*3.8. SDS-PAGE Analysis*

The total protein electrophoresis results of the different treatment groups are shown in Figure 8. The results of the band optical density analysis using Quantity One 4.0 showed that the content of

the myosin heavy chain (MHC) and actin increased between 0 to 6 days in different groups. Then, different degrees of degradation appeared in different groups after six days. At day 12, the intensity of the MHC in the AP, VP, and MAP groups was 355, 555, and 643, respectively (*p* < 0.05). The MHC, which is very easily oxidized, is crosslinked by disulfide and non-disulfide covalent bonds which contribute to the formation of high-molecular-weight polymers and aggregates [45]. The degradation of MHC was inhibited significantly by VP and MAP. The degradation of protein was probably due to MHC oxidation, and oxidative damage of proteins may have also led to protein degradation and the crosslinking and aggregation of actin, which corresponds to the results of Lu et al. [46]. The actomyosin (42 kDa) band gradually became shallow over time. The change ranges of the VP and MAP groups were significantly smaller than that of the AP group, the main reason for which being the hydrolysis of cathepsin L; the change range of the MAP group was the smallest, indicating that the activity of cathepsin L in the MAP group was inhibited. Band III (13 kDa) of the AP group at day 12 was significantly different than the other two groups, and was highly correlated with reduced actin (correlation coefficient of 0.89), which we concluded was due to the degradation product of actin.


**Figure 8.** Effects of three packaging methods (AP, VP, and MAP) on total protein degradation of grouper samples during refrigerated storage at 4 ◦C. (AP: air packaging; VP: vacuum packaging; MAP: modified atmosphere packaging).

It has not been reported in previous works that all of the intensities of band II (34 kDa) in the three groups showed an increasing trend in the first 12 days. It can be concluded that the intensity of band II, which was speculated to be an indicator protein of grouper freshness before the 12th day, had a highly linear relationship with storage time (*R*<sup>2</sup> = 0.97), as analyzed by Quantity One 4.0. However, in the VP and MAP groups, the intensity of band II decreased after 12 days. The possible reason for this is that the protein belongs to a water-soluble protein, and the rate of drip loss increases at the later stage of storage [47]. The concentration of protein decreased with water loss, due to the type of protein belonging to water-soluble proteins; such changes can also be observed in bands I and III.

This study showed that the increase of protein oxidation was reflected by the content and distribution of carbonyl and sulfhydryl content changes. Table 2 shows that the carbonyl and sulfhydryl contents of protein oxidation indexes in each group were highly correlated with protein degradation, TCA-soluble peptides, and MFI. This indicates that protein oxidation promotes the degradation process during refrigerated storage of grouper fillets. Compared with AP, VP and MAP exhibited significant positive effects with regard to reducing protein oxidation. However, the MAP group showed better effectiveness than the VP group, which may have been due to the higher residual oxygen and oxygen transmission rate in VP. When vacuum pumping cannot make a complete vacuum environment, which results in residual air, the external oxygen gradually permeates into the packaging under the effect of internal and external pressure differences.

It is also speculated that a high concentration of carbon dioxide not only controls the growth of microorganisms, but also changes the pH on the surface of the fish, thus changing the activity of protease and affecting the degradation of myofibrils.

Unlike the results of Lametsch and Lonergan, which showed that protein oxidation reduces the activity of μ-calpain cysteine [48], thus inhibiting the degradation of muscle fibril in pork, the reason here may be that the low rate of protein oxidation could enhance the sensitivity of proteolytic enzymes for myofibril [14], and the activity of protease loss is not obvious under weak oxidation. Thus, fish and other meat proteasome systems also show large differences, which also might be the reason for the difference between the experimental results. Therefore, further research is needed on the metabolic pathway of how the protein oxidation of grouper muscle affects protein degradation.


**Table 2.** Correlation analysis between protein oxidation and the degradation of grouper in three groups.

AP: air packaging; VP: vacuum packaging; MAP: modified atmosphere packaging. \* correlation \*\* strong correlation.

#### **4. Conclusions**

This study shows that the changes of carbonyl, sulfhydryl, and Ca2<sup>+</sup> ATPase activity greatly varied with prolonged storage time, and demonstrates that the degree of grouper fillet protein oxidation was increased. The amide I band absorption peak of the infrared moved towards a higher wavenumber, while the secondary structure of the α-helix gradually transformed into a random curl. It has been shown that great changes of protein structure took place in grouper fillets during refrigerated storage. By combining indexes of protein degradation, such as MFI, SDS-PAGE, and TCA-soluble peptide content, it is concluded that myofibril oxidation could promote protein degradation in grouper fillets during refrigerated storage, which could be observed in each group (AP, VP, and MAP). High-carbon-dioxide MAP played a positive role in the inhibition of myofibril degradation and oxidation for refrigerated grouper fillets.

**Author Contributions:** Conceptualization, X.Z. and J.X.; methodology, J.X.; software, X.Z.; validation, J.X.; data curation, X.Z.; writing—original draft preparation, X.Z.; writing—review and editing, W.H.; project administration, W.H.; funding acquisition, J.X.

**Funding:** This research was financially supported by National Natural Science Foundation of China (grant number: 31571914), the China Agriculture Research System (CARS-47), the Construction Project of the Public Service Platform for Shanghai Municipal Science and Technology Commission (17DZ2293400), and the Shanghai Municipal Science and Technology Project to enhance the capabilities of the platform (grant number: 19DZ2284000).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

1. Kirtil, E.; Kilercioglu, M.; Oztop, M.H. Modified atmosphere packaging of foods. In *Reference Module in Food Science*; Elsevier: Amsterdam, The Netherlands, 2016.


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