## 3 Results and Discussion  
### Proximate and Amino Acid Compositions of FBP  
The crude protein contents were 81.09 and 84.88 g/100 g on a dry basis, for SFBP and HFBP, respectively. Such levels of protein were also reported by [PERSON] et al. (2018). Compared to fish skin, fish bone usually contains lower protein, due to the presence of higher ash and fat contents [32]. The extraction rate of protein recovery from fish backbone was about 54% for both the samples, which was similar to that of cod bone gelatin (60%) [20], but higher than that of Nile perch (_Lates niloticus_) bone gelatin (<40%) [32]. The recovered protein had ash contents with values of 15.15 and 13.51 g/100 g for SFBP and HFBP, respectively. The results were comparable to the hydrolyses from Atlantic codfish (_Gadus morhua_) frames (15 g/100g) [12], but were slightly higher than the gelatin prepared from red snapper (_Lutjanus campechanus_) bone (10.32 g/100 g) [31]. The high ash contents in the recovered bone protein were most likely from an inadequate decalcification process and high mineral contents in the bones [31]. Actually, proteins with high mineral contents might also show special functional properties. A recent study found that high-mineral-content (>20%) gelatin, prepared from saithe (_Pollachius virens_) skin, exhibited higher viscoelastic behavior and foaming properties compared with commercial gelatin, which might lead it to find applications in cosmetics and foods sectors, as a potential new microencapsulating agent [33]. The fish backbones that were used in the present study did not undergo decalcification steps before the hydrothermal pretreatment. Compared with traditional alkali/acid extraction methods, hydrothermal pretreatment, by steam explosion-assisted extraction and hot-pressure extraction, was a promising process to recover protein from fish backbones, and could avoid the disadvantages of the decalcification procedure. In our previous study, no significant difference in protein recovery from fish scales was found between the hydrothermal pretreatment with or without decalcification [17]. Further studies should examine whether decalcification is actually necessary for achieving high protein recovery and good functional properties. Except for the thermal effect, as in hot-pressure extraction, there is a physical tearing effect during the steam explosion process, to overcome the key barriers limiting the degradation and extraction of the biomass [23]. As a result, similar amounts of protein could be extracted from the exploded fish bone, at a relatively low temperature. The TAA contents of FBP treated by SE and HPE pretreatment, were 749.7 and 833.7 mg/g FBP, respectively. The amino acid composition of FBP samples per 1000 total residues is shown in Table 1. Glycine was the most abundant amino acid in both the samples, making up around a third of the total residues. They were also rich in alanine (about 123 residues), proline (90.9-101.1 residues), glutamic acid (85.1-90.2 residues), hydroxproline (69.7-82.2 residues), aspartic acid (54.2-57.3 residues), and arginine (52.5-53.3 residues), but poor in histidine and tyrosine residues. Similar results were also found in gelatin hydrolyses from salmon bone [29], _Thunus orientalis_ bone [34], and barred mackerel byproducts [35]. The TAA profiles of the recovered proteins were very similar to those of type-I collagen isolated from bighead carp bones [9]. However, slight differences in TAA compositions were also found between SFBP and HFBP. Proline and hydroxproline (imino acid) accounted for \(\sim\)23% of the amino acid content of the collagen molecule [36]. In the present study, imino acid residue totaled 183.3 per 1000 total residue in SFBP, which was approximately 1.14 times higher than that of the HFBP (160.6). Accordingly, the content of imino acids contributes to the thermal stability of collagens [9].