Ultrasound accelerates sesamol grafting onto whey protein isolates

Ultrasound-assisted free-radical grafting attached sesamol to whey protein isolate as effectively as a conventional method that required 24 hours, but the ultrasound-assisted treatment reached comparable binding in 90 minutes. The study, conducted in triplicate, compared native whey protein isolate with a conventionally prepared conjugate and four ultrasound-treated formulations made at 30, 60, 90 and 120 minutes. After unbound sesamol was removed by extensive dialysis, the 90- and 120-minute ultrasound samples had binding equivalents similar to the conventional preparation, indicating that sonication accelerated the interaction without sacrificing overall binding efficiency. The treatment also altered protein structure, increased solubility and emulsifying performance, and raised antioxidant activity in laboratory assays.

Sesamol, a natural polyphenol obtained from sesame seeds, has antioxidant activity associated with its phenolic hydroxyl group, but its low water solubility and vulnerability to light, heat and oxidation restrict practical use. Whey protein isolate could provide a food-derived carrier, yet the protein itself can aggregate and lose solubility when processing changes pH, ionic strength or temperature. The researchers therefore investigated whether ultrasound could combine with a free-radical grafting reaction to connect sesamol more rapidly to the protein. Their preparation contained 10 mg/mL whey protein isolate and 2 mg/mL sesamol in phosphate buffer. Ultrasonication was performed at 240 W and 40 kHz while an ice bath limited heating, and the products were dialyzed, freeze-dried and stored before analysis.

After dialysis, the conventional conjugate contained a sesamol binding equivalent of 4.68 mg per gram of protein. Corresponding values for the ultrasound-assisted formulations were 4.48 mg/g at 30 minutes, 4.58 mg/g at 60 minutes, 4.65 mg/g at 90 minutes and 4.73 mg/g at 120 minutes. The statistically similar binding levels at 90 and 120 minutes were not significantly different from that of the 24-hour conventional treatment. The absence of a decline at 120 minutes may reflect the relative stability of sesamol’s benzodioxole structure and rapid binding that could protect it from further radical attack, although the study did not directly test either mechanism. Ultrasound was interpreted as generating radicals while mechanically unfolding protein molecules, thereby exposing more sites for sesamol attachment.

Chemical assays supported the presence of interactions between sesamol and reactive protein groups. Both conventional and ultrasound-assisted conjugates had significantly fewer detectable active thiol and free amino groups than the unmodified protein, while the losses became more pronounced as sonication time increased from 30 to 120 minutes. The authors proposed that sesamol’s hydroxyl group can participate in covalent bonding with thiol and amino residues, while mild oxidation could also contribute to the decreases. This evidence is indirect rather than definitive because the experiments did not directly identify the molecular bonds, and the authors recommended mass spectrometric confirmation in future work. The time-dependent reduction nevertheless tracked the amount of sesamol retained through dialysis, strengthening the interpretation that conjugation was a substantial mechanism.

Fourier-transform infrared spectroscopy and X-ray diffraction showed that grafting and sonication reorganized the protein’s secondary and packed structures. Relative to native whey protein isolate, the conventional conjugate had lower α-helix and β-turn content and higher β-sheet and random-coil content, consistent with partial unfolding and rearrangement. The 90- and 120-minute ultrasound samples had less random coil than the conventional conjugate, although still more than the untreated control. Their amide bands had also shifted, and changes in the spectral fingerprint region indicated alterations in hydrogen bonding and other interactions. X-ray diffraction patterns retained the two characteristic protein peaks near 10 and 20 degrees, but grafting increased their intensity and the broad peak near 20 degrees became sharper. The β-sheet peak also moved slightly toward higher angles, suggesting more compact molecular packing. Because direct thermal analysis was not performed, the researchers said this possible increase in structural order could not by itself confirm improved thermal stability.

