## 4 Discussion  
Collagens are reported to have a self-assembly ability [21], and thus are considered an important natural resource and serve as functional ingredients in foods [30; 31; 32]. The composition analysis showed that the combined moisture and collagen contents of the analyzed swim bladders were higher than 90%. Therefore, besides moisture, swim bladders were basically composed of collagens. These results are in agreement with other reports about the composition of swim bladders from other fishes [33; 34; 35]. The swim bladder with a 25-year sequence age was investigated regarding (1) the amino acid composition, (2) peptide chains, (3) triple helix structures, (4) microfibrils, and (5) fiber bundles. Peptide chains are basic structural units in collagen fibers; they are mainly composed of amino acids with a (Gly-X-Y)n repeating sequence, and hydroxyproline is the typical amino acid and frequently appears on X or Y positions [6]. According to the amino acid compositions, glycine contents were found to range from 33 to 35%; the ranges of hydroxyproline and proline were from 8 to 9% and 12 to 18%, respectively. The glycine content in the swim bladders was in agreement with the theoretical value of model collagen molecules (1/3 of total amino acids); the amino acid contents were also similar to those of pure collagen [6; 34]. It was further proved that swim bladders are basically composed of collagens. Similar Td values were observed in different swim bladders from TG-DTG analysis, indicating that the covalent linkages inside peptide chains were not affected by aging. The triple helix is considered a typical structure that is rarely found in other proteins and only appears in collagen [36; 37]. The existence of the triple helix in swim bladders was primarily revealed by DSC. The result indicated that swim bladders were found to have thermal denaturation temperatures similar to those of dry collagen [3] but different to that of the thermally denatured sample (TDSB). It was implied that the triple helix may exist in swim bladders. Furthermore, their presented Tm values were slightly different when comparisons were made within samples aged for different lengths of time. This indicates that the triple helix structures in swim bladders may be impacted by aging. Therefore, the characteristics of the triple helix were further observed by XRD and ATR-FTIR. Based on the XRD result, typical diffusion signals of the triple helix were observed. The ATR-FTIR spectra of aged swim bladders also showed similar wavenumbers to those of the FSB, indicating that the triple helixes in aged swim bladders are similar to those in native collagen [34]. The results from the secondary calculation of bands and peaks further proved the deduction that the collagens in swim bladders retain triple helix structures after years of storage. Based on the peak areas that appeared on the XRD spectrum, it can be concluded that the retained quantities of triple helix were negatively related to age. This result is in agreement with the observation from DSC, which further emphasized that the loss of the triple helix structure happened over time. The results obtained by Gaussian/Lorentz functions implied that in the aged swim bladders, the relative contents of \(\alpha\)-helixes, \(\beta\)-sheets, and random coils were found to be statistically the same as those in the FSB. The Bragg equation calculation results implied that the triple helix in aged swim bladders had a similar value to those observed in natural collagens [19; 34]. These observations implied that, after years of aging, the retained triple helix still presents natural structural characteristics. Regarding the microfibrils, despite the swim bladders being aged for years and the triple helix structures being partially lost, the cross-striated characteristics still appearedin the swim bladders. As for the swim bladder without the triple helix structure (TDSB), the cross-striated characteristics of the microfibrils were not detected.