*3.4. FT-IR Spectroscopy Analysis of Starch–Surimi Gels with Non-Setting or Setting Effect* 3.4.1. Amide Bands of Protein

The gel matrix can be analyzed by using FT-IR spectroscopy to detect functional groups associated with intramolecular and intermolecular structures [38]. The amide bands of proteins have several distinct vibrational modes, including amide I, II, and III. Amide I (1600–1700 cm−1) resulted primarily from ν (C=O) and δ(N–H), whereas amide II and III (1480–1580 cm−1; 1200–1350 cm−1) originated from ν(C–N) and δ(N–H) [39]. Among them, amide I was the most useful in reflecting secondary and tertiary structures [9]. Generally, the α-helix, random coil, β-sheet, and β-turn structures correspond to 1650– 1660 cm<sup>−</sup>1, 1660–1665 cm−1, 1665–1680 cm−1, and –1680 cm−<sup>1</sup> ranges of the amide I band, respectively [40]. Li et al. [25] discovered that the starch did not cause significant shifts in amide bands (Figure 5a, b, Table 3). Non-setting gels and setting gels both showed peak values of amide I at 1654 cm−1, suggesting that α-helix dominated the secondary structure of the protein in the starch–surimi matrix. Although starch could increase the density of the gel matrix and influence chemical interactions, it had little effect on the three-dimensional structure of proteins. In contrast, slight changes in α-helix and random coil occurred between CG and SCG, which was conducive to increasing hydrogen bonds. The α-helix of native and partially denatured proteins and β structures that formed during heating and cooling are both stabilized by hydrogen bonds [2]. Therefore, the secondary structure of surimi protein had no significant change caused by the external physical forces of starch.


**Table 3.** Secondary structure (amide I) of protein in different starch–surimi matrices.

Uppercase letters indicate significant difference (*p* < 0.05) between different heating processes, lowercase letters indicate the difference between gels with different starch content (*p* < 0.05), and values are expressed as mean ± SD.

**Figure 5.** FT-IR spectroscopy (**a**,**b**) and sulfhydryl and disulfides (**c**,**d**) regions of starch–surimi matrix with different treatment. CG0–CG12: cooking gels with starch content (0–12%); SCG0–SCG12: setting-cooking gels with starch content (0–12%).

#### 3.4.2. Tryptophan (Trp) Residue Bands and Tyrosine (Tyr) Doublet Bands

The vibrations near 760 and 1340 cm−<sup>1</sup> present the microenvironment of Trp residues. Once the Trp residues buried in the hydrophobic environment were exposed to a polar environment, the intensity of bands showed upward trends [41]. The intensity of tryptophan residue bands increased slightly with the increment of starch content in Figure 4 possibly contributing to the exposure of Trp residues [42]. The starch granules occupied the matrix space, which promoted the unfolding of protein structure and then provided impetus to the exposure of the hydrophobic core. A similar result was found in the Tyr doublet bands, which were proposed as a means for determining whether the tyrosine residue was solvent-exposed or buried [43]. If the intensity at 850 cm−<sup>1</sup> (I850) was higher than I830, this indicated that the Tyr residues at this time changed from the "buried" to the "exposed" state [44]. The distinct vibrations were exhibited differently at 830 and 850 cm−<sup>1</sup> in both

gels (Figure 5a, b). A decreased I830 and a concurrent increased I850 indicated that there were weak hydrophobic interactions among tyrosine residues. It could be demonstrated that protein solubility of CG increased due to reduced hydrophobic interactions. However, the change of hydrophobic amino acids between CG and SCG was not obvious. Therefore, the setting treatment of surimi did not hinder the effect of starch addition on the spatial structure of hydrophobic amino acids in surimi proteins.
