**3. Discussion**

From the protein pattern, we confirm that the extracted collagens were characterized as type-I collagen due to the presence of α1, α2, β and γ chains. To support this finding, earlier reports dealing with fish skin type-I collagens had a similar molecular pattern of proteins [20–22]. The SDS-PAGE protein profile further confirmed that the extracted collagen was pure and had no presence of other proteins. The pattern of the amino acid composition of blacktip reef shark skin collagen was very similar to the earlier reported fish collagens [22]. The higher amount of amino acid content in PSC was related to the extensive hydrolysis process of raw materials by pepsin, which ultimately facilitated the solubilization process of collagen in raw material and thereby releases many smaller fractions.

The maximum absorbance of collagen at 234 nm was due to higher amounts of glycine (which absorbs maxima at 216.8 nm) and peptide bonds (at 230 nm) [23]. In addition, the liberation of more peptides (which tend to absorb UV maxima at 230 nm) by pepsin treatment might be the actual reason for the higher absorption of PSC at 234 nm than ASC. Interestingly, no absorbance peak of collagens at 280 nm further confirmed that the extracted collagens were pure.

FTIR analysis confirmed that the secondary structure of collagen was not significantly altered by the different extraction procedures with acid and pepsin. Only CH2 asymmetric stretch and N-H bend coupled with C-N stretch were more pronounced in PSC than ASC, which was due to the extensive hydrolysis of collagen by pepsin. The IR absorption ratio indicated that the triple helix and high molecular structure of the two collagens were intact [24]. The amide I band in the wavelength range from 1600 to 1700 cm<sup>−</sup><sup>1</sup> was used to calculate the collagen secondary structure [25,26]. Using the Gaussian peak fitting algorithm, we confirmed that neither collagen had any significant changes in α helix, β-sheet, β-turn, triple helix and random coil, which confirms that the pepsin treatment had not significantly altered the secondary and triple helical structure of collagen and only improved the production yield. A recent study also reported a similar finding in the secondary structures of α helix, β-sheet, β-turn and triple helix by pepsin soluble collagen [27]. This finding reveals the compatibility of pepsin use in collagen extraction from blacktip reef shark skin.

Increasing ion concentration on the surface reduces the free functional groups and the hydrophilic nature, thereby reducing the solubility of collagens [28]. The maximum solubility of collagen against pH was observed at 4, which corresponded to the isoionic point of collagens; for instance, it was reported that the isoionic point of collagen ranged from pH 3 to 5 [29,30].

In the present study, the collagens were hydrolyzed at pH 7.8 by trypsin for a shorter duration (3 min) unlike pH 2.5 (3 h), due to the higher proteolytic activity of trypsin at pH 7.8 [31]. The peptide fragments observed in the present study were similar to the peptide maps generated in earlier studies [32,33]. At pH 2.5, the peptide fragments were derived from higher MW components; as evidence, the intensity of high MW bands such as α, β and γ was decreased significantly and almost absent in collagens treated at pH 7.8. To support this finding, earlier studies reported similar findings with the absence of a higher MW component after trypsin hydrolysis [34–36]. The lower amount of peptide bands observed in PSC at pH 2.5 compared to ASC was mainly attributed to the earlier hydrolysis of collagen by pepsin during extraction, which makes them more susceptible to consecutive hydrolysis by another proteolytic enzyme, trypsin, to liberate peptides less than the measurable range (below 15 kDa). In contrast, the lower amount of peptide fragments in collagens treated at pH 7.8 than at pH 2.5 were due to the extensive hydrolysis of collagen by trypsin at an optimum pH of 7.5 and thereby smaller peptides could be obtained (less than 15 kDa). To support this finding, earlier studies reported a similar observation of peptide mapping of collagens (ASC and PSC) extracted from fresh Spanish mackerel [31], and largefin longbarbel catfish [37]. The similar microstructural properties of ASC and PSC proved that the pepsin treatment did not contribute to any major changes in the microstructure, as evidenced by FTIR data earlier (Figure 3).

### **4. Materials and Methods**
