at the oil-water interface, they formed a loosely packed monolayer structure. This resulted in a relatively thin interfacial layer with limited adsorbed protein content. After introducing KGM, KGM induced a conformational shift in the 7S from an ordered to a disordered state, enhancing the interfacial adsorption capacity of the 7S-KGM composite . This allowed more 7S particles to anchor laterally at the interface, promoting the enrichment of 7S and forming a robust first layer of the interface [55] . Simultaneously, the remaining amphiphilic KGM molecules gradually diffused around the interfacial proteins. Leveraging its excellent hydrophilicity and extensibility, KGM effectively filled the gaps within the first interfacial layer and aligned longitudinally through non-covalent interactions with the proteins. This synergy built a multi-layer interfacial film, which made the composition of the interfacial layer more abundant and the structure more complete [56] . Therefore, the layer thickness and the AP were both increased. However, excessive KGM concentration could lead to crowding effect and spatial hindrance effect. Overaccumulation of KGM molecules on the interface might hinder the arrival of 7S, limiting further adsorption and distribution. The layer thickness and AP began to decrease after reaching a peak. 2.3.5 Micro-rheological properties Micro-rheology, which assessed the viscoelastic properties of turbid samples through the measurement of particles’ Brownian motion, offered a non-invasive, secure, rapid, and sensitive method for evaluating microrheological characteristics [57] . Figure 5A showed the correlation between MSD and the de-correlation time for droplets across various emulsion samples. Typically, the slope of MSD over extended de-correlation periods serves as an indicator of particle movement. For a purely viscous emulsion, the MSD curve showed a linear increase with the decorrelation time. In contrast, for a viscoelastic system, the MSD-time curve appeared nonlinear because the emulsion droplets became entrapped within a three-dimensional network structure [58] . In the case of Pickering emulsions stabilized by 7S, 7S-KGM 0.05 , and 7S-KGM 0.1 , the MSD increased in a manner that was largely linear with respect to the decorrelation time (but not purely linear). This trend indicated that the droplets had a greater freedom of movement, suggesting these emulsions displayed more viscous-like characteristics. Pickering emulsion prepared by 7S-KGM 0.15 , 7S-KGM 0.2 , and 7S-KGM 0.25 showed a pronounced plateau region and non-linear curves, indicating that they exhibited viscoelastic properties and showed shear-thinning behavior at this scale. Additionally, the emulsion prepared by 7S-KGM 0.15 composite possessed the lower plateau and the greatest extent of de-correlation time, suggesting more restricted droplet movement within this system and the formation of a particularly strong viscoelastic network. The SLB values provided insight into whether the emulsions exhibited more solid-like or liquid-like behavior. Typically, an SLB value less than 0.5 indicated a solid-like sample, an SLB of 0.5 suggested a balance between solid and liquid characteristics, an SLB between 0.5 and 1 pointed to a liquid-like sample, and an SLB greater than 1 signified the presence of sedimentation [59] . The variation in SLB values was depicted in Figure 5B. Initially, nearly all samples exhibited SLB values exceeding 1, likely due to the non-uniform distribution and instability of the freshly prepared droplets, causing some droplets to settle at the bottom of the container. Over time, as the de-correlation time increased, the SLB values for all emulsions progressively decreased and eventually stabilized. As the concentration of KGM increased, the SLB of stabilized emulsions first decreased and then increased, indicating that increasing KGM could enhance the viscoelasticity of the emulsion system. Specifically, when only 7S was present (SLB > 1), the emulsion exhibited significant instability, and experienced obvious Ostwald ripening and flocculation during the measurement process, which eventually led to the sedimentation. For the 7S-KGM 0.05 and 7S-KGM 0.10 emulsion samples (0.5 < SLB < 1), liquid behavior dominated