*3.3. Changes in Ca2*<sup>+</sup> *ATPase Activity*

Ca2<sup>+</sup> ATPase mainly concentrates in the globular heads of myosin. The hydrophobic interactions, hydration of polar residues, and hydrogen bonds influence the stability of the three-dimensional structure of the protein. Since the three-dimensional structure of the protein determines the physiological activities of the protein itself, the activities of the protein may be lost or changed because of changes in the microstructure. The Ca2<sup>+</sup> ATPase activity of actomyosin can be used as an important indicator for assessing the degree of protein denaturation, as it can indirectly reflect the integrity of myofibrillar protein [35].

Figure 3 shows that the Ca2<sup>+</sup> ATPase activity of the grouper fillets respectively decreased in all samples. The two-way analysis of variance showed that storage time and packaging methods significantly affected the Ca2<sup>+</sup> ATPase content of the grouper fillets (*p* < 0.05). On the 12th day, the content of the Ca2<sup>+</sup> ATPase activity in the AP group decreased about 75.16%. There was a significant difference between the VP and AP groups and between the AP and MAP groups (*p* < 0.01), but there was no significant difference between the VP and MAP groups (*p* > 0.05). However, the VP and MAP groups were about 45.83%, which was consistent with the existing reports of other aquatic products [36]. It has been reported that the sulfhydryl group is abundant in the center of Ca2<sup>+</sup> ATPase [21]. In our experiment, we found that the activity of Ca2<sup>+</sup> ATPase was closely related to the sulfhydryl group. The correlation coefficients of the AP, VP, and MAP groups were 0.983, 0.946, and 0.984, respectively, and the Ca2<sup>+</sup> ATPase activity was highly correlated with the change of the sulfhydryl group content. It was speculated that the oxidation of the sulfhydryl group in the active center resulted in a decrease of Ca2<sup>+</sup> ATPase activity.

**Figure 3.** Changes in Ca2<sup>+</sup> ATPase activity of grouper myofibrillar protein. (AP: air packaging; VP: vacuum packaging; MAP: modified atmosphere packaging).

#### *3.4. Trichloroacetic Acid-Soluble Peptide Analysis*

TCA-soluble peptides can reflect the degree of protein degradation. As shown in Figure 4, the TCA-soluble peptide content in all groups was obviously elevated during the entire storage period. The results showed that proteolysis occurred in succession and was similar to the trend reported by Yu et al. for grass carp fillets under refrigerated storage [37]. The initial number of TCA-soluble peptides of the grouper fillets was 0.42 μmol tyrosine/g sample, while the value reached 1.44 μmol tyrosine/g sample after six days storage for the AP group, which had the largest rate of increase among the three groups. The two-way analysis of variance revealed that the storage time and packaging methods significantly affected the TCA-soluble peptides of the grouper fillets (*p* < 0.01). The increase of TCA-soluble peptides might have initially been due to the activity of the endogenous enzyme [38]. Then, protein degradation was accelerated under the combined action of endogenous enzymes and microorganisms. The final TCA-soluble peptide content of the VP and MAP groups was significantly lower than that of the AP group (*p* < 0.05). The content of TCA-soluble peptides of the MAP group was lower than that of the VP group, but there was no significant difference between the two groups (*p* < 0.05), indicating the effective inhibition of proteolysis by VP and MAP, which might have been due to the growth of microorganisms and inhibition by the anoxic environment in the VP and MAP groups.

**Figure 4.** Changes in trichloroacetic acid (TCA)-soluble peptides of grouper myofibrillar protein (AP: air packaging; VP: vacuum packaging; MAP: modified atmosphere packaging).

#### *3.5. Myofibril Fragmentation Index Analysis*

As shown in Figure 5, the MFI maintained increasing trends among all groups. However, compared with the other two groups, the AP group rose significantly, with 97 mg/100 g being reached on day 3 (*p* < 0.05). No obvious change of the MFI in the VP and MAP groups was discovered at the corresponding time. The two-way analysis of variance revealed that storage time and packaging methods significantly affected the MFI of the grouper fillets (*p* < 0.01). There was no significant difference between the VP and MAP groups on the 12th day, but the MFI of the AP group was very significantly higher (*p* < 0.01) than those of the VP and MAP groups. This indicates that the internal integrity of myofibrillar in the AP group was the most destructive during refrigeration storage, while the destruction of the internal structure of myofibril was delayed by MAP and VP. Myofibril fragmentation refers to the phenomenon of myofibril breaking into shorter segments near the Z disk or Z line. The MFI was calculated from the percentage of myofibrils that were 1–4 sarcomeres long, which was mainly due to degradation of the connectin and actin of sarcomeric I. The MFI reflects the structural integrity of the myofibril during refrigerated storage [39].

**Figure 5.** Changes in the myofibril fragmentation index (MFI) values of grouper myofibrillar protein. (AP: air packaging; VP: vacuum packaging; MAP: modified atmosphere packaging).

