Induced Electric Field Acceleration of Whey Protein-Oligosaccharide Maillard Reaction: Structural and Functional Characteristics

Induced Electric Field Acceleration of Whey Protein-Oligosaccharide Maillard Reaction: Structural and Functional Characteristics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Induced Electric Field Acceleration of Whey Protein-Oligosaccharide Maillard Reaction: Structural and Functional Characteristics Fei Yu, Xinyu You, Taigang Zhou, Hua Jiang, Hao Zhang, Xiaoning Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8192028/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The conjugation degree and reaction rate of the Maillard reaction are key limiting factors for its application. This study introduced an induced electric field (IEF) to effectively improve the Maillard reaction and investigated the influence of oligosaccharide type. The results showed that xylooligosaccharides (XOS) led to the highest conjugation degree and the most significant change in free sulfhydryl content with whey protein isolate (WPI) compared with galactooligosaccharides (GOS) and isomaltooligosaccharides (IMO), indicating superior Maillard reaction efficiency. Moreover, the conjugation with XOS under IEF treatment decreased the particle size from approximately 400 nm to 315 nm and shifted the zeta-potential from around − 48 mV to -63 mV, suggesting improved structural unfolding and enhanced electrostatic stabilization. WPI-XOS conjugates consistently demonstrated superior performance and were thus identified as the optimal system for detailed analysis. Structural analyses, including FTIR and free sulfhydryl content, confirmed that IEF induced significant conformational changes in the protein, thereby providing a structural basis for the enhanced Maillard reaction. The IEF treatment markedly accelerated the reaction, elevating the grafting degree of WPI-XOS from 15.03% to 25.55%. Consequently, the emulsifying activity index was enhanced from 33.03 m²/g to 58.7 m²/g, and the oil-holding capacity was significantly improved from 7.21 g/g to 18.33 g/g. Accordingly, the WPI-XOS conjugate exhibited the highest antioxidant capacity among all samples.This study confirms IEF as a superior alternative to traditional thermal and other physical processing techniques for the precision-controlled glycosylation of proteins. Maillard reaction WPI Oligosaccharide Functional features Structural characteristics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction WPI is highly regarded for its substantial nutritional benefits and functional characteristics, including emulsification and gelation (Chang et al., 2023 ; Fu et al., 2024 ; K. Zhang et al., 2024 ). It has found extensive applications across the food, pharmaceutical, and cosmetic sectors (Zhao et al., 2025 ). However, the functionality of native WPI is constrained under environmental stressors, such as fluctuations in pH and temperature, as well as structural instability, which limits its utility in complex systems (Kutzli et al., 2021 ). Recently, glycosylation facilitated by the Maillard reaction has emerged as a promising approach to enhance the functional properties of proteins (F. Liu et al., 2015 ). Nonetheless, traditional thermal methods are often impeded by extended reaction times, the formation of heterogeneous products, and uncontrolled browning, which pose challenges to their scalability in industrial applications (Yan et al., 2024 ). Electric field-assisted technology, an innovative IEF-assisted thermal process, demonstrates considerable promise for promoting efficient and selective chemical reactions through the modulation of intermolecular interactions and reaction kinetics (X. Li et al., 2025 ). Prior research has shown that electric fields can expedite the conformational rearrangements of proteins and reveal reactive sites, thus improving the efficacy of covalent conjugation with saccharides (Luparelli et al., 2025 ; You et al., 2025 ). In the present investigation, WPI and functional oligosaccharides, specifically oligo-xylose (XOS), oligo-galactose (GOS), and oligo-isomaltose (IMO), were employed as a model system (Singh et al., 2025 ). This selection was strategic, as the differences in molecular size, reducing capacity, and spatial structure between XOS (a five-carbon sugar) and GOS/IMO (six-carbon sugars) allow for a systematic investigation into the influence of saccharide structure on the IEF-assisted Maillard reaction. The novel application of this technology facilitated a comprehensive assessment of the glycoconjugates' functional characteristics, encompassing emulsification, thermal stability, and pH tolerance (Altay et al., 2023 ). The results of this research will contribute to a theoretical framework for the meticulous regulation of protein glycosylation reactions and will advance the development of high-performance functional ingredients derived from whey protein (Baruah & Borgohain, 2023 ; Sinha et al., 2007 ). 2. Materials and methods 2.1 Material WPI was procured from Shanghai Yingxin Laboratory located in Shanghai, China. GOS, IMO and XOS were manufactured by Shandong Bailong Chuangyuan Biotechnology Co., Ltd., based in Dezhou, China. The water employed in this study was ultrapure water obtained from a millipore water purification system (Merck, Germany). All chemicals and reagents were of analytical grade, unless otherwise noted. 2.2 Preparation of WPI-oligosaccharide conjugates A series of oligosaccharides in different proportions (1:1, 2:1, 3:1, and 4:1 wt%) were mixed with WPI to form a solution. The selected range of protein ratios (1:1 to 4:1) was based on preliminary experiments. When the protein ratio exceeded 4:1, the viscosity of the reaction system increased significantly, potentially leading to uneven mass transfer and decreased grafting efficiency; therefore, higher ratios were not included. The WPI-oligosaccharide solution was subjected to IEF treatment at an applied voltage of 300 V and a temperature of 80℃ for 60 minutes, as previously described by (You et al., 2025 ). The control group was an untreated mixture heated under identical water bath conditions (80°C, 60 min). The optimal ratio was established by assessing the degree of grafting. Subsequently, the treated samples underwent a 24-hour dialysis process using a membrane with a molecular weight cutoff of 8,000–10,000 Da to remove unbound sugars and salts. Following dialysis, the samples were freeze-dried for 48 hours to obtain the final WPI-oligosaccharide conjugates. 2.3. Degree of grafting measurement The grafting efficiency was evaluated by measuring free amino groups using the o-phthalaldehyde (OPA) method, incorporating a minor modification to the experimental protocol outlined by (He et al., 2021 ). Distilled water served as the blank, and subsequently, 200 µL of the coupling compound solution was combined with 4 mL of the OPA reagent. Following an incubation period of 2 minutes at 35°C, the absorbance was measured at 340 nm using a UV-Vis spectrophotometer. The formula for calculation is presented as follows: \(\:\begin{array}{c}DG\left(\%\right)=\frac{{A}_{0}-{A}_{1}}{{A}_{0}}\times\:100\%\end{array}\) 1 Where A 0 and A 1 are the concentrations of free amino groups of untreated WPI and coupling agent, respectively. 2.4 Browning intensity The reacted sample solutions were subjected to dilutions of 50 and 100 times using a 1 g/L SDS solution as the diluent, followed by thorough mixing. These diluted solutions served as controls for subsequent analyses. The absorbance at 420 nm, measured using a UV-Vis spectrophotometer, was employed to assess the degree of final browning as well as the intermediate Maillard reaction. 2.5 Particle Size and Zeta Potential Analysis The experimental protocol was adapted from (Z. Li et al., 2024a ), with minor modifications. the particle size distribution and zeta potential of the samples were evaluated using dynamic light scattering (DLS). Prior to analysis, the samples were diluted with ultrapure water to a final concentration of 1 mg/mL. Subsequently, the particle size and surface zeta potential of the protein oligosaccharide solution were analyzed using a Nano ZS90 zeta potentiostat and nanoparticle size analyzer (Malvern Instruments Limited, Worcestershire, UK). Measurements were performed with a DTS 1060 cuvette at a scattering angle of 90°, employing refractive indices of 1.450 for the protein and 1.330 for the dispersion medium. 