in these emulsion systems. For the 7S-KGM 0.15 , 7S-KGM 0.2 , and 7S-KGM 0.25 emulsion samples (SLB < 0.5), the emulsion structure became more solid-like, exhibiting stronger elastic properties. This change might be due to the addition of KGM promoting more 7S particles to adsorb onto the oil droplet surface, enhancing the interfacial network structure, thereby further restricting the movement of emulsion droplets and forming a more solid-like structure. Additionally, free KGM presented in the aqueous phase might contribute to the increase in emulsion viscoelasticity through its thickening effect. The viscoelastic properties of the emulsion were closely related to its flowability, with larger SLB values corresponding to larger FI values (Figure 5C). MVI was positively correlated with the time consumed for particle movement, and EI was negativly correlated with the distance traveled by droplets in the emulsion droplets network [60] . The MVI and EI curves were shown in Figure 5D and 5E. The MVI and EI values of the 7S emulsion were the lowest, attributed to flocculation and coalescence within the 7S group, leading to emulsion instability [60] . As the concentration of KGM increased, the MVI and EI values of the emulsions first increased and then decreased, peaking at a KGM concentration of 0.15%. This phenomenon indicated that an optimal concentration of KGM could effectively promote the formation of an elastic network structure within the emulsion system, slowing particle movement over longer decorrelation times. This protected emulsion droplets from coalescence and phase separation, significantly enhancing emulsion stability. 2.3.6 Physical stability of emulsion TSI was a critical indicator for monitoring instability processes in emulsions, such as flocculation, coalescence, and sedimentation. A lower TSI value signified higher emulsion stability. The method involved monitoring the backscattered light (BS) signal to detect the uniformity of particle dispersion, migration rates, and size changes within the sample [61] . Minor fluctuations in ΔBS over time indicated high stability, whereas significant changes suggested system instability. Figure 5F and 5G showed the changes in ΔBS and TSI values over time for different emulsion samples. The negative and positive peaks were observed on the left side (bottom of the sample bottle) and right side (top of the sample bottle), indicating phase separation within the emulsion [62] . As phase separation stabilized, the distance between the scan lined progressively decreased and shifted towards the right, indicating a re-distribution of the components. The variation of ΔBS strength over time varied among different samples, indicating different instability phenomena and mechanisms. For the emulsion prepared with only 7S, the ΔBS value decreased gradually in the range of 0-27.5 mm and increased from 27.5 to 50 mm as scanning time progresses. This indicated a high ΔBS rate and the formation of a serum layer, where larger droplets destabilized the emulsion through flocculation or coalescence. This phenomenon might because the difficulty of 7S particles in forming a stable interfacial layer and smaller droplets, causing larger droplets to migrate upwards under gravity, increasing bottom transmittance [63] . As KGM concentration increased, the negative peaks of ΔBS were weaker, and the degree of ΔBS change in the emulsion gradually decreased. The ΔBS in the 7S-KGM 0.15 sample group was even almost constant. This suggested that KGM aided in forming smaller droplets alongside 7S particles, reducing the likelihood of these droplets migrating under gravity, thereby enhancing emulsion stability and preventing stratification. The increased conformational flexibility of the 7S-KGM 0.15 composite enhanced interfacial adsorption, forming a dense interfacial structure that resisted deformation of emulsion droplets during short-term storage, thus showing minimal ΔBS change. TSI values generally reflected the overall stability of the system. All samples exhibited gradually increasing TSI values over scanning time (Figure 5G). Within the scanning period, the emulsion prepared with only 7S had the highest TSI value, indicating poor stability. As KGM concentration increased, TSI values showed