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. 2026 Oct 19;40:104658. doi: 10.1016/j.fochx.2026.104658

Ultrasound-assisted free radical grafting of sesamol onto whey protein isolate: Structural modifications, functional enhancements, and antioxidant properties

Xiandan Feng 1,1, Mengting Lv 1,1, Mu Tian 1, Sining Li 1,⁎, Shanhu Tang 1,⁎
PMCID: PMC13717039  PMID: 42959313

Abstract

This study fabricated conjugates of whey protein isolate (WPI) and sesamol using an ultrasound-assisted free radical method. This approach significantly reduced the reaction time from 24 h to 90 min relative to the conventional free radical method, while achieving a comparable sesamol binding equivalent (4.65 mg/g). Structural analyses (amino acid side-chains, FTIR, XRD, surface hydrophobicity) revealed chemical modifications, as supported by reduced free thiol/amino groups and altered protein conformation under ultrasound. Molecular docking suggested potential hydrogen bonds and hydrophobic interactions between sesamol and WPI. Particle size and ζ-potential measurements revealed formation of larger, stable aggregates. Furthermore, WPI-sesamol conjugates exhibited superior functional properties, including protein solubility and emulsifying capacity, as well as enhanced antioxidant activity (DPPH• and ABTS+• scavenging), with ultrasound-assisted treatment yielding the most pronounced improvements. The ultrasound-assisted method establishes an efficient strategy for producing WPI-sesamol conjugates with superior functionality and antioxidant capacity, highlighting their potential as novel food ingredients.

Keywords: Whey protein isolate, Sesamol, Ultrasonication, Covalent interaction, Structural conformation

Highlights

  • •

    Whey protein isolate-sesamol conjugates were prepared via ultrasound-assisted method.

  • •

    Covalent conjugation with sesamol improved the crystallinity of the conjugates.

  • •

    Molecular docking showed H-bonds and hydrophobic interactions as non-covalent forces.

  • •

    Ultrasound-assisted conjugation improved solubility and emulsifying properties.

  • •

    Ultrasound-assisted conjugation increased whey protein isolate antioxidant activity.

1. Introduction

Whey proteins are milk-derived proteins mainly comprising β-lactoglobulin (18.4 kDa, pI 5.35–5.49), α-lactalbumin (14.0 kDa, pI 4.5–4.8), and bovine serum albumin (66.0 kDa, pI 4.7–5.1) (Chen et al., 2023). They are extensively used in food formulations due to their desirable nutritional value and versatile functional properties, combined with their cost-effectiveness and ready availability (Chen et al., 2023; Tian et al., 2023). Furthermore, whey proteins exhibit a broad-spectrum affinity for various bioactive compounds, including polyphenols, making them an ideal and versatile delivery platform for constructing advanced nutrient protection and delivery systems (Liang et al., 2024). However, the application of whey protein alone is often limited by its tendency to aggregate and lose solubility in response to changes in physical factors such as pH, ionic strength, or temperature during processing (Baba, McClements, & Maqsood, 2021). Therefore, developing strategies to modify whey protein structure for improved functionality has emerged as a major research focus.

Several studies have established that creating polyphenol-grafted proteins is a highly effective strategy for enhancing functional properties, particularly heat resistance, antioxidant activity, and emulsifying performance (Baba, Abdelrahman, & Maqsood, 2021; Baba, McClements, & Maqsood, 2021; Manzoor et al., 2025). These improvements arise from protein-polyphenol interactions, which occur through either non-covalent or covalent binding (Ozdal et al., 2013). In comparison with non-covalent interactions, covalent binding is particularly notable for inducing irreversible structural modifications that ensure high techno-functional stability under a wide range of environmental stresses, representing a decisive advantage for industrial applications (Liang et al., 2024; Song et al., 2024). Covalent grafting of polyphenols to proteins can be achieved via alkaline, enzymatic, or free radical-mediated methods. Alkaline treatment often causes browning and structural damage to both proteins and polyphenols, while enzymatic methods are costly and enzyme-dependent. In contrast, the free radical-mediated method offers a cost-effective and non-toxic alternative and is therefore preferred (Baba, Abdelrahman, & Maqsood, 2021; Zheng et al., 2023). Sesamol (3,4-methylenedioxyphenol) is a characteristic natural polyphenol and bioactive lignan derived from sesame seeds, with a molecular weight of 138.12 Da and a pKa of 9.79 (Majdalawieh & Mansour, 2019). Its molecule contains a phenolic hydroxyl group, which provides antioxidant activity by donating a hydrogen atom or an electron to form stable free radicals (Joshi et al., 2005). Sesamol also exhibits other health-promoting properties, such as anti-inflammatory, anti-aging, and chemopreventive effects (Majdalawieh & Mansour, 2019). These attributes highlight its potential in the food and pharmaceutical industries. Nevertheless, its practical application is hampered by low aqueous solubility and susceptibility to degradation or oxidation upon exposure to light, heat, and similar environmental factors (Basurto et al., 2018; Feng et al., 2024). Fortunately, polyphenol-protein binding can effectively elevate the solubility and stability of polyphenols (Liu et al., 2021; Song et al., 2024).

Ultrasonication is a promising technology with widespread applications in the processing and modification of proteins (Pandita et al., 2024). Ultrasound facilitates the unfolding of protein structures by combining cavitation and mechanical effects, consequently enhancing their capacity for covalent binding with polyphenols (Nwachukwu & Aluko, 2019; Pandita et al., 2024). Researchers have demonstrated that ultrasonication can not only successfully accomplish protein-polyphenol grafting, but also considerably augment the emulsifying and foaming properties of the proteins (Baba, Abdelrahman, & Maqsood, 2021; Manzoor et al., 2025; Wei et al., 2024). In particular, ultrasound-assisted free radical mediation treatment could accelerate the covalent reaction between proteins and polyphenols, leading to a shortened grafting time compared to conventional free radical reaction (Zheng et al., 2023). Although ultrasound-assisted grafting has demonstrated significant efficacy in many protein-polyphenol systems, its application to the whey protein isolate (WPI)-sesamol system remains unexplored. This is particularly important because the conventional free radical method requires prolonged reaction times that severely limit industrial scalability. Hence, a time-efficient ultrasound-assisted approach for fabricating WPI-sesamol conjugates is highly desirable.

To address this research gap, our study systematically investigated the ultrasound-assisted free radical grafting method for fabricating WPI-sesamol conjugates. The modifications were characterized by assessing the binding capacity, structural changes, physicochemical and functional properties, and antioxidant activity of the conjugates, thereby providing a theoretical foundation for understanding the synergistic mechanisms between food functional components.

2. Materials and methods

2.1. Materials

Whey protein isolate (WPI, ≥90% protein) was supplied by ShaanXi BioColex Co., Ltd. (Xi'an, China). Sesamol (98% purity), ortho-phthalaldehyde (OPA), β-mercaptoethanol, 8-anilino-1-naphthalenesulfonic acid (ANS), Folin-Ciocalteu reagent, soybean oil, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). l-ascorbic acid, sodium dodecyl sulfate (SDS), and 5,5′-dithio bis (2-nitrobenzoic acid) (DTNB) were supplied by Macklin Biotechnology Co., Ltd. (Shanghai, China). Dialysis bags (molecular weight: 8000–14,000 Da) were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). All other chemical reagents were of analytical grade and purchased from Chengdu Kelong Chemical Co., Ltd. (Chengdu, China).