2.6 Structural characterization by FTIR An adapted version of the method from (Wang et al., 2013 ), was used for the analysis. The samples, along with potassium bromide (KBr) powder, were subjected to drying in an oven, with a sample-to-KBr mass ratio established at 1:100. Subsequently, the mixture was uniformly ground and formed into tablets utilizing a tablet-pressing apparatus. The testing parameters were configured to encompass a wavenumber range of 400 to 4000 cm − 1 with a resolution of 5 and a frequency of 4 cm − 1 . The IR spectral analysis was performed using a Nicolet 10 FTIR spectrometer. 2.7 Determination of sulfhydryl content A total of 4.0 mL of Tris-Gly buffer solution was combined with 1.0 mL of the sample solution. Following this, 50 µL of Ellman's reagent (concentration of 4 mg/mL) was introduced gradually. The reaction was permitted to occur at ambient temperature for a duration of 15 minutes. Subsequently, the concentration was derived from the measured A₄₁₂ value with the equation: \(\:Free\:sulfhydryl\:content\left(\frac{\mu\:mol}{g}\right)=\frac{75.53\times\:D\times\:{A}_{412}}{C}\) 2 Where: A 412 is the absorbance value; D is the dilution of the sample; C is the sample concentration (mg/mL). 2.8 Antioxidant properties In this study, antioxidant activities were evaluated utilizing UV-Vis spectroscopy. The assessment focused on determining the scavenging capacities of DPPH, hydroxyl radicals (-OH), and ABTS radicals. The determination of DPPH was adapted from the method described by (X. Zhang et al., 2021 ), with measurements conducted at a wavelength of 517 nm. The hydroxyl radical scavenging activity was quantified in accordance with the methodology described by (W. Li et al., 2014 ), also recorded at 517 nm. Additionally, the free radical scavenging activity of ABTS was conducted at 734 nm using the method described by (Gao et al., 2019 ). 2.9 Assessment of emulsifying characteristics The measurement and analysis of emulsification characteristics were performed in accordance with the methodology outlined by (Gao et al., 2019 ). The electric field processing voltage used in this study is 300 V. A sample solution at a concentration of 2 mg/mL was combined with soybean oil in a volumetric ratio of 3:1 and subjected to homogenization at 12,000 rpm for a duration of 3 minutes. Emulsions were subsequently collected from the bottom of 50 µL centrifuge tubes at both 0 minutes and 30 minutes, and each sample was then mixed with 5 mL of a 0.1% (w/v) SDS solution. The absorbance of these samples was recorded at a wavelength of 500 nm. 2.10 Oil holding capacity Based on the literature method (Rodsamran & Sothornvit, 2018 ) with slight modifications. Ten milligrams of protein oligosaccharide conjugate sample was precisely weighed into individual centrifuge tubes, followed by the addition of 6.0 g of refined soybean oil. Vortex at 2,236 × g for 5 minutes, then allow it a 30-min quiescent period Then, the samples were centrifuged at 2,236× g for five minutes and the precipitates and centrifuge tubes were determined together gravimetrically. The oil holding capacity of the protein oligosaccharide solution was calculated as follows: \(\:\begin{array}{c}OHC\left(g/g\right)=\frac{{M}_{2}-{M}_{1}-{M}_{0}}{{M}_{0}}\end{array}\) 3 M 0 represents the weight of the sample (g), M 1 represents the weight of the centrifuge tube (g), M 2 represents the total weight of the centrifuge tube after centrifugation to remove the upper layer of oil or water (g). 2.11 Data processing and analysis The experiment was repeated three times and the resulting data were utilized to compute the mean and standard deviation. Statistical analysis was performed using SPSS software to conduct a significant anova, with a significance threshold set at P < 0.05. Furthermore, Origin 2018 software and GraphPad were employed for the generation of graphical representations. 3. Result analysis 3.1 Grafting Efficiency and Browning Intensity Figure 1 (A-C) displays the grafting efficiency, the grafting efficiency exhibited an initial increase corresponding with the rise in the mass ratio of oligosaccharides to WPI. Optimal grafting was observed at a mass ratio of 3:1 for all oligosaccharide-WPI conjugates. Among the three oligosaccharide-protein complexes studied, the XOS-WPI complex demonstrated the highest grafting degree, attaining a value of 25.55%. Furthermore, the grafting of WPI-XOS exhibited a 70.06% increase in the complexes obtained following IEF treatment. This enhancement is likely due to IEF's capacity to disrupt protein aggregation, thereby exposing the grafting sites on the protein and increasing the probability of grafting. It can be inferred that IEF treatment significantly facilitates the grafting of oligosaccharides to proteins, thereby promoting the spinodal reaction. As illustrated in Fig. 1 (D-F), the degree of browning in WPI-XOS was markedly more pronounced compared to that of WPI-GOS, WPI-IMO. This observation can be attributed to the greater reactivity of XOS, a five-carbon sugar, in contrast to GOS and IMO, which possess a six-carbon structure, resulting in a more vigorous Maillard reaction (Z. Li et al., 2024c ). 3.2 Particle Size and Zeta Potential The average particle sizes of WPI and its conjugates with oligosaccharides are depicted in Fig. 2 A. The average particle size of WPI conjugated with WPI-XOS prior to IEF treatment is measured at 400.45 nm, while the average particle size for WPI conjugated with WPI-GOS is 424.19 nm, and for WPI conjugated with WPI-IMO, it is 417.58 nm. Post-IEF treatment, a significant reduction in the mean particle size of WPI-XOS was observed, with a final diameter of 318.60 nm, while WPI-GOS to 355.89 nm, and while WPI-IMO to 331.23 nm. Following the Maillard reaction, a significant decrease in the particle sizes of the WPI-oligosaccharide conjugates is observed. This reduction may be attributed to the oligosaccharide moieties enhancing the spatial repulsive forces among protein molecules, thereby diminishing their propensity to aggregate. Conversely, the covalent bonding of WPI with oligosaccharides may enable Maillard reaction products to subsequently adsorb onto the droplet surfaces. Which further amplifies the repulsive forces between droplets, resulting in a decrease in particle size Z. Li et al. ( 2024b ). Among the various conjugates examined, the particle size of the WPI-XOS conjugate exhibited the most pronounced reduction, likely attributable to the highest degree of Maillard reaction observed and the substantial spatial repulsive forces exerted by the oligosaccharide molecules on the WPI molecules. This interaction contributed to a decrease in particle size. Furthermore, the average particle size of the complexes treated with IEF was significantly smaller compared to that of the untreated complexes. This phenomenon may be explained by the role of IEF in promoting the folding of WPI, while the oligosaccharide moieties enhance the spatial repulsive forces among protein molecules, thereby mitigating protein aggregation and inhibiting the formation of additional aggregates. Consequently, this results in a more stable protein solution characterized by improved dispersion stability (J. Yang et al., 2023 ). As depicted in Fig. 2 B, when compared to WPI, the absolute zeta potential values of the WPI-oligosaccharide conjugates show a significant increase, with WPI-XOS measuring − 47.32 ± 0.17 mV, WPI-GOS at -33.57 ± 0.56 mV, and WPI-IMO at -35.58 ± 0.32 mV. This increase is attributed to the incorporation of oligosaccharides, which, as a result of the Maillard reaction, reveal the negatively charged amino acids on the protein surface (Elias et al., 2006 ). While observed that a higher absolute zeta potential value correlates with enhanced repulsion between protein molecules, leading to a more stable dispersed state (Duan et al., 2018 ). In this study, WPI-XOS displayed the highest absolute potential, suggesting that the addition of XOS facilitated significant electrostatic repulsion among molecules, which effectively inhibited molecular aggregation and contributed to the stabilization of the solution system. Following the Maillard reaction induced by IEF, the conjugates exhibited a greater absolute potential in comparison to those that did not undergo IEF treatment. This increase is attributed to the structural modifications of the protein induced by IEF, which enhance surface negative charges and similarly augment intermolecular electrostatic repulsion. As a result, aggregation and flocculation are diminished, leading to improved dispersion stability (R. Yang et al., 2023 ). Therefore, the synergistic effects of IEF and the Maillard reaction can significantly enhance protein oligosaccharide solution stability. 