a trend of first decreasing and then increasing, with the 7S-KGM 0.15 composite -prepared emulsion having the lowest TSI value. This indicated that KGM could improve short-term storage stability. This was attributed to the thicker, harder, and interconnected 7S-KGM layers formed at the interface (Figure 3 and 4), which provideed stronger protection against interfacial wrinkling and stretching caused by droplet flocculation and coalescence. Akhtar and Dickinson [64] also found that enhanced emulsion stability depended on molecular flexibility and interfacial layer cross-linking density. Therefore, increased protein flexibility and the formation of complex interfacial structures contributed to improved emulsion stability. 2.4 DPD simulation The trajectory snapshots of the interface offered microscopic dynamic insights into how emulsion droplets formed and stabilized under varying 7S-KGM composite . These snapshots highlighted alterations in droplet size and the positional relationships of the components at the interface. In these snapshot images, 7S particles were represented by green spherical particles, while KGM polysaccharide chains were depicted as short rod-like particles. Based on the classical theory of limited coalescence [65] , during the initial emulsification phase, there were not enough colloidal particles at the interface to stabilize smaller oil droplets. As a result, the merging of emulsion droplets was restricted until the surface became adequately covered with particles. This explained the phenomenon observed in Figure 6A, where numerous small droplets existed during the initial emulsification process, and as 7S and KGM colloidal particles adsorbed onto the interface, small droplets gradually aggregated to form more stable larger droplets. Moreover, in our simulation system, smaller-sized particles have lower adsorption energy, making it easy for 7S particles to displace from the interface and disperse into the continuous phase. Consequently, individual 7S particles, due to inadequate interfacial activity, failed to effectively adsorb and envelop the droplets, resulting in many 7S particles remaining dispersed in the aqueous phase and eventually forming larger oil droplet aggregates. Upon adding 0.15% KGM, the KGM polysaccharide chains could randomly distribute at the interface and attract more 7S particles through non-covalent interactions, anchoring them uniformly within the interfacial layer, thereby stabilizing the emulsion droplets together. Meanwhile, the outer layer of KGM chains filled in gaps in the first interfacial layer, increasing interfacial adsorption density, consistent with SEM results. As a result, the number of formed emulsion droplets increased, droplet size decreased, and droplet flocculation was reduced. However, when the KGM concentration reached 0.25%, significant aggregation occured between adjacent droplets. Combined with the results of Figure S1, it indicated that excessive KGM chains diffused near the interface, competing for interface sites with 7S particles and reducing the mobility of 7S particles, easily displacing the originally saturated 7S particles on the interface. This led to 7S particles being squeezed and accumulating together, forming protein aggregates, which diminished interfacial adsorption capacity. DPD simulations showed that only an appropriate concentration of KGM could synergistically improve the adsorption behavior of 7S particles, forming smaller and more stable droplets. These results aligned well with earlier experimental findings (Figure 2, 3, 4), thereby confirming the accuracy of the simulations. Figure 6B showed the radial distribution function (RDF, g(r)) for every material, which indicated the likelihood of finding a molecule at a specific distance from the droplet’s center. This analysis helped to characterize the positioning of substances relative to the oil phase, interface, or aqueous phase [66] . The RDF peak for 7S particles was observed in the aqueous phase, suggesting that these particles tend to be hydrophilic. When the system contained 0.15% concentration of KGM, the RDF peak of KGM and 7S particles almost simultaneously appeared near the oil-water interface, suggesting that KGM and 7S particles could synergistically stabilize