2.2. Preparation of the WPI-sesamol conjugates

The WPI-sesamol conjugates were prepared using the ultrasound-assisted free radical grafting method (Zheng et al., 2023). Briefly, 1 g of WPI was dissolved in 49 mL of 20 mmol/L phosphate buffer (pH 7.2) and hydrated in the dark for 2 h. Subsequently, 0.03% (w/v) sodium azide was added to inhibit microbial growth. Then, 1 mL of 5 mol/L hydrogen peroxide and 0.25 g of ascorbic acid were introduced, and the reaction proceeded under magnetic stirring for 2 h. Separately, sesamol was dissolved in 2 mL of 30% (v/v) ethanol and then diluted with 48 mL of 20 mmol/L phosphate buffer (pH 7.2). The sesamol solution was mixed with an equal volume of the WPI solution to achieve final concentrations of 10 mg/mL for WPI and 2 mg/mL for sesamol. The mixture was immediately subjected to ultrasonic treatment (SB-5200DT, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) at 240 W and 40 kHz for different durations (30, 60, 90, and 120 min). The entire ultrasonication process was conducted in an ice bath to avoid the thermal effects of ultrasound. After ultrasonication, the samples were dialyzed against deionized water using dialysis membranes (molecular weight cut-off: 8000–14,000 Da) for 48 h to remove unbound sesamol, with deionized water replaced at 6-h intervals. Finally, the samples were pre-frozen and vacuum-lyophilized for 48 h, and the resulting powders were stored in a desiccator for further use. The WPI-sesamol conjugates prepared with ultrasonic treatment for 30, 60, 90, and 120 min were designated as U/WPI-SE-30, U/WPI-SE-60, U/WPI-SE-90, and U/WPI-SE-120, respectively. The mixture of WPI and sesamol was continuously stirred and incubated for 24 h using the conventional free radical grafting method (without ultrasonic treatment), serving as the C/WPI-SE control. Additionally, native WPI was treated under the same conventional conditions but without sesamol and was labeled as the WPI control.

2.3. Binding equivalents of sesamol

The Folin-Ciocalteu assay was employed to determine the sesamol binding equivalents according to the method of Zhang et al. (2022). Aliquots of 0.5 mL of 10-fold diluted WPI-sesamol conjugates were added to 2.5 mL of 0.2 mol/L Folin-Ciocalteu reagent. After thorough mixing, the mixture was incubated protected from light for 5 min. Then, 2 mL of 7.5% (w/v) sodium carbonate was added, and the reaction mixture was further incubated in a water bath at 40 °C for 60 min in the dark. Finally, the absorbance of resulting solution was recorded at 760 nm using a UV spectrophotometer (AOE Instruments Co., Ltd., Shanghai, China). The phenolic contents of the conjugates were determined using a sesamol standard curve (0–0.10 mg/mL) and were expressed as sesamol equivalents.

2.4. Determination of amino acid side-chain groups

2.4.1. Active thiol group

The active thiol groups were measured according to the method of Zheng et al. (2023) with slight modifications. Specifically, 0.5 mL of the WPI-sesamol conjugates was mixed with 4.5 mL of Tris-glycine buffer (pH 8.0) containing 0.5% (w/v) SDS, 0.086 mol/L Tris, 0.092 mol/L glycine, and 0.004 mol/L EDTA. Afterward, 0.5 mL of 4 mg/mL DTNB (dissolved in Tris-glycine buffer, pH 8.0) was added, and the mixture was incubated for 60 min in the dark. The absorbance was recorded at 412 nm. A blank control was prepared by replacing the conjugate sample with an equal volume of Tris-glycine buffer (pH 8.0). The active thiol groups (μmol/g protein) were calculated using Eq. (1):

Active thiol groupμmol/gprotein=73.53×A×DC (1)

where coefficient 73.53 is derived from 106/(1.36 × 104), accounting for the molar absorption coefficient of 1.36 × 104 L/(mol•cm) and the factor of 106 for converting mol to μmol and mg to g, A is the absorbance at 412 nm, D is the dilution factor, and C is the protein concentration (mg/mL).

2.4.2. Free amino group

The free amino content was determined using the OPA method (Liu et al., 2025). An OPA solution was formulated by dissolving 80 mg OPA in 2 mL of methanol, then adding 5 mL of 20% (w/v) SDS solution, 200 μL of β-mercaptoethanol, and 50 mL of 0.1 mol/L borax solution, followed by dilution to 100 mL with deionized water. For the assay, 50 μL of the WPI-sesamol conjugates was mixed with 1 mL OPA reagent and subsequently incubated at 37 °C for 2 min prior to absorbance measurement at 340 nm. The free amino groups (nmol/mg) were calculated from a standard curve plotted with l-lysine.

2.5. Fourier-transform infrared spectroscopy (FTIR) analysis

Following the method described in our previous study (Ni et al., 2025), the FTIR spectra were acquired using a Perkin Elmer spectrophotometer (Massachusetts, USA). In brief, the freeze-dried WPI-sesamol conjugates were mixed with KBr at a mass ratio of 1:100 (w/w), and spectral scans were conducted over the range of 4000–400 cm−1 at a resolution of 4 cm−1.

2.6. X-ray diffraction (XRD) analysis

The crystalline properties of the WPI-sesamol conjugates were analyzed using a Bruker D6 PHASER diffractometer (Germany) by employing Cu-Kα radiation (λ = 1.5406 Å) under the operational conditions of 40 kV and 40 mA (Yan et al., 2023). The measurements were performed across a 2θ interval of 5°–40° with a scanning speed of 2°/min.

2.7. Determination of surface hydrophobicity

Surface hydrophobicity was assessed based on the method of Kato and Nakai (1980) with some modifications, using ANS as a fluorescent probe. Specifically, 4 mL of the conjugates (0.2 mg/mL in deionized water) and 20 μL of 8 mmol/L ANS solution (dissolved in 10 mmol/L phosphate buffer, pH 7.0) were combined. After thorough vortexing, the mixture was left to stand for 3 min. Fluorescence intensity was monitored at 390 nm excitation and 470 nm emission, with a slit width of 5 nm.

2.8. Molecular docking analysis

The molecular structures of β-lactoglobulin (uniprot ID: P02754), α-lactalbumin (PDB ID: 1HFZ), and sesamol (PubChem CID: 68289) were retrieved from the UniProt database (https://www.uniprot.org/), RCSB Protein Data Bank (https://www.rcsb.org/), and PubChem database (https://pubchem.ncbi.nlm.nih.gov/), respectively. Molecular docking of WPI (with its primary proteins β-lactoglobulin and α-lactalbumin) and sesamol was performed using Molecular Operating Environment (MOE) 2019. Energy minimization was conducted using the AMBER14:EHT force field. Ligand placement was performed using the Triangle Matcher method to generate 30 poses, which were scored using the London dG function, followed by rescoring with GBVI/WSA dG. The mode with the lowest binding energy calculated by MOE 2019 was selected as the optimal binding mode for subsequent visualization and analysis using PyMOL.

2.9. Determination of particle size and ζ-potential

Measurements for particle size and ζ-potential were obtained with a Zetasizer Nano ZS90 analyzer (Malvern Instruments Ltd., UK) at 25 °C. After 100-fold dilution in deionized water, the samples were measured using refractive index values of 1.46 for the protein particle and 1.33 for the dispersed phase.

