Enhancing the Extraction, Recovery, and Purification of Short‐Chain Peptides From Bee Pupae Using Ultrasound at Different Frequencies

Bee pupae are a protein‐rich apicultural by‐product with strong potential as a source of short‐chain bioactive peptides. This study investigated the effect of ultrasound at different frequencies on the extraction, hydrolysis, recovery, and purification of peptides from bee pupae. Defatted bee pupae powder was pretreated using single‐frequency ultrasound (20, 40, and 60 kHz) and combined‐frequency ultrasound (20/40 and 20/40/60 kHz), followed by Alcalase hydrolysis and membrane ultrafiltration. Ultrasound pretreatment significantly improved soluble protein extraction, degree of hydrolysis, short‐chain peptide recovery, molecular‐weight distribution, and functional properties of the final peptide fractions. The highest soluble protein concentration was obtained at 20/40 kHz (28.8 mg mL −1 ), compared with 18.1 mg mL −1 in the control. Under the same condition, the degree of hydrolysis reached 26.1% after 120 min, higher than the control (18.6%) and all single‐frequency treatments. Dual‐frequency ultrasound also yielded the highest recovery of peptides below 3 kDa (48.1% of total peptide solids) and the highest short‐chain peptide yield (28.0% of starting powder). In addition, the purified peptides showed a clear shift toward lower molecular weights, with the lowest weighted average molecular weight observed at 20/40 kHz (1065 Da). Functional evaluation further confirmed improved antioxidant activity, solubility, and foaming capacity, with DPPH, ABTS, FRAP, solubility, and foaming capacity reaching 68%, 490 μmol TE g −1 , 815 μmol Fe 2+ g −1 , 90.2%, and 32.8%, respectively. Overall, dual‐frequency ultrasound provided the most effective pretreatment for producing low‐molecular‐weight, functionally enhanced peptides from bee pupae, highlighting its potential as a green and scalable strategy for peptide valorization.