High-intensity ultrasound-assisted recovery of type II collagen from porcine trachea: Enhanced recovery and multiscale structural organization.

Porcine trachea is an underutilized co-product with high potential as a source of type II collagen (CII). This study evaluated high-intensity ultrasound (HIU) as a pretreatment to enhance collagen extraction efficiency while preserving structural integrity. Collagen obtained by acid (PTC-A), pepsin (PTC-P), and ultrasound-pepsin treatment (PTC-U) was compared. Under optimized conditions (23.75 W/cm2, 15 min), HIU increased collagen yield to 19.97 % and reduced extraction time by approximately 58 % compared to conventional methods. Acid extraction (PTC-A) yielded only 1.82 % and predominantly recovered type I collagen, whereas pepsin-assisted extraction (PTC-P) achieved 19.91 % and enabled enrichment of cartilage-derived CII. The combined treatment (PTC-U) further improved collagen content to 80.51 % (vs. 75.04 % for PTC-P). Amino acid analysis confirmed a typical CII profile, with glycine (173-208 residues/1,000 residues) and high imino acid content (proline + hydroxyproline: ∼270 residues/1,000 residues), supporting triple-helix stability. Scanning electron microscopy and synchrotron radiation X-ray tomographic microscopy revealed a compact lamellar (layer-like) three-dimensional architecture with reduced porosity (77.97 ± 2.75 %), compared to PTC-A (85.70 ± 0.29 %) and PTC-P (81.82 ± 0.30 %). Circular dichroism, Fourier transform infrared spectroscopy, and synchrotron radiation wide-angle X-ray scattering confirmed preservation of the native triple-helical structure. All samples exhibited comparable denaturation temperatures (∼39.7-39.9 °C) based on micro-differential scanning calorimetry. Proteomic analysis further verified that acid extraction yielded type I collagen, whereas HIU-assisted extraction enriched type II collagen.