2.10. Determination of functional properties

2.10.1. Protein solubility

The protein solubility was determined following the method previously used in our team (Feng et al., 2024). Briefly, a WPI suspension was centrifuged at 10,000×g for 20 min. The protein concentration in the resulting supernatant was determined using the Biuret method. The solubility was calculated as the percentage of protein remaining in the supernatant relative to the total protein pre-centrifugation.

2.10.2. Emulsifying properties

The emulsification activity index (EAI) and emulsion stability index (ESI) of the samples were determined according to previous publication (Wei et al., 2024) with slight modifications. A 15 mL protein solution was mixed with 5 mL of soybean oil and homogenized (Scientz Biotechnology Co. Ltd., Ningbo, China) at 12,000 rpm for 3 min. Following, 50 μL aliquots were taken from the bottom of the homogenate at 0 and 10 min, and each was diluted in 5 mL of a 0.1% (w/v) SDS solution. The diluted emulsions were vortexed, and their optical density was measured at 500 nm. The EAI and ESI were then determined according to the Eqs. (2), (3):

EAIm2/g=2×2.303×A0×DFC×f×10000 (2)
ESImin=A0A0−A10×10 (3)

where A0 is the optical density of the emulsion at 0 min, A10 is the optical density of the emulsion at 10 min, DF is the dilution factor (101), C is the protein concentration (g/mL), f was the oil volume fraction (0.25).

2.11. Determination of antioxidant capacity

2.11.1. DPPH radical scavenging activity

The DPPH• scavenging capacity was assessed as previously described (Zheng et al., 2023). A mixture containing the protein sample and an equal volume of 0.2 mmol/L DPPH (in 95% ethanol) was prepared, followed by incubation at room temperature for 30 min in the dark compartment. Absorbance was subsequently recorded at 517 nm. The scavenging activity was calculated using the following Eq. (4):

Scavenging rate%=Ab−As−AcAb×100 (4)

where Ac is the absorbance of 95% ethanol with protein samples, As is the absorbance of the protein samples, Ab is the absorbance of 95% ethanol with DPPH solution.

2.11.2. ABTS+ radical scavenging activity

The ABTS+• scavenging assay was performed using the method reported by Siddhuraju (2006). The ABTS+• working solution was prepared by reacting 10 mg ABTS with 2.6 mL of 2.45 mmol/L potassium persulfate for 16 h in the dark and diluting with deionized water to an absorbance of 0.70 ± 0.02 at 734 nm. For the assay, a mixture of 0.2 mL protein sample and 4 mL ABTS+• working solution was prepared and then allowed to react for 60 min at room temperature in the dark compartment. The absorbance of resulting mixture was read at 734 nm. The ABTS+• scavenging activity was calculated with the following Eq. (5):

Scavenging rate%=A1−A2A1×100 (5)

where A1 is the absorbance of ABTS+• solution without protein samples, A2 is the absorbance of ABTS+• solution with protein samples.

2.12. Statistical analysis

All experiments were conducted in triplicate, and the results were expressed as mean ± standard deviation (SD). Data were processed using SPSS 21.0 software by one-way analysis of variance (ANOVA). A P-value of less than 0.05 was deemed statistically significant.

3. Result and discussion

3.1. The binding capacity of sesamol

The sesamol contents of WPI-sesamol conjugates were quantified to estimate the apparent amount of sesamol associated with WPI after extensive dialysis. As depicted in Fig. 1A, the sesamol binding equivalent of C/WPI-SE samples was 4.68 mg/g. The corresponding values in U/WPI-SE conjugates were 4.48, 4.58, 4.65 and 4.73 mg/g for 30, 60, 90 and 120 min of ultrasonication time, respectively, indicating effective interaction between sesamol and WPI. The observed increase in sesamol binding equivalents with the extension of sonication time in U/WPI-SE conjugates is likely attributed to the combined mechanical and cavitation action of the ultrasound field. This process promotes both free hydroxyl radical generation and protein molecular unfolding, thus increasing the reaction possibility of WPI and sesamol (Zhang et al., 2022; Zheng et al., 2023). In contrast, Zheng et al. (2023) reported a decrease in the binding degree of ovalbumin and epigallocatechin gallate when ultrasonic time exceeded 90 min. They attributed this reduction to protein denaturation and aggregation, as well as polyphenol decomposition, caused by prolonged ultrasonication, which ultimately impaired effective binding. These discrepant results likely arise from the distinct nature of the proteins and polyphenols involved, along with the ultrasonication parameters. Notably, unlike Zheng et al. (2023), we observed no decline in sesamol binding equivalents within 120 min. This is likely due to the stable benzodioxole structure of sesamol, which is less prone to ultrasound-induced degradation than epigallocatechin gallate. Moreover, rapid conjugation with WPI may protect sesamol from radical attack. The U/WPI-SE conjugates obtained after 90–120 min of ultrasonication exhibited no significant difference (P > 0.05) relative to those produced by conventional free radical methods. These results suggested that ultrasonication significantly accelerated the interaction between WPI and sesamol, and shortened the reaction time from 24 h to 90 min without compromising the binding efficiency.

Fig. 1.

Fig. 1

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Sesamol binding equivalent (A), active sulfhydryl group (B) and free amino group (C) of WPI-sesamol conjugates. C/WPI-SE: WPI-sesamol conjugates prepared by conventional free radical method. U/WPI-SE-30, U/WPI-SE-60, U/WPI-SE-90, and U/WPI-SE-120: WPI-sesamol conjugates prepared by ultrasound-assisted free radical grafting method with ultrasound durations of 30, 60, 90, and 120 min, respectively. WPI: whey protein isolate; SE: sesamol. Error bars represent the standard deviation (n = 3). Bars with lowercase letters (a–d) differ significantly.

3.2. Active thiol and free amino groups

The thiol and amino groups of proteins readily react with phenolic compounds, resulting in changes in the contents of these groups (Ozdal et al., 2013). The results of ultrasound-assisted sesamol binding on the active thiol and free amino content of WPI are shown in Fig. 1B and C. In contrast with WPI control, the values of active thiol and free amino groups of both C/WPI-SE and U/WPI-SE conjugates were remarkably decreased (P < 0.05). This reduction is suggested to be partially attributable to the covalent grafting of sesamol, in which its hydroxyl groups may participate in forming C—S or C—N bonds with the thiol (SH) and amino (NH2) groups of the proteins. A plausible mechanism for this grafting is supported by previous reports indicating that oxidized phenolic compounds are capable of reacting with nucleophilic residues in proteins, such as amino groups, tryptophan, cysteine, and histidine (Yan et al., 2023). The interaction between WPI and sesamol hindered the binding of DTNB to thiol groups and OPA to amino groups, respectively, resulting in reduced detectable levels of both active thiol and free amino groups (Feng et al., 2024). This observation is consistent with the previous publication that the free thiol and amino groups were lost when WPI was covalently modified with tea polyphenols (Zheng et al., 2022). It should be noted that mild oxidation of thiol and amino residues may also partially account for the observed decreases. Nevertheless, the positive correlation with dialysis-retained sesamol content suggests that covalent conjugation remains a significant mechanism, although the evidence is indirect. Concerning the ultrasound-assisted conjugates, the loss of active thiol and free amino groups in the samples was more pronounced (P < 0.05) as ultrasound time increased from 30 to 120 min, suggesting that ultrasonication effectively promoted the binding of sesamol to WPI (Wei et al., 2024; Zheng et al., 2023). Ultrasound-induced mechanical shear and cavitation effects could cause the proteins to unfold and expose the hidden thiol and amino groups buried inside the protein molecules, which provide more available grafting sites for sesamol to proteins (Manzoor et al., 2025; Wei et al., 2024).

