*2.1. Preparation of ASC and PSC*

The yields (dry weight basis, 19.07% and 19.61% respectively) of ASC and PSC were in highly agreement with former reports [42,43]. Comparison of the collagens extract from different fish have been reported, including the skin of paper nautilus (55.2%) [44], bigeye snapper (1.59%) [45], deep-sea redfish (10.3%) [46], Japanese sea bass (40.7%) [47], yellow sea bream (40.1%) [48], and grass carp (45.3%) [43]. In contrast, the yield of collagen from tilapia was different from these reported species. The results suggest that some discrepancies might exist among these fish species. In addition, the yield of ASC was a little bit lower than the yield of PSC, this might be due to the disadvantage of the solubility of cross-links formed through the reaction of aldehyde with lysine and hydroxylysine at telopeptide helical sites [49], which also can be explained by the results of Fourier transform infrared (FTIR) analysis. Besides the effects on solubility, the pepsin that is used in the extraction of PSC might bring other changes, such as the stability and biocompatibility of the resultant collagens.

#### *2.2. Amino Acid Composition*

Table 1 shows the amino acid composition of ASC and PSC, which is expressed as residues per 1000 total amino acid residues. According to Table 1, glycine is the most important component in ASC and PSC, with 322 and 343 residues, accounting for about one-third of the total amino acid residues. It is slightly higher than common aquatic organisms carp skin (311 residues), cod skin (308 residues), squid skin (269 residues) [50,51], and very similar to land mammals' bovine skin (320 residues),

bovine skin (334 residues), and porcine skin (326 residues) [52,53]. Glycine is the most important amino acid in collagen. All members of the collagen family have a tripeptide (Gly-X-Y) repetitive structure, which plays an important role in the formation of the triple-helix structure. The tripeptides (Gly-X-Y) are repeatedly arranged on each chain of collagen, accounting for about 20–30% of all tripeptide structures. The X position of Gly-X-Y is often occupied by proline, which is consistent with the result in Table 1 (115 and 106 proline residues in ASC and PSC).

**Table 1.** The amino acid composition of acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from Nile tilapia skin.


The existence of the triple-helix structure is the most direct evidence to distinguish collagen from gelatin. [54]. As is known, electrospinning is a method of stretching a polymer solution to fibers that have a diameter of about several hundred nanometers by electrostatic force. Due to its wide applicability, high efficiency, and simplicity, electrospinning is widely used in the field of tissue engineering scaffold materials.

In addition, it is worth noting that the content of hydroxyproline in tilapia skin is ASC (70 residues) and PSC (86 residues). As a characteristic component of collagen, the content of collagen in raw materials can be measured by the ratio of hydroxyproline. No cystine and tryptophan were detected in both the ASC and PSC of tilapia skin collagen, which was consistent with the characteristics of type I collagen.
