*2.12. Statistical Analysis*

Three replicates were used for all samples with three parallel tests. SPSS 8.0 (SPSS, Chicago, IL, USA) was used for statistical analysis. Univariate ANOVA was used to determine the statistical differences in different treatment groups. Duncan's multiple range test was used to determine the significant difference between the averages (*p* < 0.05). The data were expressed as the mean and standard deviation (SD), and Origin 8.5 (OriginLab, Northampton, MA, USA) was used for illustration.

#### **3. Results and Discussion**

#### *3.1. Changes in Total Sulfhydryl and Disulfide Bonds*

Figure 1 shows the changes of sulfhydryl and disulfide bonds in each group. As shown in Figure 1, the content of the total sulfhydryl groups in each treatment group presented a downward trend with the extension of storage time, which is similar to trends found in other studies on grouper (He et al., 2018). The sulfhydryl content of the AP group decreased to 49.05 nmol/mg of protein on the sixth day, which was significantly different compared with the VP and MAP groups. In contrast, the differences

between the VP and MAP groups were not significant. The sulfydryl content in the VP group was slightly lower than that in the MAP group after nine days.

**Figure 1.** Changes in sulfhydryl (**top**) and disulfide (**bottom**) bond contents of grouper myofibrillar protein (AP: air packaging; VP: vacuum packaging; MAP: modified atmosphere packaging). Different lower-case letters in different groups from same day indicate a significant difference (*p* < 0.05).

The sulfhydryl group of myofibrils can be oxidized by reactive oxygen species (ROS) that produce disulfide bonds, sulfonic acid, and other oxidation products during refrigerated storage [29]. Protein oxidation can be reflected by the loss of the sulfhydryl group [30], which contains a disulfide bond; this is a common oxidation product that can gradually increase with the oxidation of the sulfhydryl group. The disulfide bond content in each group increased gradually with the oxidation of the sulfhydryl group (Figure 1), and the content of the AP group increased significantly, from 44.5 to 82.5 nmol/mg protein. It is worth noting that the disulfide bond contents of the AP and MAP groups decreased slightly starting from the ninth day, which may have been caused by the degeneration of myofibril.

As shown by the two-way analysis of variance, the storage time and packaging methods significantly affected the total sulfhydryl and disulfide bonds of the grouper fillets (*p* < 0.01). There was a significant difference between the VP and AP groups, and between the AP and MAP groups (*p* < 0.01); however, there was no significant difference between the VP and MAP groups (*p* > 0.05).

#### *3.2. Changes in Carbonyl Content and Hydrophobicity*

Protein carbonylation is one of the most important changes in the oxidation of muscle proteins, especially the extensive modifications caused by the oxidation of myofibrils. In particular, from the formation of carbonyl compounds [31], changes in the content of carbonyl compounds are usually used to represent the oxidation rate of proteins. In Figure 2, it can be seen that the change of carbonyl content in each group increased with the cold storage time. The initial carbonyl content of the fresh fish was increased 10 times in the AP group on day 6. The two-way analysis of variance revealed that protein carbonylation was significantly affected by storage time and packaging methods (*p* < 0.05).

The specific binding of bromophenol blue with myofibrillar is considered to be a simple and reliable method for the determination of surface hydrophobicity. The hydrophobicity of the protein surface can be determined by the degree to which bromophenol blue binds specifically to myofibrillar [20]. Due to the conformational changes induced by hydroxyl radicals, oxidized myofibrillar protein undergoes extensive exposure of hydrophobic groups [32]. This phenomenon was confirmed in our experiment. The degree to which bromophenol blue bound specifically to myofibrillar became serious in all three groups, which showed no significant differences in the first six days (*p* > 0.05). In the AP group, the content reached 102.98 μg, which was slightly higher than those of the other two groups. However, the VP group was slightly higher than the MAP group on day 15. The change of the surface hydrophobicity of the protein may have been caused by the entry of nonpolar amino acid molecules into hydrophobic clusters [33]. The expansion or rearrangement of protein molecules could lead to a change of the secondary and tertiary structures of the proteins. VP and MAP significantly delayed the oxidation of protein in grouper meat, similar to what has been found in other fish studies [34]. The two-way analysis of variance showed that the storage time and packaging methods significantly affected the carbonyl content and hydrophobicity of the grouper fillets (*p* < 0.01). There was a significant difference between the VP and AP groups, and between the AP and MAP groups (*p* < 0.01); however, there was no significant difference between the VP and MAP groups (*p* > 0.05).

**Figure 2.** Changes in carbonyl content (bar graph) and protein surface hydrophobicity (expressed as Bound BPB, line graph) of grouper myofibrillar protein. (AP: air packaging; VP: vacuum packaging; MAP: modified atmosphere packaging; BPB: bromophenol blue).