3.3. FTIR spectroscopy and X-ray diffraction

FTIR spectroscopy serves as a sensitive probe for changes in protein conformations (Wei et al., 2024). As depicted in Fig. 2A, the WPI control showed prominent peaks at 3272 cm−1 (amide A), 1631 cm−1 (amide I) and 1520 cm−1 (amide II). However, all WPI-sesamol conjugates underwent notable changes in the shape and positions of their FTIR peaks relative to WPI, suggesting that sesamol binding to WPI may have induced a secondary structural rearrangement and conformational transition in the proteins. For the C/WPI-SE conjugates, the amide I (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O stretching) and amide II (N—H bending and C—N stretching) bands blue-shifted to 1634 cm−1 and 1526 cm−1, respectively, from 1631 cm−1 and 1520 cm−1 in the WPI control. This can be attributed to the conformational changes upon conjugation between WPI and sesamol, as supported by the concomitant decrease in active thiol and free amino groups (Fig. 1B and C). Similar findings were reported by Guo et al. (2023), who found a shift to higher wavenumbers for the amide I and II bands after rutin bound to WPI. In contrast, Liu et al. (2021) documented a red shift in these bands following the binding of epigallocatechin gallate or quercetin to WPI. This discrepancy is primarily attributable to the distinct structures and varying phenolic hydroxyl content of the polyphenols involved. Notably, the U/WPI-SE-120 conjugates exhibited an even more pronounced shift in the amide II band (to 1529 cm−1), indicating that polyphenol association coupled with ultrasonication induced more extensive protein structural changes. The intense physical forces generated by ultrasound promote a more profound and rapid partial unfolding of WPI, thereby exposing a greater number of reactive sites for subsequent conjugation with sesamol. Furthermore, the binding of sesamol led to significant changes in the fingerprint region (1200–800 cm−1) of all WPI-sesamol conjugates, which suggests an alteration in hydrogen bonds, π-π stacking, dipole-dipole interactions, or other non-covalent interactions (Zheng et al., 2023). The secondary structure content (α-helix, β-sheet, β-turn, and random coil) obtained from amide I deconvolution is as plotted in Fig. 2B. Compared to the WPI control, grafting of sesamol via the conventional free radical method resulted in a decrease in α-helix and β-turn content but an increase in β-sheet and random coil content, indicating partial stretching and rearrangement of the WPI structure. Similar trends were observed for conjugates between β-lactoglobulin and dicaffeoylquinic acid (Song et al., 2024). The ultrasound-assisted conjugates (90 and 120 min) exhibited similar α-helix, β-sheet and β-turn content, but a lower (P < 0.05) random coil content compared with C/WPI-SE conjugates, although the random coil content remained higher (P < 0.05) than that of the WPI control. These structural changes suggest that turbulence and shear forces arising from cavitation induced protein structural rearrangement.

Fig. 2.

Fig. 2

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The FTIR spectra (A), secondary structure content (B), XRD patterns (C) and surface hydrophobicity (D) of WPI-sesamol conjugates. C/WPI-SE: WPI-sesamol conjugates prepared by conventional free radical method. U/WPI-SE-30, U/WPI-SE-60, U/WPI-SE-90, and U/WPI-SE-120: WPI-sesamol conjugates prepared by ultrasound-assisted free radical grafting method with ultrasound durations of 30, 60, 90, and 120 min, respectively. WPI: whey protein isolate; SE: sesamol. Error bars represent the standard deviation (n = 3). Bars with lowercase letters (a–e) differ significantly.

The changes in the XRD patterns of WPI after binding with sesamol were evaluated by monitoring the crystallinity. The XRD spectra of the WPI-sesamol conjugates were comparable to those of the WPI control (Fig. 2C). Two characteristic peaks around 10° and 20° were observed in the diffractograms of WPI control, as well as C/WPI-SE and U/WPI-SE conjugates, commonly corresponding to the packing arrangements of α-helix and β-sheet, respectively (Mohammadi et al., 2023). After grafting of sesamol to WPI, the diffraction peak intensity of WPI-sesamol conjugates increased. This aligned with the findings presented by Wang et al. (2025) who discovered the increased diffraction peak intensity of β-casein and β-lactoglobulin after interaction with rutin. Yan et al. (2021) and Han et al. (2025) reported that the increase in the intensity of diffraction peak was due to the formation of new chemical bonds between proteins and polyphenols, enhancing the regular arrangement of protein molecules. The broad WPI diffraction peaks around 20° transitioned to sharp peaks, the intensity of which became increasingly pronounced and correlated directly with a higher sesamol grafting degree in the U/WPI-SE conjugates. According to Yu et al. (2015), a lower diffraction intensity corresponds to a smaller crystal size of proteins; however, the present XRD data alone do not permit a definitive conclusion regarding changes in crystal size. Additionally, the β-sheet peak of the conjugates shifted slightly to higher 2θ angles compared to WPI control. Such a shift may be related to a decrease in interlayer spacing, suggesting a possible increase in structural order (Mohammadi et al., 2023). This decreased interlayer spacing implies a more compact and ordered crystalline arrangement, which is thermodynamically favorable and has been hypothesized to enhance thermal stability (Li, Gao, et al., 2025); however, direct thermal analysis is required for confirmation. In conclusion, the ultrasound-assisted radical grafting of sesamol onto WPI promoted protein unfolding and subsequent rearrangement into a more ordered molecular packing, as reflected by the increased XRD peak intensity and the shift of the β-sheet peak to higher 2θ angles.

3.4. Surface hydrophobicity

The surface hydrophobicity, determined by the number and disposition of hydrophobic groups on the protein surface, is closely related to protein emulsification (Ali, 2019; Guo et al., 2023). As presented in Fig. 2D, C/WPI-SE conjugates showed a significant (P < 0.05) increase in surface hydrophobicity relative to the WPI control. Sesamol conjugation likely promoted the exposure of internal hydrophobic domains on the WPI surface, thereby creating more hydrophobic binding sites for ANS. In addition, the binding with phenolic compounds might cause a loosening of protein structure due to the introduced steric hindrance and hydrophobicity. This structural alteration permits ANS to access previously inaccessible hydrophobic sites in the protein interior, thus increasing the fluorescence intensity in the conjugates (Ali, 2019). This result aligns with the observations of Sun et al. (2022), who reported increased surface hydrophobicity in egg white proteins after conjugation with various polyphenols. On the contrary, Liu et al. (2025) reported a decrease in the surface hydrophobicity of pea protein following covalent conjugation with four polyphenols. This discrepancy can likely be explained by differences in protein and polyphenol types, as well as reaction conditions. Following ultrasonic treatment, the surface hydrophobicity exhibited a time-dependent trend, characterized by an initial increase to a peak followed by a decrease. The initial rise (30–90 min) is attributed to the ultrasound-induced unfolding of the protein structure, which exposed buried hydrophobic groups (Li, Peng, et al., 2025). However, with prolonged sonication beyond the peak, the increased attachment of sesamol likely shielded these hydrophobic groups, thereby hindering their contact with ANS probes and resulting in the observed decline.