the emulsion interface. However, when the KGM concentration increased to 0.25%, the RDF peaks of KGM and 7S particles shifted towards the aqueous phase, indicating that excess KGM weakened the ability of 7S particles to stabilize the interface, leading to more 7S particles dispersing into the aqueous phase. The distance from the zero point to the interface accurately represented the size of the emulsion droplet. The droplet size of emulsion stabilized by only 7S particle was 7.155 Å, the droplet size of emulsion stabilized by 7S-KGM 0.15 was 5.785 Å, and the droplet size of emulsion stabilized by 7S-KGM 0.25 was 6.565 Å. This indicated that an appropriate amount of KGM helped to form smaller and more stable droplets, while excess KGM tends to cause droplet re-aggregation. The RDF analysis firstly uncovered the size, shape, and relative positioning of the substances when 7S particles and KGM polysaccharide chains co-stabilized emulsion droplets, further elucidating the mechanism by which the 7S-KGM composite stabilized the emulsion interface. 3. Conclusion In the food industry, incorporating polysaccharides as natural stabilizers is essential, yet their impact on protein adsorption and interfacial film microstructure is often underappreciated. This study integrated experimental techniques with DPD simulations to elucidate the dynamic adsorption mechanism of 7S particles and KGM sugar chains at the oil-water interface from the subunit-level, highlighting their influence on emulsion stability. The findings showed that low concentrations of KGM improved the wettability of the 7S-KGM composite, promoting rapid particle adsorption and rearrangement. This resulted in the formation of a dense, viscoelastic multilayer at the interface, which enhanced emulsion stability. However, at higher concentrations, excessive competitive adsorption of KGM displaced 7S particles from the interface, decreasing their contact area with interface and gradually forming particle aggregates. In summary, this work highlighted the essential role of polysaccharides in optimizing interfacial design and enhancing the stability of Pickering emulsions. It provided valuable insights into the dynamic adsorption behavior and stabilization mechanisms of emulsions that are co-stabilized by proteins and polysaccharides. 4. Experimental Section 4.1 Materials Defatted soybean was provided by Yuwang Industrial Co., Ltd (Shandong, China). The 7S protein was extracted from defatted soy flour according to the method of Huang et al. [67] . The obtained 7S protein content was 98.02% ± 0.50% tested by the Dumas combustion method. Konjac glucomannan (KGM, 95%, 15000 mPa.s) and Medium chain triglycerides (MCT) was provided by Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). Nile Blue and Nile Red were provided by Jingbo Biology Co., Ltd. (Xi’an, China). All other reagents were analytical grade. 4.2 Preparation of the 7S-KGM composite The 7S and KGM solutions were prepared using a phosphate buffer solution (PBS, 0.01 M, pH 7.0) at 25°C. These solutions were then combined in specific ratios and gently stirred for 4 h to create PPI-KGM composites with varying KGM concentrations (0%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%). The 7S concentration was 1 wt%. The prepared protein and composite were respectively categorized as 7S, 7S-KGM 0.05 , 7S-KGM 0.1 , 7S-KGM 0.15 , 7S-KGM 0.2 , and 7S-KGM 0.25 in the following study. 4.3 Structural characteristics of 7S-KGM composite 4.3.1 Fourier-transform infrared (FTIR) spectroscopy The FTIR Spectra of samples was acquired at 500-4000 cm −1 in 32 scans at a resolution of 4 cm −1 using a FTIR spectrometer (Nicolet iS10, Thermo Fisher Scientific, USA). 4.3.2 Intrinsic fluorescence The Hitachi RF-6000 fluorescence spectrophotometer (Tokyo, Japan) was used to measure intrinsic fluorescence. The excitation was set at a wavelength of 280 nm, with emissions detected within the range of 300 to 450 nm [68] . 