Surface hydrophobicity rose significantly after conventional sesamol conjugation, consistent with partial unfolding and greater exposure of hydrophobic regions. Ultrasound produced a non-linear response: hydrophobicity increased from 30 through 90 minutes and then declined at 120 minutes. The early rise was attributed to cavitation-driven exposure of buried hydrophobic groups, while the later decrease was interpreted as increasing attachment of sesamol shielding those groups from the fluorescent probe. Molecular docking provided a computational picture consistent with these observations. The lowest-energy sesamol poses had predicted binding energies of −4.182 kcal/mol for β-lactoglobulin and −3.586 kcal/mol for α-lactalbumin. Docking predicted hydrogen bonds with Trp 35 in β-lactoglobulin and Asp 83 in α-lactalbumin, together with hydrophobic contacts involving several residues. These calculations identify plausible interactions, but they do not demonstrate the binding geometry present in the experimental conjugates.

Conjugation also changed the colloidal behavior of the protein. Native whey protein isolate had an average particle size of 476.67 nm, whereas the conventional conjugate measured 669.00 nm. Ultrasound-treated samples ranged from 547.33 nm at 30 minutes to 637.67 nm at 90 minutes and 628.00 nm at 120 minutes, consistent with the formation of larger protein–sesamol structures. At the same time, the magnitude of negative ζ-potential generally increased with sonication, reaching −20.73 mV for the 90-minute formulation compared with −14.20 mV for the conventional conjugate. Greater electrostatic repulsion can inhibit particle approach and aggregation, so the study characterized the ultrasound products as larger but more colloidally stable aggregates. The 120-minute sample had a slightly smaller particle size than the 90-minute sample, but the difference was not significant.

Protein solubility improved in every conjugated formulation, rising from 86.78% for native whey protein isolate to 89.62% for the conventional conjugate and as much as 95.27% after 120 minutes of ultrasound. The 30-minute ultrasound treatment did not significantly outperform the conventional method, whereas treatments lasting 60–120 minutes produced significantly higher solubility. The authors linked this result to disruption of non-covalent interactions and exposure of hydrophilic residues, together with hydrogen bonding between water and sesamol hydroxyl groups. The conjugates also displayed better emulsifying activity and emulsion stability than native whey protein isolate. The 120-minute sample outperformed the conventional conjugate, while the 60- and 90-minute formulations produced values similar to it. Exposed hydrophobic regions can favor protein adsorption at oil–water interfaces, and increased negative surface charge can create an electrostatic barrier against droplet coalescence.

The antioxidant assays showed that sesamol modification increased the ability of whey protein isolate to scavenge two laboratory radical systems. Native protein scavenged 25.09% of DPPH radicals and 45.61% of ABTS+ radicals, compared with 32.02% and 82.43% for the conventional conjugate. With 120 minutes of ultrasound, the corresponding values rose to 48.92% and 84.34%, both significantly above the conventional preparation. DPPH scavenging increased progressively across the ultrasound series, while ABTS values varied less consistently. Greater attachment of phenolic hydroxyl groups was the main proposed explanation, supplemented by the exposure of antioxidant amino acid residues during protein unfolding. These results measure chemical radical-scavenging activity in laboratory assays; they do not establish effects in animals or humans, and the conjugates were not tested for safety, toxicity, sensory performance or performance through industrial processing. The findings therefore support ultrasound as a faster laboratory route to functional whey protein–sesamol conjugates, while leaving direct molecular confirmation and scale-up validation for future research.

Subject of Research: Ultrasound-assisted sesamol grafting modifies whey protein structure and functional properties

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

Article References: Feng, X., Lv, M., Tian, M., Li, S., & Tang, S. (2026). Ultrasound-assisted free radical grafting of sesamol onto whey protein isolate: Structural modifications, functional enhancements, and antioxidant properties. Food Chemistry: X, 40, Article 104658. https://doi.org/10.1016/j.fochx.2026.104658

Image Credits: AI Generated

DOI: 10.1016/j.fochx.2026.104658

Keywords: whey protein isolate, sesamol, ultrasound, free-radical grafting, protein conjugates, polyphenols, protein structure, food functionality, emulsifying properties, antioxidant activity, Ultrasound-assisted, free