3.5. Molecular docking

Molecular docking analysis was employed to elucidate the interactions between the proteins and polyphenols (Zhong et al., 2021). Since β-lactoglobulin and α-lactalbumin are the most abundant proteins in WPI, accounting for ∼48% and ∼ 19% of the total whey protein, respectively, molecular docking was performed in this study to investigate their interactions with sesamol. Fig. 4A and B show the lowest energy conformers obtained from molecular docking of sesamol with β-lactoglobulin (ΔG = −4.182 kcal/mol) and α-lactalbumin (ΔG = −3.586 kcal/mol), respectively. These results indicated that sesamol likely interacts with specific regions on the protein surfaces. The optimal docked poses of sesamol show that its binding sites on β-lactoglobulin and α-lactalbumin are situated at the interface of α-helices, β-sheets, and random coils within the respective protein structures (Fig. 4A and B). Specifically, molecular docking results reveal that sesamol forms hydrogen bonds with Trp 35 of β-lactoglobulin and Asp 83 of α-lactalbumin. This suggests that the formation of hydrogen bonds may contribute to the stability of WPI-sesamol conjugates. Moreover, hydrophobic interactions were observed between sesamol and β-lactoglobulin (involving Trp 35, Val 59, Leu 172, Cys 176, Tyr 36, and Tyr 58) as well as α-lactalbumin (involving Leu 3 and Leu 85). These hydrophobic interactions are consistent with the significantly increased surface hydrophobicity of the conjugates (Fig. 2D), supporting that sesamol binding exposes hydrophobic domains on the protein surface. Together, the formation and stability of the WPI-sesamol conjugates may be influenced by the combined action of hydrogen bonding and hydrophobic interactions.

Fig. 4.

Fig. 4

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Docking conformations of sesamol with β-lactoglobulin (A) and α-lactalbumin (B) in 3D and 2D views.

3.6. Particle size and ζ-potential

The particle size is a key indicator of the formation and stability of polyphenol-protein conjugates in solution (Liu et al., 2025). As outlined in Table 1, the average particle size of the WPI-sesamol conjugates considerably increased (P < 0.05) compared to the WPI control (476.67 nm). A previous publication also reported an increase in the particle size of pea protein after conjugation with gallic acid, ferulic acid, catechin or kaempferol via the free radical method (Liu et al., 2025). This event could be due to the interaction between polyphenols and proteins to form larger conjugated compounds, as well as due to aggregation of proteins (Liu et al., 2025). Zhou et al. (2020) and Xu et al. (2024) stated that the oxidation and cross-linking of polyphenols in protein-polyphenol covalent conjugates contribute to an increased average diameter and a stable network structure. Hence, the varying degrees of binding between polyphenols and reactive groups of proteins may lead to the formation of aggregates with larger particle sizes. It could be noted that the average particle sizes of U/WPI-SE conjugate samples significantly increased (P < 0.05) with prolonged sonication time from 30 to 120 min. This observed increase in particle sizes, which paralleled the aforementioned sesamol binding equivalents (Fig. 1A), is likely due to the ultrasonic treatment enhancing the conjugation of sesamol to proteins and the subsequent formation of larger conjugates. The U/WPI-SE-90 and U/WPI-SE-120 treatments showed no observable difference (P > 0.05) but a slight decrease in the particle size compared to the C/WPI-SE treatment.

Table 1.

Particle size, ζ-potential and protein solubility of WPI-sesamol conjugates.

Samples Particle size (nm) ζ-potential (mV) Protein solubility (%)
WPI 476.67 ± 32.62d −11.63 ± 0.15a 86.78 ± 1.58d
C/WPI-SE 669.00 ± 33.87a −14.20 ± 0.76b 89.62 ± 0.86c
U/WPI-SE-30 547.33 ± 42.10c −16.10 ± 0.26bc 91.81 ± 2.35bc
U/WPI-SE-60 592.50 ± 26.50bc −18.40 ± 1.35cd 93.31 ± 0.89ab
U/WPI-SE-90 637.67 ± 53.00ab −20.73 ± 1.46d 93.34 ± 1.77ab
U/WPI-SE-120 628.00 ± 28.36ab −20.00 ± 2.43d 95.27 ± 0.91a
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Data are expressed as mean value ± standard deviation (n = 3). C/WPI-SE: WPI-sesamol conjugates prepared by conventional free radical method. U/WPI-SE-30, U/WPI-SE-60, U/WPI-SE-90, and U/WPI-SE-120: WPI-sesamol conjugates prepared by ultrasound-assisted free radical grafting method with ultrasound durations of 30, 60, 90, and 120 min, respectively. WPI: whey protein isolate; SE: sesamol. Values with lowercase letters (a-d) in the same column differ significantly.

The ζ-potential reflects the surface charge of suspended particles and serves as an essential parameter of colloidal stability in protein systems. In general, a lower absolute value of ζ-potential indicates that the protein tends to flocculate or coagulate, whereas a higher absolute value reveals stronger electrostatic repulsion and greater stability against aggregation (Manzoor et al., 2025). The conjugation of WPI with sesamol led to a significant increase in the absolute ζ-potential value (Table 1). A similar observation for whey-quercetin conjugates was reported by Baba, Abdelrahman, and Maqsood (2021). The electrostatic interaction between WPI and polyphenols, where homologous charges repel each other, contributes positively to the stability of the solution system (Wei et al., 2024). The absolute ζ-potential values of the U/WPI-SE conjugates rose (P < 0.05) with prolonged ultrasonic time, suggesting that ultrasonication led to protein unfolding and an increase in intermolecular repulsion (Guo et al., 2023). Interestingly, all U/WPI-SE samples manifested higher (P < 0.05) absolute ζ-potential than C/WPI-SE samples, except for the U/WPI-SE-30 samples, which displayed a similar ζ-potential value (P > 0.05) to C/WPI-SE samples. These results suggested that ultrasound-assisted free radical method was more effective in improving the stability of the WPI-sesamol conjugates compared to free-radical method alone. Ultrasonic treatment generates cavitation bubbles that undergo violent collapse, creating localized high shear forces near particle surfaces, which promotes the dissociation of positively charged ions from negatively charged surfaces, resulting in an enhanced net negative charge (Jara-Quijada et al., 2025). The elevated negative charge strengthens electrostatic repulsion among particles, effectively preventing their approach and aggregation; as a result, the stability of proteins is markedly improved (Manzoor et al., 2025).

3.7. Functional properties

3.7.1. Protein solubility

Solubility is the fundamental indicator for assessing the functional properties of proteins (Guo et al., 2023). High protein solubility is a critical prerequisite for enabling other functionalities, such as emulsification, foaming, and gelation (Manzoor et al., 2025). As listed in Table 1, all WPI-sesamol conjugates exhibited a significant augmentation (P < 0.05) in the protein solubility compared to WPI control. This suggested that sesamol effectively triggered the partial unfolding of WPI molecules, resulting in increased exposure of their internal hydrophilic residues. Additionally, the hydroxyl groups in sesamol facilitated hydrogen bonding with water molecules, which in turn improved the overall hydration properties and solubility of proteins (Guo et al., 2023). The ultrasound-assisted treatment for 30 min had no obvious effect (P > 0.05) on the protein solubility, while extending the ultrasound duration to 60–120 min resulted in markedly higher solubility (P < 0.05) with respect to the conventional free radical treatment alone. The micro-streaming and shear effects generated by ultrasound cavitation disrupt non-covalent interactions between protein molecules, such as hydrogen bonds and hydrophobic interactions, which lead to the exposure of more hydrophilic amino acid residues and, ultimately, enhance the protein solubility (Guo et al., 2023; Wei et al., 2024). An important observation was the positive correlation between the solubility of the WPI-sesamol conjugates and the absolute ζ-potential. This correlation suggests that conjugates with a higher net charge exhibited greater electrostatic repulsion, which prevented aggregation and thereby enhanced their solubility.

