## 3 Results and Discussion
### Ftir  
FTIR spectroscopy can be used to characterize the changes of functional groups and secondary structures of protein. As shown in Figure 2, all the samples exhibited typical amide vibrations at amide A, amide B, amide I, amide II, and amide III, with similar spectral patterns, indicating their similar chemical compositions. The amide A region, at 3292\(-\)3340 cm\({}^{-1}\), was found in all the samples, representing the N-H stretching vibration coupled with hydrogen bonding [28]. SFBP showed a higher amplitude than HFBP, suggesting greater free amino groups were caused by the SE pretreatment. The amide I region (1600-1700 cm\({}^{-1}\)), involving mainly the C=O stretching vibration, was found at 1628 cm\({}^{-1}\) and 1633 cm\({}^{-1}\) for the SFBP and HFBP samples, respectively, which was indicative of the presence of a \(\beta\)-sheet secondary structure in the recovered bone proteins. This phenomenon might be attributed to the disruption of the native structure in bone materials during the SE and HPE pretreatment process. After enzymatic hydrolysis, a shift towards higher wavenumbers was found in the peak at 1654 cm\({}^{-1}\) and 1657 cm\({}^{-1}\), for the SFBP-H and HFBP-H samples, respectively, indicating the disruption of some ordered \(\beta\)-sheet structures.  
The amide III region (1220-1340 cm\({}^{-1}\)) is associated with C-N stretching vibrations, and O=C-N and N-H bending. Due to atmospheric water vapor interference in the amide I region of FTIR, the deconvolution of the amide III region is recommended for more accurate analysis of the protein secondary structure [26]. The relative contents of the microstructural components could be obtained based on the \(\beta\)-sheet (1220-1250 cm\({}^{-1}\)), random coil (1250-1270 cm\({}^{-1}\)), \(\beta\)-turn (1270-1295 cm\({}^{-1}\)), and \(\alpha\)-helix (1295-1330 cm\({}^{-1}\)) [26]. As shown in Table 2, the secondary structures of protein in the SFBP and HFBP samples were mainly \(\beta\)-sheet (about 52%) and random coil (about 33%). It is widely known that the main structure of the collagen molecule in native collagen materials is a triple superhelix [43]. Thus, the transformation of an \(\alpha\)-helix to a random coil structure is related to an employed pretreatment that causes a higher disorder of the molecular structure [33]. Interestingly, the contents of the \(\beta\)-sheet reduced to 41.72% and 35.62%, respectively, and the contents of the \(\beta\)-turn increased to about 20% in both the SFBP-H  
Figure 1: Degree of hydrolysis (**a**) and nitrogen recovery (**b**) of SFBP and HFBP by Flavourzyme. SFBP: fish bone protein prepared through steam explosion-assisted extraction; HFBP: fish bone protein prepared through hot-pressure extraction.  
and HFBP-H samples, after 3 h of hydrolysis. This was consistent with the results obtained from amide I. It is worth noting that the percent of \(\beta\)-sheets, before enzymatic hydrolysis, seemed similar for both the samples that underwent the different pretreatments, while after enzymatic hydrolysis, these values showed an obvious difference. The difference may result from the variance of the cleavage sites exposed during the SE and HPE processes, which requires more study.