3.3 FTIR Figure 3 presents the infrared spectra of WPI and its conjugates with three oligosaccharides, analyzed both prior to and following IEF treatment. The distinct peaks observed in the spectra are indicative of various functional groups, with the stretching vibrations of O-H groups detected within the 3150–3600 cm − 1 range. The analysis indicates that the peaks corresponding to both untreated WPI and WPI-oligosaccharide conjugates exposed to an electric field exhibit greater intensity compared to those of untreated WPI within this spectral range. This enhancement is likely attributable to the oligosaccharides, which possess multiple hydroxyl groups and carbon-hydrogen bonds. Additionally, this finding implies that effective covalent interactions have been established between the protein and the oligosaccharides (Yao et al., 2023 ). The changes in the 1650–1660 cm − 1 region reflect the transformation from disordered to ordered structures, optimizing functional active sites. After the Maillard reaction, the amide I band shows minimal change in absorption, while the amide II band undergoes a slight blue shift. This phenomenon is more pronounced after IEF treatment. This phenomenon might arise from the condensation between the protein’s free amino group and the reducing sugar’s carbonyl group during the Maillard reaction (Murray & Ettelaie, 2004 ). Collectively, the qualitative and quantitative FTIR analyses demonstrate that IEF treatment induces a dynamic reorganization of protein secondary structure, characterized by a reduction in α-helix and an increase in β-sheet content. This restructuring not only provides a favorable conformational basis for the Maillard reaction but also serves as the core mechanism for the observed enhancement in functional properties, such as emulsification. 3.4 Analysis of changes in sulfhydryl content Sulfhydryl groups are recognized as highly reactive functional groups within protein molecules, significantly influencing protein aggregation and dispersion (Bryant & McClements, 1998 ). As depicted in Fig. 4 , the content of free thiol groups in the conjugated forms of WPI, WPI-XOS, WPI-GOS, and WPI-IMO, measured without the application of IEF electric field-assisted treatment, were determined to be 11.57, 21.35, 15.31, and 27.01 µmol/g, respectively. Conversely, the concentrations of free thiol groups in the conjugates of WPI, WPI-XOS, WPI-GOS, and WPI-IMO following IEF treatment were measured at 15.59, 32.60, 24.84, and 29.29 µmol/g, respectively. The free sulfhydryl content of the Maillard reaction products demonstrated significant variations when compared to WPI. Typically, structural modifications in proteins lead to the gradual exposure of sulfhydryl groups that were previously concealed within the protein structure (Han et al., 2018 ). However, during the Maillard reaction, elevated temperature conditions induce protein denaturation and aggregation, thereby enhancing the chemical reactivity of sulfhydryl groups on the protein surface. This phenomenon can promote the interconversion between sulfhydryl groups and disulfide bonds (X. Dong et al., 2020 ). It is suggested that the electric field can influence the structure, thereby leading to this result. Importantly, the sulfhydryl content of WPI-oligosaccharide conjugates exceeds that of WPI, which can be attributed to the structural alterations in these conjugates that result in the continuous exposure of free sulfhydryl groups that were once buried within the protein molecules (Jiang et al., 2023 ). 3.5 Antioxidant properties As depicted in Fig. 5 A, compared to using WPI alone, the combination of WPI and oligosaccharides significantly enhanced free radical scavenging activity. The WPI-XOS exhibited the highest antioxidant capacity among the three models evaluated: DPPH, ABTS, and hydroxyl radicals (-OH), with respective values of 57.65%, 60.41%, and 25.36%. This enhancement is attributed to the products and intermediates generated during the Maillard reaction, which function as hydrogen donors, thereby augmenting the scavenging activity against the three types of free radicals (K. Chen et al., 2019 ). A similar conclusion can be drawn from Fig. 5 B, however, it is important to note that the products derived from the IEF assisted Maillard reaction markedly improved antioxidant capacity (S. Dong et al., 2012 ). Furthermore, the degree of grafting and the extent of browning suggest that WPI-XOS produced a greater quantity of browning products than WPI-GOS and WPI-IMO, which can explain why WPI-XOS has better antioxidant properties (Jia et al., 2020 ). 3.6 Emulsification analysis The observed enhancement in emulsifying properties is a direct result of IEF treatment, with effects significantly superior to conventional thermal treatment at the same temperature. This is primarily attributed to the IEF-induced unfolding of protein structure and the enhancement of its interfacial adsorption capacity, rather than a mere thermal effect. Emulsifying activity pertains to the capacity of proteins to engage with oil/water interfaces, which is commonly assessed through EAI and ESI (Tian et al., 2022 ). As depicted in Fig. 6 , both EAI and ESI exhibited an increase for WPI and WPI-oligosaccharide complexes that had not been subjected to IEF treatment. At treatment voltages of 300 V, the emulsifying properties of WPI and WPI-GOS,WPI-IMO.WPI-XOS complexes were recorded at 33.03 m²/g, 37.03 m²/g, 42.42 m²/g, and 38.60 m²/g, respectively. This phenomenon could be ascribed to the influence of IEF on the structural configuration of WPI, augmenting its propensity to adsorb at the oil-water interface (P. Liu et al., 2023 ). IEF-treated WPI exhibited superior emulsion stability compared to its untreated counterpart, suggesting that IEF treatment is beneficial for the emulsifying characteristics of WPI-oligosaccharide conjugates. This enhancement may stem from IEF treatment increasing the protein's affinity for the interface, thereby exposing hydrophobic groups. The elevation in β-folding induced by IEF facilitates the formation of a robust film at the oil-water interface, while the incorporation of XOS introduces a steric hindrance barrier, resulting in reduced particle sizes and thinner interface layers, which disrupts the stability of oil droplets within the emulsion. Consequently, IEF treatment can significantly improve emulsification, emulsion stability, and the stability of protein solutions for WPI-oligosaccharide conjugates. 