3.7.2. Emulsifying property

Typically, the emulsifying activity index (EAI) and emulsifying stability index (ESI) reflect the capability of proteins to form and stabilize emulsions, which are crucial indexes for evaluating the emulsifying profiles of proteins (Manzoor et al., 2025). Fig. 3 illustrates the EAI and ESI of WPI-sesamol conjugates obtained with and without ultrasonic assistance during free radical conjugation. Compared with WPI control, WPI-sesamol conjugates had higher (P < 0.05) EAI and ESI values. The improved emulsifying property was closely associated with the changes of protein structure after binding with polyphenols. According to Zheng et al. (2022) and Liu et al. (2025), the covalent grafting of polyphenols onto proteins induced protein rearrangement to form an exposed hydrophobic domain, thereby significantly enhancing the affinity of the protein to the oil-water interface. Furthermore, the negative charge imparted by polyphenols increased the interfacial charge density of the protein-coated droplets, providing a strong electrostatic barrier against coalescence and thereby further enhancing the emulsion stability (Liu et al., 2025). It was noted that C/WPI-SE conjugates showed similar values (P > 0.05) to U/WPI-SE-60 and U/WPI-SE-90 samples, but exhibited lower (P < 0.05) levels compared to U/WPI-SE-120 samples. The application of ultrasound not only exposed hydrophobic regions but also altered the interfacial composition by incorporating additional polar and non-polar moieties, and these modifications positively contributed to improved emulsification performance (Baba, Abdelrahman, & Maqsood, 2021; Yücetepe et al., 2019). Likewise, Baba, Abdelrahman, and Maqsood (2021) also demonstrated that ultrasonication notably enhanced the emulsifying properties of camel whey-quercetin conjugates.

Fig. 3.

Fig. 3

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Emulsifying activity index (EAI) and emulsifying stability index (ESI) of WPI-sesamol conjugates. C/WPI-SE: WPI-sesamol conjugates prepared by conventional free radical method. U/WPI-SE-30, U/WPI-SE-60, U/WPI-SE-90, and U/WPI-SE-120: WPI-sesamol conjugates prepared by ultrasound-assisted free radical grafting method with ultrasound durations of 30, 60, 90, and 120 min, respectively. WPI: whey protein isolate; SE: sesamol. Error bars represent the standard deviation (n = 3). Bars with different lowercase letters (a–e) differ significantly.

3.8. Antioxidant activity

The scavenging abilities of DPPH• combined with ABTS+• were employed to estimate the antioxidant properties of WPI-sesamol conjugates. As summarized in Table 2, WPI showed scavenging activities of 25.09% for DPPH radicals and 45.61% for ABTS+ radicals, respectively. This antioxidant effectiveness is attributed to amino acid residues such as tryptophan, methionine and cysteine in the proteins (Razzak & Cho, 2022). As expected, both DPPH• and ABTS+• scavenging activities of WPI were significantly enhanced (P < 0.05) after sesamol modification, which aligns with the findings for ovalbumin-epigallocatechin gallate conjugates (Zheng et al., 2023). This phenomenon can be attributed to the incorporation of phenolic hydroxyl groups from sesamol onto the surface of WPI, thereby improving the antioxidant capacity of WPI (Sun et al., 2022; Yan et al., 2021). Evidently, the ultrasound-assisted protein-sesamol conjugates showed time-dependent DPPH• and ABTS+• clearance rates over a sonication duration ranging from 30 to 120 min. The observed disparity in antioxidant activity among the ultrasonic-treated samples was primarily governed by the differential extent of sesamol grafted onto WPI (Zheng et al., 2023), and this assertion was supported by quantitative analysis of phenolic content (Fig. 1). Notably, the U/WPI-SE-120 samples (48.92% of DPPH and 84.34% of ABTS) showed more (P < 0.05) potent antioxidant activity than the C/WPI-SE samples (DPPH: 32.02% and ABTS: 82.43%). Ultrasonic mechanical effects loosened the protein structure and exposed more reactive binding sites on protein backbones, thus facilitating the attachment of additional hydroxyl groups from sesamol (Li, Peng, et al., 2025). This increased phenolic binding was responsible for the elevated antioxidant activity. Additionally, the exposure of amino acids (e.g., tryptophan) induced by ultrasonic treatment also endowed the samples with high antioxidant activity (Wei et al., 2024).

Table 2.

DPPH• and ABTS+• scavenging activity of WPI-sesamol conjugates.

Samples DPPH• scavenging activity (%) ABTS+• scavenging activity (%)
WPI 25.09 ± 0.47d 45.61 ± 0.76d
C/WPI-SE 32.02 ± 2.09bc 82.43 ± 1.59b
U/WPI-SE-30 27.89 ± 0.90cd 78.81 ± 0.32c
U/WPI-SE-60 33.53 ± 4.06b 79.76 ± 0.32c
U/WPI-SE-90 44.83 ± 3.97a 81.78 ± 1.39b
U/WPI-SE-120 48.92 ± 0.46a 84.34 ± 0.91a
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Data are expressed as mean value ± standard deviation (n = 3). C/WPI-SE: WPI-sesamol conjugates prepared by conventional free radical method. U/WPI-SE-30, U/WPI-SE-60, U/WPI-SE-90, and U/WPI-SE-120: WPI-sesamol conjugates prepared by ultrasound-assisted free radical grafting method with ultrasound durations of 30, 60, 90, and 120 min, respectively. WPI: whey protein isolate; SE: sesamol. Values with lowercase letters (a–d) in the same column differ significantly.

4. Conclusion

In this study, ultrasound-assisted free radical grafting was used to prepare WPI-sesamol conjugates and characterize their structural and functional properties, as well as antioxidant activity. Ultrasound-assisted treatment for 90 min achieved a sesamol binding equivalent comparable to the conventional free radical method. Conjugation of sesamol led to a reduction in active thiol and free amino groups of WPI, an effect that intensified with prolonged ultrasonication. FTIR, XRD, and surface hydrophobicity results suggested that the combined effects of sesamol grafting and ultrasonication induced more pronounced and rapid unfolding of WPI, resulting in secondary structural changes and a structural rearrangement toward a more ordered conformation. Molecular docking suggested potential hydrogen bonding and hydrophobic interactions between sesamol and WPI. Particle size and ζ-potential analyses confirmed that ultrasound-enhanced sesamol conjugation facilitated the formation of larger yet colloidally stable aggregates, and improved the solubility and emulsifying properties of WPI along with its antioxidant activity, likely attributable to the enhanced sesamol binding. These findings collectively support the formation of covalent WPI-sesamol conjugates, and future mass spectrometric confirmation is recommended. In conclusion, the ultrasound-assisted approach provides an efficient strategy for the synthesis of novel protein-phenol conjugates, which could be applied for stabilizing oil-in-water emulsions and encapsulating bioactive compounds in industry.

