*3.10. Cell Proliferation*

Cell proliferation experiments were performed using a method described by Golser et al. [51] with slight modification. The collagen sample was dissolved in 0.5 mol/L acetic acid and cast into cell culture wells at a final concentration of 0.5 mg/mL. After the collagen was fully dried, the plates were sterilized via UV treatment and seeded with the NIH3T3 fibroblasts. NIH3T3 fibroblasts seeded in the plates without collagen served as a negative control. After adding Dulbecco's Modified Eagle Medium (DMEM) cell culture medium to the plates, the cells were cultured in an incubator at 37 ◦C. At cell seeded times of 1, 2, 3, 4, and 5 days, 3-(4,5-dimethylthiazol-2-y)-2,5-diphenyltetrazolium bromide (MTT) solution was added to the cell culture medium and incubated for 4 h at 37 ◦C. The plates were removed, and the culture medium was aspirated. After adding dimethyl sulfoxide (DMSO) for 10 min, the absorbance (OD value) was measured at 490 nm.

#### *3.11. Statistical Analyses*

All data in this study are expressed as average ± standard deviation of at least three measurements. One-way ANOVAs coupled with Dunnett's test was performed using SPSS 13.0 to determine the statistical difference at 95% confidence level.

#### **4. Conclusions**

Our study, for the first time, introduced electrodialysis for the extraction of fish skin collagen (*T. flavidus*). This cost-effective technique was found to be superior to traditional dialysis with its advanced

efficiency (2 h/trial), large capacity (100 L/trial), high extraction yield (67.3 ± 1.3 g/100 g DW), and better environmental sustainability (6 L waste water/L sample). SDS-PAGE, amino acid composition analysis, and spectrophotometric characterization demonstrated that *T. flavidus* collagen extracted by electrodialysis primarily consisted of type I collagen with a purity of 96.1 ± 1.3%. ICP-MS analysis demonstrated that the heavy metals of *T. flavidus* collagen were less than the Chinese national standards. ELISA analysis indicated the safety of *T. flavidus* collagen. Notably, the collagen appeared to have better thermal stability than other fish species, with Tmax and Td of 41.8 ± 0.35 and 28.4 ± 2.5 ◦C, respectively. This observation can be attributed to its relative higher imino acid content (246 ± 0.04 residues/1000 amino acid residues). Cell proliferation experiment demonstrated that the cell proliferation rates of the experimental groups were slightly higher than those of the negative control group, but there was no significant difference. In addition, both NaCl level and pH condition were found to affect the relative solubility of *T. flavidus* collagen. All evidence suggests that electrodialysis is a promising technique for fish collagen and that *T. flavidus* skin collagen could serve as an alternative source of collagen to meet the increasing demand from academia and industry.

**Author Contributions:** J.C. completed the study design, most of the experiments and manuscript preparation; M.L. and R.Y. took part in data analysis; K.B., G.W., R.T., S.S., and N.X. took part in experiments and data analysis.

**Funding:** This work was gratefully supported by National Natural Science Foundation of China (41676129 & 41106149), and the Scientific Research Foundation of the Third Institute of Oceanography, SOA (2016007).

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