**1. Introduction**

Collagen is the major structural component of various extracellular matrices in mammalian connective tissues, such as the skin, corneas, cartilage, bone, and blood vessel [1]. Collagen is also widely used in biomaterials, drugs, foods, and cosmetics [2]. Its high biocompatibility as well as the ability to support cell growth and differentiation has made it an important matrix for cell biology, cosmetics, and regenerative medicine [3,4]. Collagen is also widely used as a gelatin precursor in the food industry for formulating emulsions, foams, colloids, and biodegradable films [5]. Annual sales of collagen and its derivatives in the global market have reached billions of dollars [6]. Despite high feasibility and biocompatibility, these mammalian origins potentially limit practical application of collagen due to sociocultural beliefs (e.g., in Muslim countries and India). In addition, the mammalian origins can further restrain collagen application by increasing additional sanitary costs for industrial production because of extensive consumer concerns regarding transmissible diseases from porcine and bovine [7,8].

Fish, a popular dietary ingredient, is a great alternative source of commercial collagen. In addition to its worldwide acceptance by different sociocultural belief systems, fish is less likely to carry mammalian-borne diseases, thus requiring lower sanitary costs for industrial production of collagen [9]. More interestingly, the rapid development of the fishing industry has resulted in a huge amount of collagen-rich by-products, including skin, scales, and bones (50.2–117 million metric tons) [10]. Pufferfish aquaculture is a thriving industry in China, producing more than 14,000 metric tons in 2013 [11]. Studies on fish suggest that pufferfish skin is rich in type I collagen and may serve as an appropriate source of collagen to replace their mammalian counterparts.

While promising, industrial production of fish collagen is limited by the low efficiency, low productivity, and low sustainability of the purification process. Crude fish collagen extract is mainly purified by dialysis through a passive diffusion process in the industry. This method usually takes 3–4 days to isolate pure collagen and often processes less than 1 L of samples [12,13]. Moreover, dialysis of crude fish collagen extract can produce a relatively large amount of acetified wastewater (20 L dialysate/L sample) [14–16], leading to severe environmental stress. An advanced purification technique with better economic and environmental outcome is therefore needed to take full advantage of collagen-rich fish by-products.

Electrodialysis, as an active diffusion technique, appears to be an excellent solution to improve the efficiency, productivity, and sustainability of fish collagen purification. This method can purify charged proteins/peptides by ion-exchange membranes through a stimulated diffusion process under the influence of electric potential difference [17]. A recent study on marine protein hydrolysate showed that electrodialysis could process 3 L of sample within a relative short time (6 h) while requiring just a small amount of dialysis buffer (1 L/L sample) [18].

The aim of this study was to assess the practicality of electrodialysis for the isolation and purification of collagen derived from yellowbelly pufferfish (*Takifugu flavidus*) skin. Considering that this technique has not been applied in the fishing industry, we hypothesized that the application of electrodialysis could not only improve economic and environmental outcomes of fish collagen production but also retain the physicochemical properties of *T. flavidus* collagen. Extraction efficiency, productivity, and wastewater production were also determined in this study, and the quality of *T. flavidus* collagen was systemically evaluated by electrophoresis, spectrophotometric characterization, thermal properties, and solubility.

#### **2. Results and Discussion**