3.7 Oil-holding capacity The oil-holding capacity of proteins pertains to their ability to absorb and retain oils through interactions with lipid molecules. This property is particularly significant in the food industry, as it influences oil distribution and emulsification (Y. Zhang et al., 2021 ). The oil-retention capability of proteins is closely linked to their structural configurations. Figure 7 highlights the oil retention characteristics of WPI and its three oligosaccharide derivatives, both prior to and following IEF treatment. Among the WPI and oligosaccharide conjugates that did not undergo IEF treatment, WPI-XOS demonstrated the highest oil-holding capacity, with an increase of 97.35% relative to WPI. This was succeeded by WPI-IMO, which exhibited a 62.50% increase compared to WPI, and WPI-GOS, which showed a 35.78% increase compared to WPI. The enhanced oil-holding capacity of WPI-XOS is attributed to the Maillard reaction, which may facilitate the unfolding of protein side chains, thereby enabling the formation of strong interactions with oil droplets and enhancing their adsorption capacity (H.-J. Zhang et al., 2012 ). Furthermore, as illustrated in the figure, both WPI and the WPI-oligosaccharide conjugate demonstrated enhanced oil retention following IEF treatment, with WPI-XOS showing a 1.54-fold increase in oil retention relative to the untreated WPI-XOS conjugate. The interactions between proteins and lipids are characterized by hydrophobic bonds, electrostatic bonds, and hydrogen bonds, with hydrophobic bonds being particularly crucial for the stability of protein-lipid complexes. This enhancement may be attributed to the exposure of hydrophobic bonds in WPI due to the electromagnetic field, which facilitates the formation of a three-dimensional network structure (Y. Chen et al., 2024 ). Consequently, proteins that possess a greater number of hydrophobic regions tend to exhibit superior oil holding capacity. 4. Conclusion In summary, this investigation effectively demonstrated that IEF constitutes a potent and controllable physical processing method to expedite the Maillard reaction between WPI and oligosaccharides. The application of IEF significantly accelerated the reaction kinetics and improved grafting efficiency, yielding values 1.8 to 2.5 times greater than those achieved through conventional thermal treatment, while concurrently mitigating excessive browning. Functional assessments indicated a marked enhancement in emulsifying activity, reaching 58.7 m²/g, which corresponds to a 68.3% increase relative to native WPI. Therefore, the IEF-assisted Maillard reaction is a promising strategy for producing high-quality protein-polysaccharide conjugates. Despite these promising results, this study was conducted on a laboratory scale. Future research should focus on scaling up the IEF process for industrial applications and evaluating the performance of these conjugates in real food model systems, such as emulsions, beverages, and encapsulation systems, to fully assess their commercial potential. Moreover, the energy consumption and economic feasibility of the IEF process compared to traditional methods warrant detailed investigation. Declarations Funding Declaration This study was supported by grants from the 2023 Shandong Province Key R&D Program (Major Science and Technology Innovation Project) (2023CXGC010414). Shandong Province small and medinm-sized scientific and techmological enterprises inmovation capacity improvement project（2025TSGCCZZB0391）. Corresponding author Hua Jiang will handle correspondence for your article at all stages of the refereeing and publication process and also post-publication. This responsibility includes answering any future queries about your results, data, methodology and materials. Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution F.Y: Conceptualization, Data curation, Writing - original draft; H.J: Funding acquisition, Project administration; T.Z: Resources, Software; X.Y: Validation, Methodology; H.Z: Formal analysis, Investigation; X.Z: Writing - review & editing; C.W: Supervision, Visualization. Data Availability All data supporting the findings of this study are available within the paper. References Altay, I., Queiroz, L. S., Silva, N. F. N., Feyissa, A. H., Casanova, F., Sloth, J. J., & Mohammadifar, M. A. (2023). Effect of Moderate Electric Fields on the Physical and Chemical Characteristics of Cheese Emulsions. 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1","display":"","copyAsset":false,"role":"figure","size":95235,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the ratio of WPI to oligosaccharide on grafting and browning\u003c/p\u003e\n\u003cp\u003eA–C represent the grafting degrees of XOS-WPI, GOS-WPI, and IMI-WPI conjugates after IEF treatment and without IEF treatment, respectively. D–F represent the browning degrees of the conjugates of XOS-WPI, GOS-WPI, and IMI-WPI that have undergone IEF treatment and those that have not.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8192028/v1/0236e16a76c7b7c4cebeaf31.png"},{"id":97987424,"identity":"2e7ecf03-bed0-4d14-85fb-8a4dfdd04ccb","added_by":"auto","created_at":"2025-12-11 14:05:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":64887,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size (A) and potential maps (B) of WPI and WPI-oligosaccharide complexes.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8192028/v1/7a6668c6feb7a98b8f5141a7.png"},{"id":97987433,"identity":"4fe35a4c-b4ba-4ad4-9076-9a72c9df856e","added_by":"auto","created_at":"2025-12-11 14:05:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34636,"visible":true,"origin":"","legend":"\u003cp\u003eFourier transform infrared spectra of WPI and three WPI-oligosaccharide conjugates before and after IEF treatment.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8192028/v1/6352888e299623864f69dc1c.png"},{"id":97987423,"identity":"bb233afd-0d5b-4178-a8d7-04efa6561799","added_by":"auto","created_at":"2025-12-11 14:05:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":55819,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in sulfhydryl content of WPI and WPI-oligosaccharide conjugates.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8192028/v1/e0b5c5f970cd1c30a970466d.png"},{"id":98424189,"identity":"6d24ab85-aa69-4912-90f3-684e7d07fbc4","added_by":"auto","created_at":"2025-12-17 16:33:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":40243,"visible":true,"origin":"","legend":"\u003cp\u003eUntreated (A) and IEF treatment (B) antioxidant activity of WPI and four WPI-oligosaccharide conjugates. Values are expressed as mean ± standard deviation; means with different letters differ significantly (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8192028/v1/b2e13c5c85e00bb266ce4fe1.png"},{"id":98423935,"identity":"62881784-1c5c-4ac2-a0c5-08096fdd684a","added_by":"auto","created_at":"2025-12-17 16:32:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":46361,"visible":true,"origin":"","legend":"\u003cp\u003eEmulsifying capacity and emulsion stability of WPI and WPI-oligosaccharide conjugates before and after IEF treatment.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8192028/v1/95512fb629c1ed1b61702c01.png"},{"id":97987428,"identity":"25c51b88-9688-4b18-aa47-c832e306bd0c","added_by":"auto","created_at":"2025-12-11 14:05:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":31914,"visible":true,"origin":"","legend":"\u003cp\u003eOil-holding properties of WPI and WPI-oligosaccharide conjugates prepared in different ways.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8192028/v1/bf2a4cc9b574f750ea0838e5.png"},{"id":98629110,"identity":"31716d96-dd59-4e37-abdf-d5002540c247","added_by":"auto","created_at":"2025-12-19 17:13:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1061995,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8192028/v1/b444649d-60df-48d7-b917-efa217076efe.pdf"}],"financialInterests":"Competing interest reported. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.","formattedTitle":"Induced Electric Field Acceleration of Whey Protein-Oligosaccharide Maillard Reaction: Structural and Functional Characteristics","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWPI is highly regarded for its substantial nutritional benefits and functional characteristics, including emulsification and gelation (Chang et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Fu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; K. Zhang et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). It has found extensive applications across the food, pharmaceutical, and cosmetic sectors (Zhao et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, the functionality of native WPI is constrained under environmental stressors, such as fluctuations in pH and temperature, as well as structural instability, which limits its utility in complex systems (Kutzli et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Recently, glycosylation facilitated by the Maillard reaction has emerged as a promising approach to enhance the functional properties of proteins (F. Liu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Nonetheless, traditional thermal methods are often impeded by extended reaction times, the formation of heterogeneous products, and uncontrolled browning, which pose challenges to their scalability in industrial applications (Yan et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eElectric field-assisted technology, an innovative IEF-assisted thermal process, demonstrates considerable promise for promoting efficient and selective chemical reactions through the modulation of intermolecular interactions and reaction kinetics (X. Li et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Prior research has shown that electric fields can expedite the conformational rearrangements of proteins and reveal reactive sites, thus improving the efficacy of covalent conjugation with saccharides (Luparelli et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; You et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In the present investigation, WPI and functional oligosaccharides, specifically oligo-xylose (XOS), oligo-galactose (GOS), and oligo-isomaltose (IMO), were employed as a model system (Singh et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This selection was strategic, as the differences in molecular size, reducing capacity, and spatial structure between XOS (a five-carbon sugar) and GOS/IMO (six-carbon sugars) allow for a systematic investigation into the influence of saccharide structure on the IEF-assisted Maillard reaction. The novel application of this technology facilitated a comprehensive assessment of the glycoconjugates' functional characteristics, encompassing emulsification, thermal stability, and pH tolerance (Altay et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The results of this research will contribute to a theoretical framework for the meticulous regulation of protein glycosylation reactions and will advance the development of high-performance functional ingredients derived from whey protein (Baruah \u0026amp; Borgohain, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sinha et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Material\u003c/h2\u003e\n \u003cp\u003eWPI was procured from Shanghai Yingxin Laboratory located in Shanghai, China. GOS, IMO and XOS were manufactured by Shandong Bailong Chuangyuan Biotechnology Co., Ltd., based in Dezhou, China. The water employed in this study was ultrapure water obtained from a millipore water purification system (Merck, Germany). All chemicals and reagents were of analytical grade, unless otherwise noted.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Preparation of WPI-oligosaccharide conjugates\u003c/h2\u003e\n \u003cp\u003eA series of oligosaccharides in different proportions (1:1, 2:1, 3:1, and 4:1 wt%) were mixed with WPI to form a solution. The selected range of protein ratios (1:1 to 4:1) was based on preliminary experiments. When the protein ratio exceeded 4:1, the viscosity of the reaction system increased significantly, potentially leading to uneven mass transfer and decreased grafting efficiency; therefore, higher ratios were not included. The WPI-oligosaccharide solution was subjected to IEF treatment at an applied voltage of 300 V and a temperature of 80℃ for 60 minutes, as previously described by (You et al., \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e). The control group was an untreated mixture heated under identical water bath conditions (80\u0026deg;C, 60 min). The optimal ratio was established by assessing the degree of grafting. Subsequently, the treated samples underwent a 24-hour dialysis process using a membrane with a molecular weight cutoff of 8,000\u0026ndash;10,000 Da to remove unbound sugars and salts. Following dialysis, the samples were freeze-dried for 48 hours to obtain the final WPI-oligosaccharide conjugates.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Degree of grafting measurement\u003c/h2\u003e\n \u003cp\u003eThe grafting efficiency was evaluated by measuring free amino groups using the o-phthalaldehyde (OPA) method, incorporating a minor modification to the experimental protocol outlined by (He et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Distilled water served as the blank, and subsequently, 200 \u0026micro;L of the coupling compound solution was combined with 4 mL of the OPA reagent. Following an incubation period of 2 minutes at 35\u0026deg;C, the absorbance was measured at 340 nm using a UV-Vis spectrophotometer. The formula for calculation is presented as follows:\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\begin{array}{c}DG\\left(\\%\\right)=\\frac{{A}_{0}-{A}_{1}}{{A}_{0}}\\times\\:100\\%\\end{array}\\)\u003c/span\u003e\u003c/span\u003e 1\u003c/p\u003e\n \u003cp\u003eWhere A\u003csub\u003e0\u003c/sub\u003e and A\u003csub\u003e1\u003c/sub\u003e are the concentrations of free amino groups of untreated WPI and coupling agent, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Browning intensity\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe reacted sample solutions were subjected to dilutions of 50 and 100 times using a 1 g/L SDS solution as the diluent, followed by thorough mixing. These diluted solutions served as controls for subsequent analyses. The absorbance at 420 nm, measured using a UV-Vis spectrophotometer, was employed to assess the degree of final browning as well as the intermediate Maillard reaction.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Particle Size and Zeta Potential Analysis\u003c/h2\u003e\n \u003cp\u003eThe experimental protocol was adapted from (Z. Li et al., \u003cspan class=\"CitationRef\"\u003e2024a\u003c/span\u003e), with minor modifications. the particle size distribution and zeta potential of the samples were evaluated using dynamic light scattering (DLS). Prior to analysis, the samples were diluted with ultrapure water to a final concentration of 1 mg/mL. Subsequently, the particle size and surface zeta potential of the protein oligosaccharide solution were analyzed using a Nano ZS90 zeta potentiostat and nanoparticle size analyzer (Malvern Instruments Limited, Worcestershire, UK). Measurements were performed with a DTS 1060 cuvette at a scattering angle of 90\u0026deg;, employing refractive indices of 1.450 for the protein and 1.330 for the dispersion medium.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Structural characterization by FTIR\u003c/h2\u003e\n \u003cp\u003eAn adapted version of the method from (Wang et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e), was used for the analysis. The samples, along with potassium bromide (KBr) powder, were subjected to drying in an oven, with a sample-to-KBr mass ratio established at 1:100. Subsequently, the mixture was uniformly ground and formed into tablets utilizing a tablet-pressing apparatus. The testing parameters were configured to encompass a wavenumber range of 400 to 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a resolution of 5 and a frequency of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The IR spectral analysis was performed using a Nicolet 10 FTIR spectrometer.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Determination of sulfhydryl content\u003c/h2\u003e\n \u003cp\u003eA total of 4.0 mL of Tris-Gly buffer solution was combined with 1.0 mL of the sample solution. Following this, 50 \u0026micro;L of Ellman\u0026apos;s reagent (concentration of 4 mg/mL) was introduced gradually. The reaction was permitted to occur at ambient temperature for a duration of 15 minutes. Subsequently, the concentration was derived from the measured A₄₁₂ value with the equation:\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Free\\:sulfhydryl\\:content\\left(\\frac{\\mu\\:mol}{g}\\right)=\\frac{75.53\\times\\:D\\times\\:{A}_{412}}{C}\\)\u003c/span\u003e\u003c/span\u003e 2\u003c/p\u003e\n \u003cp\u003eWhere: A\u003csub\u003e412\u003c/sub\u003e is the absorbance value; D is the dilution of the sample; C is the sample concentration (mg/mL).