**Background:** Totoaba (Totoaba macdonaldi) is a large croaker native to the upper Gulf of California, Mexico, currently listed as vulnerable by the IUCN and commercially farmed under licensed Wildlife Conservation Management Units. Processing totoaba for meat generates by-products, including swim bladders, whose market potential is unknown. The dried swim bladder of wild totoaba (maw) is valued in traditional Chinese medicine, but swim bladders from small, farmed fish are not highly prized in that market. However, these by-products may contain beneficial compounds, particularly collagen. Collagen is a dominant fibrous protein widely used in food, cosmetics, pharmaceuticals, and tissue engineering. While livestock are the primary commercial sources, concerns over zoonotic disease transmission and cost have driven interest in aquatic alternatives. Collagen has been isolated from fish skin and swim bladders of various species, with pepsin-soluble collagen (PSC) methods generally yielding higher amounts than acid-soluble collagen (ASC) methods. No prior research had characterized the biochemical composition or collagen properties of the totoaba swim bladder.
**Methods:** Twenty-four farmed totoaba (average body weight 2.57 ± 0.264 kg; 3 years old) were sampled from three 8,000 L tanks maintained at 27 ± 1 °C with a 12:12 light:dark photoperiod. Swim bladders were manually removed, cleaned, and subjected to proximate analysis (moisture, protein, lipid, ash). Amino acid composition was analyzed using RP-UHPLC after PITC derivatization. Collagen was extracted using pepsin digestion in 0.5 M acetic acid at 4 °C for 24 h, followed by NaCl precipitation, dialysis, and lyophilization. The extracted collagen (TSBC) was characterized by SDS-PAGE, UV-Vis spectroscopy, FTIR, X-ray diffraction (XRD), circular dichroism (CD) spectroscopy, protein solubility, and zeta potential measurements. Collagen hydrolysates were prepared using Alcalase® and papain, ultrafiltered (≤3 kDa), and tested for DPPH radical scavenging activity at 3.2 mg mL⁻¹.
**Key Results:** The swim bladder represented 2% of total body weight and contained 67.43 ± 1.24% moisture, 95.72 ± 1.07% crude protein (dry weight), 2.46 ± 0.18% lipids, and 0.88 ± 0.06% ash. Nineteen amino acids were identified, with glycine most abundant (29.19%), followed by alanine (12.26%), proline (12.10%), arginine (11.58%), glutamic acid (9.61%), hydroxyproline (5.43%), and aspartic acid (5.13%). Only 12% of amino acids were essential, while 71% were functional amino acids. The PSC yield was 68.18 ± 1.62% (dry weight basis), comparable to Gulf corvina (69%) and significantly higher than miiuy croaker (8%), yellowfin tuna (12%), and catla (61%). TSBC protein content was 96.34 ± 1.19%, ash 0.83 ± 0.09%, with no detectable fat. SDS-PAGE revealed two α chains (α1 at 126 kDa, α2 at 116 kDa) in a 2:1 ratio, plus β and γ chains, confirming type I collagen ([α1(I)]₂α2(I)). UV-Vis showed maximum absorption at 228 nm with no peak at 280 nm, indicating high purity. FTIR showed characteristic amide bands (A: 3280 cm⁻¹, B: 3071 cm⁻¹, I: 1629 cm⁻¹, II: 1543 cm⁻¹, III: 1237 cm⁻¹) with an amide III/1454 cm⁻¹ ratio of 1.05, confirming triple helix preservation. XRD showed peaks at 7.7° and 20.02°, consistent with collagen triple helical structure. CD spectroscopy showed a weak positive peak at 222 nm and negative peak at 197 nm, with thermal denaturation temperature (Td) of approximately 32.5 °C. Imino acid content was 205/1000 residues, with proline hydroxylation of 40.65% and lysine hydroxylation of 14.43%. Solubility was highest at pH 2–4, lowest around pH 6, with slight increase at pH 7–10. The isoelectric point (pI) was pH 5.4. DPPH radical scavenging activity at 3.2 mg mL⁻¹ was 37% higher (p < 0.05) for papain hydrolysate (HCP) than Alcalase® hydrolysate (HCA), though both were lower than ascorbic acid.
**Clinical Implications:** This study provides the first characterization of collagen from farmed totoaba swim bladder, demonstrating high yield (68%) and preservation of native triple helix structure with good thermal stability (32.5 °C), approaching that of mammalian collagen. The high protein and low lipid content, along with abundance of functional amino acids (71%), suggest potential as a functional food supplement. The collagen's thermal stability makes it a promising alternative to mammalian collagen for food, cosmetic, and biomedical applications, reducing reliance on livestock sources and associated zoonotic risks. The partial antioxidant activity of collagen hydrolysates indicates potential for bioactive peptide development, though further research is needed to isolate and identify specific active peptides. Utilizing this by-product could reduce waste and add economic value to totoaba aquaculture operations.