4.3.3 Surface hydrophobicity (H 0 ) H 0 was measured using 8-anilino-1-naphthalenesulfonic acid (ANS) method. ANS solution (8.0 Mm, 30 μL) was vortexed with samples (4.0 mL). The fluorescence spectrophotometer (FL6500, PerkinElmer, USA.) was used to measure fluorescence emission spectra. The test was conducted at 400-600 nm with an emission wavelength of 380 nm. 4.3.4 Three-phase contact angle (θ) To determine the contact angle θ of the samples, a Theta Flex goniometer (Biolin, Sweden) was utilized. The sample pieces were positioned in a clear glass container filled with MCT oil. The Baseline circle method was applied to calculate the θ. 4.4 Interface characteristics of 7S-KGM composite 4.4.1 Dynamic surface tension and interfacial adsorption kinetics Video-based optical goniometer (DSA 100, Krüss GmbH, Hamburg, Germany) was used to measure interfacial tension. Initially, a 5 μL drop at the tip was created through syringe needle. Measurements were taken over 10,800 s, and the interfacial pressure (π, mN/m) was determined through Eq. (1). \(\pi=\gamma_{0}-\gamma\) (1) γ 0 and γ are the interfacial tension of the water and sample with MCT oil, respectively. The rates of diffusion, penetration, and rearrangement were assessed through Eq. (2) and (3). \(\pi=2{C_{0}K_{B}T\left(\text{Dt}\text{/}3.14\right)}^{1/2}\) (2) \(ln(\frac{\pi_{f}-\pi_{t}}{\pi_{f}-\pi_{0}})=-k_{i}\text{t\ }\) (3) C 0 is the protein concentration, K B is Boltzmann constant, T is temperature, Dt is diffusion coefficient, t is time, \(\pi_{f}\) , \(\pi_{0}\) and \(\pi_{t}\) are surface pressure at final, initial and any time, respectively. \(k_{i}\) is the first-order rate constant. 4.4.2 Quartz crystal microbalance with dissipation monitoring (QCM-D) To create a hydrophobic surface layer on the chips, they were immersed in 1-hexadecanethiol for a period of 16 h. A baseline signal was then established using Milli-Q water through a Q-Sense instrument (Biolin Scientific AB, Sweden). Adsorption measurements, including changes in frequency (Δf) and energy dissipation (ΔD), were conducted with the samples. Following this, the chip surfaces underwent a rinse with Milli-Q water once more. The acquired data were processed and analyzed via the QSense Dfind software, presenting results in terms of layer thickness, elastic modulus, mass, and viscosity. 4.5 Emulsion characteristics of 7S-KGM composite 4.5.1 Preparation of emulsions The O/W emulsion was obtained by homogenizing 18 mL sample and 2 ml MCT oil at 12,000 rpm for 2 min through Ultraturrax T18 homogenizer (IKA, Staufen, Germany). 4.5.2 Emulsifying activity index (EAI) and emulsifying stability index (ESI) EAI and ESI were using the method of Pearce and Kinsella [69] . The emulsions (50 μL) were dispersed into SDS solution (5 mL, 0.1%), and Microplate Reader (Tecan, Austria) was used to measure solutions (kept for 0 min and 30 min) at 500 nm. EAI and ESI were calculated through Eq. (4), (5). \(EAI\ (m^{2}/g)=\frac{2\times 2.303\times n\times A_{0}}{C\times(1-\varphi)\times 10,000}\)(4) \(ESI\ (\%)=\frac{A_{30}}{A_{0}}\times 100\) (5) where A 0 and A 30 are the absorbance of sample at 0 min and 30 min, respectively, C is the protein concentration (g/mL), n is the dilution factor and φ is the oil volume fraction (v/v). 4.5.3 Confocal laser scanning microscopy (CLSM) A Leica SP8 CLSM (Leica Microsystems GmbH, Germany) was used with 633 nm of excitation wavelength and 488 nm. Nile red and Nile blue were utilized to stain oil and protein components, respectively. Droplet size distribution was analyzed using the ImageJ software. 4.5.4 In situ characterization of interfacial particle The procedure followed the methodology reported by Zhang et al. [53] . In summary, different 7S-KGM composite solutions served as the aqueous phase. A mixture of styrene and benzoyl peroxide was prepared to form a styrene-benzoyl peroxide system. Emulsions were created following the protocol outlined in 4.5.1, then subjected to degassing with nitrogen for 10 min, followed by polymerization at 65°C for a duration of 24 h. The resultant polystyrene microspheres were characterized using a scanning electron microscope (SEM) (S4800, Hitachi, Japan). 