*2.7. Cell Proliferation*

The greater the optical density (OD) value indicted, the greater is the number of cells. As shown in Figure 5, the OD value of the negative control and experimental group increased with culture time. However, at different points in time, the OD values of the experimental groups were slightly higher than those of the negative control group, although the difference was not significant. This may be because the cells were cultured at 37 ◦C, which was above the denaturation temperature of 28.4 ± 2.5 ◦C for the collagen samples. When the collagen sample was kept at 37 ◦C for a long time, the collagen degraded, leading to loss of collagen activity. In the next stage of research, one important aim of our research will be to improve the thermal stability of collagen.

**Figure 5.** MTT assay detecting the effects of collagen sample on cell proliferation of NIH3T3 cell.

#### *2.8. Extraction of T. flavidus Collagen*

To our knowledge, this is the first time electrodialysis was introduced as part of fish collagen purification. The application of electrodialysis significantly increased the extraction yield of fish collagen. Our experiments showed that 67.3 ± 1.3 g of *T. flavidus* collagen could be produced from 100 g of dry skin, which is much higher than the yields of *T. rubripes* collagen (10.7–44.7 g/100 g dried weight, DW) extracted by traditional dialysis [23,30]. This discernible difference could be partially attributed to the variations in collagen contents between the two *Takifugu* species. It is also likely due to electrodialysis-mediated improvements of extraction efficiency as the extraction yield obtained from this study was significantly higher than those reported by previous dialysis research on tilapia (27.2 g/100 g DW) [46] and grass carp (46.6 g/100 g DW) [16].

This study showed that electrodialysis produced more than 10 kg of collagen samples in one purification effort by purifying 100 L of crude collagen solution. It also concentrated the purification step from a traditional 96-h dialysis [13–15] into a 2-h period. Considering that the traditional method could not purify 100 L of crude collagen solution in one effort [12–14], the application of electrodialysis is estimated to significantly reduce instrument and labor time for large-scale production. This demonstrates that electrodialysis has a huge advantage over traditional dialysis for translation into industrial use.

In addition to improving purification efficiency and productivity, electrodialysis is a cost-effective technique with better environmental sustainability relative to the traditional method. In our study, to produce 10 kg of *T. flavidus* collagen, electrodialysis consumed 45.0 L of acetic acid and produced 600 L of waste water (Table 3). In comparison, traditional dialysis is estimated to use 400 times more acetic acid and 600 m of additional dialysis bag [12,14,15] while producing 600 times more waste water to purify the same amount of collagen extract. In total, it would take US\$15 to produce 10 kg of collagen using electrodialysis extraction. This indicates a strong potential of the electrodialysis technique to be used in industrial applications.


**Table 3.** Expenses and instrumental requirements for producing 10 kg of *T. flavidus* collagen by electrodialysis compared to traditional dialysis.

Expenses and essential instruments for traditional dialysis was estimated based on the studies of Liu et al. (2012), Matmaroh et al. (2011), Nagai and Suzuki (2000), and Ogawa et al. (2004).