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Antioxidant properties\u003c/h2\u003e\n \u003cp\u003eIn this study, antioxidant activities were evaluated utilizing UV-Vis spectroscopy. The assessment focused on determining the scavenging capacities of DPPH, hydroxyl radicals (-OH), and ABTS radicals. The determination of DPPH was adapted from the method described by (X. Zhang et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e), with measurements conducted at a wavelength of 517 nm. The hydroxyl radical scavenging activity was quantified in accordance with the methodology described by (W. Li et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e), also recorded at 517 nm. Additionally, the free radical scavenging activity of ABTS was conducted at 734 nm using the method described by (Gao et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 Assessment of emulsifying characteristics\u003c/h2\u003e\n \u003cp\u003eThe measurement and analysis of emulsification characteristics were performed in accordance with the methodology outlined by (Gao et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). The electric field processing voltage used in this study is 300 V. A sample solution at a concentration of 2 mg/mL was combined with soybean oil in a volumetric ratio of 3:1 and subjected to homogenization at 12,000 rpm for a duration of 3 minutes. Emulsions were subsequently collected from the bottom of 50 \u0026micro;L centrifuge tubes at both 0 minutes and 30 minutes, and each sample was then mixed with 5 mL of a 0.1% (w/v) SDS solution. The absorbance of these samples was recorded at a wavelength of 500 nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10 Oil holding capacity\u003c/h2\u003e\n \u003cp\u003eBased on the literature method (Rodsamran \u0026amp; Sothornvit, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e) with slight modifications. Ten milligrams of protein oligosaccharide conjugate sample was precisely weighed into individual centrifuge tubes, followed by the addition of 6.0 g of refined soybean oil. Vortex at 2,236 \u0026times; g for 5 minutes, then allow it a 30-min quiescent period Then, the samples were centrifuged at 2,236\u0026times; g for five minutes and the precipitates and centrifuge tubes were determined together gravimetrically. The oil holding capacity of the protein oligosaccharide solution was calculated as follows:\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\begin{array}{c}OHC\\left(g/g\\right)=\\frac{{M}_{2}-{M}_{1}-{M}_{0}}{{M}_{0}}\\end{array}\\)\u003c/span\u003e\u003c/span\u003e 3\u003c/p\u003e\n \u003cp\u003eM\u003csub\u003e0\u003c/sub\u003e represents the weight of the sample (g), M\u003csub\u003e1\u003c/sub\u003e represents the weight of the centrifuge tube (g), M\u003csub\u003e2\u003c/sub\u003e represents the total weight of the centrifuge tube after centrifugation to remove the upper layer of oil or water (g).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.11 Data processing and analysis\u003c/h2\u003e\n \u003cp\u003eThe experiment was repeated three times and the resulting data were utilized to compute the mean and standard deviation. Statistical analysis was performed using SPSS software to conduct a significant anova, with a significance threshold set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Furthermore, Origin 2018 software and GraphPad were employed for the generation of graphical representations.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Result analysis","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.1 Grafting Efficiency and Browning Intensity\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (A-C) displays the grafting efficiency, the grafting efficiency exhibited an initial increase corresponding with the rise in the mass ratio of oligosaccharides to WPI. Optimal grafting was observed at a mass ratio of 3:1 for all oligosaccharide-WPI conjugates. Among the three oligosaccharide-protein complexes studied, the XOS-WPI complex demonstrated the highest grafting degree, attaining a value of 25.55%. Furthermore, the grafting of WPI-XOS exhibited a 70.06% increase in the complexes obtained following IEF treatment. This enhancement is likely due to IEF's capacity to disrupt protein aggregation, thereby exposing the grafting sites on the protein and increasing the probability of grafting. It can be inferred that IEF treatment significantly facilitates the grafting of oligosaccharides to proteins, thereby promoting the spinodal reaction. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (D-F), the degree of browning in WPI-XOS was markedly more pronounced compared to that of WPI-GOS, WPI-IMO. This observation can be attributed to the greater reactivity of XOS, a five-carbon sugar, in contrast to GOS and IMO, which possess a six-carbon structure, resulting in a more vigorous Maillard reaction (Z. Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024c\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Particle Size and Zeta Potential\u003c/h2\u003e\u003cp\u003eThe average particle sizes of WPI and its conjugates with oligosaccharides are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. The average particle size of WPI conjugated with WPI-XOS prior to IEF treatment is measured at 400.45 nm, while the average particle size for WPI conjugated with WPI-GOS is 424.19 nm, and for WPI conjugated with WPI-IMO, it is 417.58 nm. Post-IEF treatment, a significant reduction in the mean particle size of WPI-XOS was observed, with a final diameter of 318.60 nm, while WPI-GOS to 355.89 nm, and while WPI-IMO to 331.23 nm. Following the Maillard reaction, a significant decrease in the particle sizes of the WPI-oligosaccharide conjugates is observed. This reduction may be attributed to the oligosaccharide moieties enhancing the spatial repulsive forces among protein molecules, thereby diminishing their propensity to aggregate. Conversely, the covalent bonding of WPI with oligosaccharides may enable Maillard reaction products to subsequently adsorb onto the droplet surfaces. Which further amplifies the repulsive forces between droplets, resulting in a decrease in particle size Z. Li et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). Among the various conjugates examined, the particle size of the WPI-XOS conjugate exhibited the most pronounced reduction, likely attributable to the highest degree of Maillard reaction observed and the substantial spatial repulsive forces exerted by the oligosaccharide molecules on the WPI molecules. This interaction contributed to a decrease in particle size. Furthermore, the average particle size of the complexes treated with IEF was significantly smaller compared to that of the untreated complexes. This phenomenon may be explained by the role of IEF in promoting the folding of WPI, while the oligosaccharide moieties enhance the spatial repulsive forces among protein molecules, thereby mitigating protein aggregation and inhibiting the formation of additional aggregates. Consequently, this results in a more stable protein solution characterized by improved dispersion stability (J. Yang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, when compared to WPI, the absolute zeta potential values of the WPI-oligosaccharide conjugates show a significant increase, with WPI-XOS measuring \u0026minus;\u0026thinsp;47.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 mV, WPI-GOS at -33.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56 mV, and WPI-IMO at -35.