4.5.5 Interface layer thickness and adsorbed protein content (AP) The spherical polystyrene latex beads were diluted to 0.1 wt% and stored at 25°C. The 900 μL of the sample solution was mixed with 100 μL of the latex suspension and allowed to equilibrate for 30 min, enabling adsorption of the emulsifiers onto the surface of the latex beads. The thickness of the interfacial layer was characterized by measuring the difference in average particle size of the polystyrene latex beads before and after sample adsorption using laser particle size analyzer (Mastersizer 3000E, Malvern Panalytical, UK). Freshly emulsions underwent centrifuged at a speed of 1650 rpm for 30 min. This procedure led to the division of the emulsions into a dispersed phase and a clear aqueous layer. Using a syringe, the clear aqueous phase located at the bottom was carefully removed. The interfacial AP were calculated through Eq. (6). \(\text{AP\ }\left(\%\right)=\frac{(C_{0}-C_{1})\times 100}{C_{0}}\) C 0 and C 1 were the initial protein concentration in emulsion and the initial protein concentration in the supernatant after centrifugation. 4.5.6 Micro-rheological properties The prepared emulsions were measured at glass cuvette for 2 h through microrheometer Rheolaser Master (Formulaction, I’Union, France). The collected measurements were processed and analyzed using the software (Rheosoft Master 1.4.0.0). 4.5.7 Physical stability of emulsion The MultiScan (MS 20 Dispersion Stability Analyzer, Germany) was used to measure the physical stability of emulsion for 3h. The analysis software was used to obatain the Delta backscattering (ΔBS) and Turbiscan stability index (TSI). 4.6 Dissipative particle dynamics (DPD) simulations Simulations were conducted within cubic cells measuring 200 × 200 × 20 ų to replicate the formation mechanism. Within this setup, the proportion of 7S particles was maintained at a constant 1%, whereas KGM proportions varied at 0%, 0.15%, and 0.25%. An oil-to-water ratio of 1:9 was established for these simulations. Conservative forces served as the predominant influence in this system. A summary of these parameters can be found in Table S1. Additional settings were guided by Ye et al.’s study (2025), and the Material Studio 8.0 software facilitated the simulation process. To model the intricate interfacial dynamics of 7S particles in varying concentrations of KGM, the DPD simulation was employed. The simulation environment comprised four elements: 7S particles, KGM, water, and oil, as illustrated in Scheme 1. Of which, the 7S particle representation (consisting of 93 beads) followed the method of Zhang et al. (2023). Meanwhile, KGM, water, and oil were each represented by singular, simplified bead models. The two-dimensional boxes (200 × 200 × 20 ų) were used to simulate the formation process. The relative percentage of 7S was 1%, and KGM were set to 0%, 0.15%, and 0.25%. The ratio of O/W was 1:9. Conservative forces served as the predominant influence in this system, and summarized in Table. S1. The setting of other parameters refers to the research of Ye et al. [32] . The simulation software was Material Studio 8.0. 4.7. Statistical analysis For each experiment, triplicate trials were conducted, with the results presented as the mean ± standard deviation. The statistical evaluation of the collected data was performed using SPSS® software (version 22.0, SPSS Inc., Chicago, Illinois, USA). A one-way ANOVA was applied for data analysis, followed by Duncan’s multiple range test to compare various groups, setting the threshold for statistical significance at P < 0.05. Acknowledgements Grants from the National key R&D Plan (2022YFF1100603) funded this research. Conflict of Interests The authors declare no conflict of interests. Data Availability Statement Data available on request from the authors Supporting Information Supporting Information is available from the Wiley Online Library or from the author. References [1] Clements, D. (2005). Food Emulsions : principles, practices and techniques. 