CRediT authorship contribution statement

Xiandan Feng: Software, Methodology, Investigation, Data curation. Mengting Lv: Software, Methodology, Investigation. Mu Tian: Methodology, Data curation. Sining Li: Writing – review & editing, Writing – original draft, Supervision, Funding acquisition, Conceptualization. Shanhu Tang: Writing – review & editing, Supervision, Project administration, Funding acquisition.

Declaration of competing interest

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.

Acknowledgments

This work is supported by the Fundamental Research Funds for the Central Universities (ZYN2026133) and Open Project Program of Panxi Crops Research and Utilization Key Laboratory of Sichuan Province (No. SZKF202406) and the Sichuan Provincial Science and Technology Program (No. 2025YFHZ0298).

Contributor Information

Sining Li, Email: ruoly123@126.com.

Shanhu Tang, Email: stang01@126.com.

Data availability

Data will be made available on request.

References

  1. Ali M. Chemical, structural and functional properties of whey proteins covalently modified with phytochemical compounds. Journal of Food Measurement and Characterization. 2019;13:2970–2979. doi: 10.1007/s11694-019-00217-1. [DOI] [Google Scholar]
  2. Baba W.N., Abdelrahman R., Maqsood S. Conjoint application of ultrasonication and redox pair mediated free radical method enhances the functional and bioactive properties of camel whey-quercetin conjugates. Ultrasonics Sonochemistry. 2021;79 doi: 10.1016/j.ultsonch.2021.105784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baba W.N., McClements D.J., Maqsood S. Whey protein-polyphenol conjugates and complexes: Production, characterization, and applications. Food Chemistry. 2021;365 doi: 10.1016/j.foodchem.2021.130455. [DOI] [PubMed] [Google Scholar]
  4. Basurto M.A.S., Martínez A.C., Tostado E.C., Bah M., Camacho R.R., Llano S.L.A. Study of the interactions occurring during the encapsulation of sesamol within casein micelles reformed from sodium caseinate solutions. Journal of Food Science. 2018;83:2295–2304. doi: 10.1111/1750-3841.14293. [DOI] [PubMed] [Google Scholar]
  5. Chen G.Q., Qu Y.R., Gras S.L., Kentish S.E. Separation technologies for whey protein fractionation. Food Engineering Reviews. 2023;15:438–465. doi: 10.1007/s12393-022-09330-2. [DOI] [Google Scholar]
  6. Feng X.D., Li S.N., Tang S.H., Wu W.J. Insight into the effect of sesamol on the structural and gel properties of yak myofibrillar proteins. International Journal of Biological Macromolecules. 2024;282 doi: 10.1016/j.ijbiomac.2024.137039. [DOI] [PubMed] [Google Scholar]
  7. Guo N., Ye S., Zhou G.H., Zhang Y.M., Zhang F.Y., Xu J.J.…Wang Z.Y. Effect of ultrasound treatment on interactions of whey protein isolate with rutin. Ultrasonics Sonochemistry. 2023;95 doi: 10.1016/j.ultsonch.2023.106387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Han P., Ma F., Wang Z.P., Zhou X., Zheng K., Lu S.L.…Dong J. Characteristics of ultrasound-induced sesamol-soybean peptide composite particles (SSPPs) and SSPP-stabilized Pickering emulsions. International Journal of Biological Macromolecules. 2025;318 doi: 10.1016/j.ijbiomac.2025.145367. [DOI] [PubMed] [Google Scholar]
  9. Jara-Quijada E., Lagos-Muñoz V., Guzmán-Bizama F., Jorquera-Palacios A., Muñoz-Muñoz P., Jarpa-Parra M.…González-Cavieres L. Enhancement of polyphenol bio-accessibility from maqui residue through ultrasound-assisted O/W nano-emulsions stabilized with oat protein and sodium alginate. Food Bioscience. 2025;71 doi: 10.1016/j.fbio.2025.107239. [DOI] [Google Scholar]
  10. Joshi R., Kumar M.S., Satyamoorthy K., Unnikrisnan M.K., Mukherjee T. Free radical reactions and antioxidant activities of sesamol: Pulse radiolytic and biochemical studies. Journal of Agricultural and Food Chemistry. 2005;53:2696–2703. doi: 10.1021/jf0489769. [DOI] [PubMed] [Google Scholar]
  11. Kato A., Nakai S. Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins. Biochimica et Biophysica Acta. 1980;624:13–20. doi: 10.1016/0005-2795(80)90220-2. [DOI] [PubMed] [Google Scholar]
  12. Li H.C., Gao F.Z., Wang X.N., Han Y.K., Lei W.Z., Gao Z.P. Ultrasonic cavitation-driven construction of hydroxypropyl-β-cyclodextrin and whey protein co-assembled nanoparticles: Loading, sustained-release delivery and interaction mechanisms for EGCG. Food Chemistry. 2025;493 doi: 10.1016/j.foodchem.2025.146081. [DOI] [PubMed] [Google Scholar]
  13. Li S.S., Peng S.Q., Wei L., Li J.Y., Lu Y.Y., Ma X.B., Hu F.L. Ultrasound-induced modifications in structure, antioxidant activity and functionality of whey protein isolate-propolis polyphenol conjugates. Food Chemistry. 2025;492 doi: 10.1016/j.foodchem.2025.145596. [DOI] [PubMed] [Google Scholar]
  14. Liang X.P., Zhou L.L., Zhang Y.T., Yang B.H., McClements D.J., Jin Z.Y., Chen L. Enhancing the water dispersibility and intestinal targeting of pterostilbene using tannic acid-whey protein conjugates. Food Research International. 2024;196 doi: 10.1016/j.foodres.2024.115083. [DOI] [PubMed] [Google Scholar]
  15. Liu H.Y., Zhang X.Y., Hou R.Y., Chen G.J., Fang Y.J., Zhang J.C.…Liu L.L. Ultrasonic assisted preparation of covalent bonding pea protein and polyphenol conjugate in emulsion delivery system. International Journal of Biological Macromolecules. 2025;287 doi: 10.1016/j.ijbiomac.2024.138442. [DOI] [PubMed] [Google Scholar]
  16. Liu X.J., Song Q.B., Li X., Chen Y.X., Liu C., Zhu X.…Huang J.B. Effects of different dietary polyphenols on conformational changes and functional properties of protein-polyphenol covalent complexes. Food Chemistry. 2021;361 doi: 10.1016/j.foodchem.2021.130071. [DOI] [PubMed] [Google Scholar]
  17. Majdalawieh A.F., Mansour Z.R. Sesamol, a major lignan in sesame seeds (Sesamum indicum): Anti-cancer properties and mechanisms of action. European Journal of Pharmacology. 2019;15(855):75–89. doi: 10.1016/j.ejphar.2019.05.008. [DOI] [PubMed] [Google Scholar]
  18. Manzoor M.F., Waseem M., Diana T., Wang R., Ahmed Z., Ahmed I.A.M.…Zeng X.A. Ultrasound-assisted modification to improve the red pepper seed protein isolate structural, functional, and antioxidant properties. International Journal of Biological Macromolecules. 2025;309 doi: 10.1016/j.ijbiomac.2025.143154. [DOI] [PubMed] [Google Scholar]
  19. Mohammadi A., Kashi P.A., Kashiri M., Bagheri A., Chen J.S., Ettelaie R.…Shahbazi M. Self-assembly of plant polyphenols-grafted soy proteins to manufacture a highly stable antioxidative Pickering emulsion gel for direct-ink-write 3D printing. Food Hydrocolloids. 2023;142 doi: 10.1016/j.foodhyd.2023.108851. [DOI] [Google Scholar]