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 mV. This increase is attributed to the incorporation of oligosaccharides, which, as a result of the Maillard reaction, reveal the negatively charged amino acids on the protein surface (Elias et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). While observed that a higher absolute zeta potential value correlates with enhanced repulsion between protein molecules, leading to a more stable dispersed state (Duan et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this study, WPI-XOS displayed the highest absolute potential, suggesting that the addition of XOS facilitated significant electrostatic repulsion among molecules, which effectively inhibited molecular aggregation and contributed to the stabilization of the solution system. Following the Maillard reaction induced by IEF, the conjugates exhibited a greater absolute potential in comparison to those that did not undergo IEF treatment. This increase is attributed to the structural modifications of the protein induced by IEF, which enhance surface negative charges and similarly augment intermolecular electrostatic repulsion. As a result, aggregation and flocculation are diminished, leading to improved dispersion stability (R. Yang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, the synergistic effects of IEF and the Maillard reaction can significantly enhance protein oligosaccharide solution stability.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 FTIR\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the infrared spectra of WPI and its conjugates with three oligosaccharides, analyzed both prior to and following IEF treatment. The distinct peaks observed in the spectra are indicative of various functional groups, with the stretching vibrations of O-H groups detected within the 3150\u0026ndash;3600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range. The analysis indicates that the peaks corresponding to both untreated WPI and WPI-oligosaccharide conjugates exposed to an electric field exhibit greater intensity compared to those of untreated WPI within this spectral range. This enhancement is likely attributable to the oligosaccharides, which possess multiple hydroxyl groups and carbon-hydrogen bonds. Additionally, this finding implies that effective covalent interactions have been established between the protein and the oligosaccharides (Yao et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The changes in the 1650\u0026ndash;1660 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e region reflect the transformation from disordered to ordered structures, optimizing functional active sites. After the Maillard reaction, the amide I band shows minimal change in absorption, while the amide II band undergoes a slight blue shift. This phenomenon is more pronounced after IEF treatment. This phenomenon might arise from the condensation between the protein\u0026rsquo;s free amino group and the reducing sugar\u0026rsquo;s carbonyl group during the Maillard reaction (Murray \u0026amp; Ettelaie, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Collectively, the qualitative and quantitative FTIR analyses demonstrate that IEF treatment induces a dynamic reorganization of protein secondary structure, characterized by a reduction in α-helix and an increase in β-sheet content. This restructuring not only provides a favorable conformational basis for the Maillard reaction but also serves as the core mechanism for the observed enhancement in functional properties, such as emulsification.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Analysis of changes in sulfhydryl content\u003c/h2\u003e\u003cp\u003eSulfhydryl groups are recognized as highly reactive functional groups within protein molecules, significantly influencing protein aggregation and dispersion (Bryant \u0026amp; McClements, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the content of free thiol groups in the conjugated forms of WPI, WPI-XOS, WPI-GOS, and WPI-IMO, measured without the application of IEF electric field-assisted treatment, were determined to be 11.57, 21.35, 15.31, and 27.01 \u0026micro;mol/g, respectively. Conversely, the concentrations of free thiol groups in the conjugates of WPI, WPI-XOS, WPI-GOS, and WPI-IMO following IEF treatment were measured at 15.59, 32.60, 24.84, and 29.29 \u0026micro;mol/g, respectively. The free sulfhydryl content of the Maillard reaction products demonstrated significant variations when compared to WPI. Typically, structural modifications in proteins lead to the gradual exposure of sulfhydryl groups that were previously concealed within the protein structure (Han et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, during the Maillard reaction, elevated temperature conditions induce protein denaturation and aggregation, thereby enhancing the chemical reactivity of sulfhydryl groups on the protein surface. This phenomenon can promote the interconversion between sulfhydryl groups and disulfide bonds (X. Dong et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is suggested that the electric field can influence the structure, thereby leading to this result. Importantly, the sulfhydryl content of WPI-oligosaccharide conjugates exceeds that of WPI, which can be attributed to the structural alterations in these conjugates that result in the continuous exposure of free sulfhydryl groups that were once buried within the protein molecules (Jiang et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Antioxidant properties\u003c/h2\u003e\u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, compared to using WPI alone, the combination of WPI and oligosaccharides significantly enhanced free radical scavenging activity. The WPI-XOS exhibited the highest antioxidant capacity among the three models evaluated: DPPH, ABTS, and hydroxyl radicals (-OH), with respective values of 57.65%, 60.41%, and 25.36%. This enhancement is attributed to the products and intermediates generated during the Maillard reaction, which function as hydrogen donors, thereby augmenting the scavenging activity against the three types of free radicals (K. Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A similar conclusion can be drawn from Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, however, it is important to note that the products derived from the IEF assisted Maillard reaction markedly improved antioxidant capacity (S. Dong et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, the degree of grafting and the extent of browning suggest that WPI-XOS produced a greater quantity of browning products than WPI-GOS and WPI-IMO, which can explain why WPI-XOS has better antioxidant properties (Jia et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Emulsification analysis\u003c/h2\u003e\u003cp\u003eThe observed enhancement in emulsifying properties is a direct result of IEF treatment, with effects significantly superior to conventional thermal treatment at the same temperature. This is primarily attributed to the IEF-induced unfolding of protein structure and the enhancement of its interfacial adsorption capacity, rather than a mere thermal effect. Emulsifying activity pertains to the capacity of proteins to engage with oil/water interfaces, which is commonly assessed through EAI and ESI (Tian et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, both EAI and ESI exhibited an increase for WPI and WPI-oligosaccharide complexes that had not been subjected to IEF treatment. At treatment voltages of 300 V, the emulsifying properties of WPI and WPI-GOS,WPI-IMO.WPI-XOS complexes were recorded at 33.03 m\u0026sup2;/g, 37.03 m\u0026sup2;/g, 42.42 m\u0026sup2;/g, and 38.60 m\u0026sup2;/g, respectively. This phenomenon could be ascribed to the influence of IEF on the structural configuration of WPI, augmenting its propensity to adsorb at the oil-water interface (P. Liu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). IEF-treated WPI exhibited superior emulsion stability compared to its untreated counterpart, suggesting that IEF treatment is beneficial for the emulsifying characteristics of WPI-oligosaccharide conjugates. This enhancement may stem from IEF treatment increasing the protein's affinity for the interface, thereby exposing hydrophobic groups. The elevation in β-folding induced by IEF facilitates the formation of a robust film at the oil-water interface, while the incorporation of XOS introduces a steric hindrance barrier, resulting in reduced particle sizes and thinner interface layers, which disrupts the stability of oil droplets within the emulsion. Consequently, IEF treatment can significantly improve emulsification, emulsion stability, and the stability of protein solutions for WPI-oligosaccharide conjugates.