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Figure 1. The FT-IR spectra (A), Intrinsic fluorescence (B), Surface hydrophobicity (C), and W ettability (D) of 7S-KGM composite with different KGM concentration. Figure 2. Surface tension (A), Surface pressure (B) versus time (s) at the oil-water interface for 7S-KGM composite, plot of ln [(π7200-πt)/(π7200-π0)] versus time (s) for the interfacial molecule (C). Figure 3. ΔD (A), Δf (B), ΔD-Δf curve (C), mass and viscosity (D), thickness and elastic modulus (E) of adsorption processes of 7S-KGM composite with different KGM concentration. The EAI and ESI (F) of 7S-KGM composite with different KGM concentration. Figure 4. CLSM images (Oil droplets (A), protein (B), average droplet size (C)) of the fresh emulsions, SEM images (D, E) of polymerized styrene-in-water Pickering emulsions that were stabilized by 7S-KGM composite with different KGM concentration. Layer thickness (F) and AP (G) of emulsion stabilized by 7S-KGM composite with different KGM concentration. Figure 5. The MSD curve (A), SLB (B), FI (C), MVI (D), EI (E), delta backscattering images (F) and TSI (G) of the emulsions stabilized by 7S-KGM composite with different KGM concentration. Figure 6. (A) Snapshot images of the DPD trajectory file that describe the formation and stabilization of emulsion with different 7S-KGM composite at different simulation progress (5%, 15%, 50%, and 100% completion). (B) Numerical variation of Radial Distribution Function of emulsion droplets with different 7S-KGM composite. Table 1. The secondary structure content of 7S-KGM composite with different KGM concentration. 7S 17.53±0.04 a 31.29±0.11 c 35.69±0.04 a 15.49±0.12 e 7S-KGM 0.05 15.66±0.10 bc 32.97±0.06 b 35.68±0.08 a 15.69±0.05 d 7S-KGM 0.10 15.52±0.11 c 33.06±0.06 b 35.63±0.10 a 15.79±0.09 d 7S-KGM 0.15 13.18±0.18 e 33.20±0.05 a 25.57±0.07 d 28.05±0.18 a 7S-KGM 0.20 14.26±0.09 d 31.04±0.14 c 32.37±0.15 c 22.33±0.10 b 7S-KGM 0.25 15.78±0.06 b 30.48±0.15 d 34.97±0.08 b 18.77±0.11 c Note: Different letters in the same column represent a significant difference of different samples (P < 0.05). Table 2. The adsorption kinetics at the oil-water interface of 7S-KGM composite with different KGM concentration. 7S 0.18±0.01 d -2.80±0.10 c -21.19±0.41 c 7S-KGM 0.05 0.23±0.03 bc -2.81±0.04 c -22.54±0.37 b 7S-KGM 0.10 0.26±0.01 b -3.03±0.05 b -22.91±0.50 b 7S-KGM 0.15 0.32±0.04 a -3.18±0.08 a -24.86±0.16 a 7S-KGM 0.20 0.19±0.02 cd -2.56±0.13 d -19.59±0.48 d 7S-KGM 0.25 0.15±0.01 e -2.94±0.07 b -17.31±0.35 e Note: Different letters in the same column represent a significant difference of different samples (P < 0.05). Table 3. Slopes of ΔD-Δf curve from the third overtone for 7S-KGM composite with different KGM concentration during adsorption and desorption processes. 7S 0.452±0.003 d 0.115±0.003 e 0.819±0.013 d 0.082±0.002 e 7S-KGM 0.05 0.491±0.004 c 0.153±0.008 d 0.844±0.010 c 0.106±0.005 d 7S-KGM 0.10 0.516±0.002 b 0.181±0.006 b 0.864±0.008 b 0.115±0.003 c 7S-KGM 0.15 0.584±0.001 a 0.197±0.001 a 0.905±0.015 a 0.134±0.002 a 7S-KGM 0.20 0.492±0.005 c 0.175±0.001 c 0.872±0.013 b 0.129±0.001 b 7S-KGM 0.25 0.427±0.004 e 0.154±0.003 d 0.732±0.017 e 0.116±0.002 c Note: Different letters in the same column represent a significant difference of different samples (P < 0.05). Information & Authors Information Version history V1 Version 1 23 January 2025 Peer review timeline Published International Journal of Biological Macromolecules Version of Record 1 Jul 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords co-stabilized emulsion dissipative particle dynamic interfacial behavior interfacial microstructure konjac glucomannan β-conglycinin Authors Affiliations Yanan Guo Northeast Agricultural University View all articles by this author Tianfu Cheng Northeast Agricultural University View all articles by this author Shuo Zhang Northeast Agricultural University View all articles by this author Haotian Liu 0000-0001-9398-9062 Northeast Agricultural University View all articles by this author Ramnarain Ramakrishna North Dakota State University View all articles by this author Lianzhou Jiang Northeast Agricultural University View all articles by this author Zhongjiang Wang Northeast Agricultural University View all articles by this author Zengwang Guo 0000-0002-3996-7214 [email protected] Northeast Agricultural University View all articles by this author Metrics & Citations Metrics Article Usage 255 views 118 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Yanan Guo, Tianfu Cheng, Shuo Zhang, et al. Formation and properties of β-conglycinin particle-Konjac Glucomannan co-stabilized Pickering emulsion: Dynamic adsorption behavior and complex interface microstructure. Authorea . 23 January 2025. DOI: https://doi.org/10.22541/au.173759969.99359333/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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