#### *3.6. Free Amino Acid Analysis*

The concentrations of FAAs in grouper fillets on the first, sixth, and twelfth days of refrigerated storage are shown in Table 1. The degradation of protein was the main reason for the variation of FAAs [37], so the degree of protein degradation could be reflected by the changes of the FAA content. A similar trend was also reported by Shi et al. for grass carp [40]. Table 1 shows that the content of FAA rapidly increased with storage time in the AP group, which might have been due to the degradation of protein caused by protein oxidation. However, the content of histidine in the grouper fillets was far below that found in grass carp, as reported by Yu et al., during refrigerated storage, which might be ascribed to the muscle composition of grouper. Aspartic acid, glutamic acid, and glycine are the main flavor contributors in the fillets, and the change of glycine was the most obvious among all groups. Glycine in the AP group increased from 91 mg/100 g on day 0 to 153 mg/100 g on day 6, then it dropped to 78 mg/100 g on day 12. The corresponding contents in the VP and MAP groups were 88 and 108 mg/100 g, respectively. The total FAA contents in the AP group were significantly higher (*p* < 0.05) compared with the other groups on day 6, and this phenomenon indicates that the degree of protein degradation was the greatest in the AP group. The number of FAAs was closely related to the storage time (*p* < 0.05), and the packaging method had no significant effect on it, as shown by the two-way analysis of variance (*p* > 0.05). The final FAA content was reduced among all groups, which when compared to day 6, might have been due to the degradation of enzymes caused by microbial growth [41].

**Table 1.** Changes in free amino acid (FAA) content (mg/100 g) of grouper muscle during refrigerated storage at 4 ◦C.


Results are expressed as means in mg per 100 g of sample with standard errors. Different lower-case letters (a, b, <sup>c</sup> and d) in different groups for same amino acid indicate a significant difference (*p* < 0.05). AP: air packaging group; VP: vacuum packaging group; MAP: modified atmosphere packaging group.

#### *3.7. FTIR Analysis*

The secondary structure of the protein was composed of an α-helix, a β-sheet, a β-turn, and a random coil. The function of a protein and its biochemical properties change with the variation of the secondary structure, which is due to the oxidation of the protein. As one of the main methods of studying the secondary structure of proteins, FTIR can be used to analyze changes in protein structure and the spatial distribution of proteins [42]. The amide I band of a protein (from 1600 to 1700 cm−<sup>1</sup> of mid-infrared spectroscopy) can reveal a wealth of information about the constituents of the secondary structure in proteins. The peaks at the wavenumbers of 1600–1640, 1640–1650, 1650–1660, and 1660–1700 cm−<sup>1</sup> are for the β-sheet, random coil, α-helix, and β-turn, respectively [43]. As shown in Figure 6, the second-order, second-derivative, mid-infrared spectra of the Gaussian fitting drawn by PeakFit (PeakFit v 412, Systat Software Inc., USA) was used to analyze the protein secondary structure changes of each group at days 0 and 12.

Figure 6 shows that the peaks of each packaging group had a certain weakening on the 12th day. The change from the range of 1650–1660 cm−<sup>1</sup> was significant compared with that of day 0, indicating that the levels of the random coil had increased. As shown in Figure 6A–D, the absorption peak of the spectrum shifted to the high-wavenumber area. The reason for this phenomenon was reported as the hydrogen bond of the protein structure being destroyed during refrigerated storage [44]. Since the spectrum diagram of the MAP group on the 12th day was most similar to that on day 0, compared to the other treatment groups, it indicates that the destruction of the secondary structure of the protein was inhibited in refrigerated grouper fillets in the MAP group.

**Figure 6.** *Cont.*

**Figure 6.** The second-order, second-derivative, mid-infrared spectra by PeakFit. (**A**: 0 days; **B**: AP group on the 12th day; **C**: VP group on the 12th day; **D**: MAP group on the 12th day).

The change in the secondary structure for all groups is shown in Figure 7. The random coil increased, and the α-helix content decreased during refrigerated storage, which might have been due to the breakage of the hydrogen bonds. Further, the surface hydrophobicity increased, and a disordered state gradually became present in the protein. The random coil increased by 34.8% on the 12th day compared to day 0 in the AP group, which was significantly higher than that of the VP and MAP groups (*p* > 0.05). The results showed that the protein structure was partly inhibited by VP and MAP. The β-sheet of each group also increased to a certain extent (*p* > 0.05), due to the change of the peptide chain folding structure caused by the gradual formation of sulfhydryl oxidation and disulfide bonds.

**Figure 7.** Changes in secondary structure contents of grouper protein during refrigerated storage (**A**: the change in the random coil content under different treatments, **B**: the change in the α-helix content under different treatments, **C**: the change in the β-turn content under different treatments, **D**: the change in the β-sheet content under different treatments). (Air: air packaging; VP: vacuum packaging; MAP: modified atmosphere packaging).