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Oil-holding capacity\u003c/h2\u003e\u003cp\u003eThe oil-holding capacity of proteins pertains to their ability to absorb and retain oils through interactions with lipid molecules. This property is particularly significant in the food industry, as it influences oil distribution and emulsification (Y. Zhang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The oil-retention capability of proteins is closely linked to their structural configurations. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e highlights the oil retention characteristics of WPI and its three oligosaccharide derivatives, both prior to and following IEF treatment. Among the WPI and oligosaccharide conjugates that did not undergo IEF treatment, WPI-XOS demonstrated the highest oil-holding capacity, with an increase of 97.35% relative to WPI. This was succeeded by WPI-IMO, which exhibited a 62.50% increase compared to WPI, and WPI-GOS, which showed a 35.78% increase compared to WPI. The enhanced oil-holding capacity of WPI-XOS is attributed to the Maillard reaction, which may facilitate the unfolding of protein side chains, thereby enabling the formation of strong interactions with oil droplets and enhancing their adsorption capacity (H.-J. Zhang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, as illustrated in the figure, both WPI and the WPI-oligosaccharide conjugate demonstrated enhanced oil retention following IEF treatment, with WPI-XOS showing a 1.54-fold increase in oil retention relative to the untreated WPI-XOS conjugate. The interactions between proteins and lipids are characterized by hydrophobic bonds, electrostatic bonds, and hydrogen bonds, with hydrophobic bonds being particularly crucial for the stability of protein-lipid complexes. This enhancement may be attributed to the exposure of hydrophobic bonds in WPI due to the electromagnetic field, which facilitates the formation of a three-dimensional network structure (Y. Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consequently, proteins that possess a greater number of hydrophobic regions tend to exhibit superior oil holding capacity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn summary, this investigation effectively demonstrated that IEF constitutes a potent and controllable physical processing method to expedite the Maillard reaction between WPI and oligosaccharides. \u0026nbsp;The application of IEF significantly accelerated the reaction kinetics and improved grafting efficiency, yielding values 1.8 to 2.5 times greater than those achieved through conventional thermal treatment, while concurrently mitigating excessive browning. Functional assessments indicated a marked enhancement in emulsifying activity, reaching 58.7 m\u0026sup2;/g, which corresponds to a 68.3% increase relative to native WPI. Therefore, the IEF-assisted Maillard reaction is a promising strategy for producing high-quality protein-polysaccharide conjugates. Despite these promising results, this study was conducted on a laboratory scale. Future research should focus on scaling up the IEF process for industrial applications and evaluating the performance of these conjugates in real food model systems, such as emulsions, beverages, and encapsulation systems, to fully assess their commercial potential. Moreover, the energy consumption and economic feasibility of the IEF process compared to traditional methods warrant detailed investigation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the 2023 Shandong Province Key R\u0026amp;D Program (Major Science and Technology Innovation Project) (2023CXGC010414).\u003c/p\u003e\n\u003cp\u003eShandong Province small and medinm-sized scientific and techmological enterprises inmovation capacity improvement project（2025TSGCCZZB0391）.\u003c/p\u003e\n\u003ch2\u003eCorresponding author\u003c/h2\u003e\n\u003cp\u003eHua Jiang will handle correspondence for your article at all stages of the refereeing and publication process and also post-publication. This responsibility includes answering any future queries about your results, data, methodology and materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eF.Y: Conceptualization, Data curation, Writing - original draft; H.J: Funding acquisition, Project administration; T.Z: Resources, Software; X.Y: Validation, Methodology; H.Z: Formal analysis, Investigation; X.Z: Writing - review \u0026amp; editing; C.W: Supervision, Visualization.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAltay, I., Queiroz, L. 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Recent Advances, Challenges, and Functional Applications of Protein Chemical Modification in the Food Industry. \u003cem\u003eFoods\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(16), 2784. https://doi.org/10.3390/foods14162784\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Maillard reaction, WPI, Oligosaccharide, Functional features, Structural characteristics","lastPublishedDoi":"10.21203/rs.3.rs-8192028/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8192028/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe conjugation degree and reaction rate of the Maillard reaction are key limiting factors for its application. This study introduced an induced electric field (IEF) to effectively improve the Maillard reaction and investigated the influence of oligosaccharide type. The results showed that xylooligosaccharides (XOS) led to the highest conjugation degree and the most significant change in free sulfhydryl content with whey protein isolate (WPI) compared with galactooligosaccharides (GOS) and isomaltooligosaccharides (IMO), indicating superior Maillard reaction efficiency. Moreover, the conjugation with XOS under IEF treatment decreased the particle size from approximately 400 nm to 315 nm and shifted the zeta-potential from around \u0026minus;\u0026thinsp;48 mV to -63 mV, suggesting improved structural unfolding and enhanced electrostatic stabilization. WPI-XOS conjugates consistently demonstrated superior performance and were thus identified as the optimal system for detailed analysis. Structural analyses, including FTIR and free sulfhydryl content, confirmed that IEF induced significant conformational changes in the protein, thereby providing a structural basis for the enhanced Maillard reaction. The IEF treatment markedly accelerated the reaction, elevating the grafting degree of WPI-XOS from 15.03% to 25.55%. Consequently, the emulsifying activity index was enhanced from 33.03 m\u0026sup2;/g to 58.7 m\u0026sup2;/g, and the oil-holding capacity was significantly improved from 7.21 g/g to 18.33 g/g. Accordingly, the WPI-XOS conjugate exhibited the highest antioxidant capacity among all samples.This study confirms IEF as a superior alternative to traditional thermal and other physical processing techniques for the precision-controlled glycosylation of proteins.\u003c/p\u003e","manuscriptTitle":"Induced Electric Field Acceleration of Whey Protein-Oligosaccharide Maillard Reaction: Structural and Functional Characteristics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 14:05:52","doi":"10.21203/rs.3.rs-8192028/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"719be220-0c87-42e6-bbc5-1dbcb6466ce4","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-19T14:39:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 14:05:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8192028","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8192028","identity":"rs-8192028","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}