  20. Ni X.X., Li M.T., Huang Z.W., Wei Y.Y., Duan C.Y., Li R.X.…Yu R.R. Study on the regulation of tea polyphenols on the structure and gel properties of myofibrillar protein from Neosalanx taihuensis. Food Chemistry: X. 2025 doi: 10.1016/j.fochx.2025.102243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nwachukwu I.D., Aluko R.E. Structural and functional properties of food protein-derived antioxidant peptides. Journal of Food Biochemistry. 2019;43 doi: 10.1111/jfbc.12761. [DOI] [PubMed] [Google Scholar]
  22. Ozdal T., Capanoglu E., Altay F. A review on protein–phenolic interactions and associated changes. Food Research International. 2013;51:954–970. doi: 10.1016/j.foodres.2013.02.009. [DOI] [Google Scholar]
  23. Pandita G., Sharma S., Oommen I.E., Madaan N., Bhosale Y., Nagy V.…Kovács B. Comprehensive review on the potential of ultrasound for blue food protein extraction, modification and impact on bioactive properties. Ultrasonics Sonochemistry. 2024;111 doi: 10.1016/j.ultsonch.2024.107087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Razzak M.A., Cho S.J. Molecular characterization of capsaicin binding interactions with ovalbumin and casein. Food Hydrocolloids. 2022;133 doi: 10.1016/j.foodhyd.2022.107991. [DOI] [Google Scholar]
  25. Siddhuraju P. The antioxidant activity and free radical-scavenging capacity of phenolics of raw and dry heated moth bean (Vigna aconitifolia) (Jacq.) Marechal seed extracts. Food Chemistry. 2006;99:149–157. doi: 10.1016/j.foodchem.2005.07.029. [DOI] [Google Scholar]
  26. Song G.S., Li F., Shi X.T., Liu J.Y., Cheng Y., Wu Y.H.…Gong J.Y. Characterization of ultrasound-assisted covalent binding interaction between β-lactoglobulin and dicaffeoylquinic acid: Great potential for the curcumin delivery. Food Chemistry. 2024;441 doi: 10.1016/j.foodchem.2024.138400. [DOI] [PubMed] [Google Scholar]
  27. Sun J., Zhang F., Liu T.M., Jing H., Huang Y.Q., Obadi M., Xu B. Ultrasound-enhanced egg white proteins conjugated with polyphenols: The structure of the polyphenols on their functional properties. LWT- Food Science and Technology. 2022;164 doi: 10.1016/j.lwt.2022.113600. [DOI] [Google Scholar]
  28. Tian R., Han X., Tian B., Li G.L., Sun L.N., Tian S.F.…Wang S. Effects of covalent binding of different polyphenols on structure, rheology and functional properties of whey protein isolate. LWT-Food Science and Technology. 2023;184 doi: 10.1016/j.lwt.2023.114968. [DOI] [Google Scholar]
  29. Wang H.L., Xiang L.W., Zhang Z.W., Li G.Q., Li P., Chen W.T.…Lin S.F. Elucidating the interaction mechanism of rutin with β-casein and β-lactoglobulin: A comprehensive analysis using multi-spectroscopy, molecular docking, and molecular dynamic simulations. Food Chemistry. 2025;476 doi: 10.1016/j.foodchem.2025.143411. [DOI] [PubMed] [Google Scholar]
  30. Wei X.L., Zhou C.X., Luo D.H., Jiang G.L., Zhao Z.L., Wang W.D.…Dou Z.M. Insighting the effect of ultrasound-assisted polyphenol non-covalent binding on the functional properties of myofibrillar proteins from golden threadfin (Nemipterus virgatus) Ultrasonics Sonochemistry. 2024;109 doi: 10.1016/j.ultsonch.2024.106988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Xu Q.D., Yu Z.L., He Q., Zeng W.C. Establishing a novel ternary complex of soybean protein isolated-tannic acid-magnesium ion and its properties. Food Chemistry. 2024;446 doi: 10.1016/j.foodchem.2024.138861. [DOI] [PubMed] [Google Scholar]
  32. Yan S.Z., Wang Q., Yu J.Y., Li Y., Qi B.K. Soy protein interactions with polyphenols: Structural and functional changes in natural and cationized forms. Food Chemistry: X. 2023;19(2023) doi: 10.1016/j.fochx.2023.100866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yan X.H., Gao Y.F., Liu S.C., Zhang G.H., Zhao J.X., Cheng D.…Gong D.M. Covalent modification by phenolic extract improves the structural properties and antioxidant activities of the protein isolate from Cinnamomum camphora seed kernel. Food Chemistry. 2021;352 doi: 10.1016/j.foodchem.2021.129377. [DOI] [PubMed] [Google Scholar]
  34. Yu L., Yang W., Sun J., Zhang C., Bi J., Yang Q. Preparation, characterisation and physicochemical properties of the phosphate modified peanut protein obtained from Arachin Conarachin L. Food Chemistry. 2015;170:169–179. doi: 10.1016/j.foodchem.2014.08.047. [DOI] [PubMed] [Google Scholar]
  35. Yücetepe A., Saroğlu Ö., Özçelik B. Response surface optimization of ultrasound-assisted protein extraction from Spirulina platensis: Investigation of the effect of extraction conditions on techno-functional properties of protein concentrates. Journal of Food Science and Technology. 2019;56:3282–3292. doi: 10.1007/s13197-019-03796-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhang S.R., Li X.Q., Yan X.J., McClements D.J., Ma C.C., Liu X.B., Liu F.G. Ultrasound-assisted preparation of lactoferrin-EGCG conjugates and their application in forming and stabilizing algae oil emulsions. Ultrasonics Sonochemistry. 2022;89 doi: 10.1016/j.ultsonch.2022.106110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zheng J.C., Xiao N., Li Y., Xie X.N., Li L. Free radical grafting of whey protein isolate with tea polyphenol: Synthesis and changes in structural and functional properties. LWT--Food Science and Technology. 2022;153 doi: 10.1016/j.lwt.2021.112438. [DOI] [Google Scholar]
  38. Zheng Y.M., Chen B.Y., Huang X.X., Teng H., Ai C., Chen L. Ultrasound-assisted free radical modification on the structural and functional properties of ovalbumin-epigallocatechin gallate (EGCG) conjugates. Ultrasonics Sonochemistry. 2023;95 doi: 10.1016/j.ultsonch.2023.106396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhong Y.J., Zhao J.C., Dai T.T., McClements D.J., Liu C.M. The effect of whey protein-puerarin interactions on the formation and performance of protein hydrogels. Food Hydrocolloids. 2021;113 doi: 10.1016/j.foodhyd.2020.106444. [DOI] [Google Scholar]
  40. Zhou S.D., Lin Y.F., Xu X., Meng L., Dong M.S. Effect of non-covalent and covalent complexation of (−)-epigallocatechin gallate with soybean protein isolate on protein structure and in vitro digestion characteristics. Food Chemistry. 2020;309 doi: 10.1016/j.foodchem.2019.125718. [DOI] [PubMed] [Google Scholar]

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Data Availability Statement

Data will be made available on request.

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