RESEARCH ARTICLE. Introduction. Chang-Feng Chi, Bin Wang, Zhong-Rui Li, Hong-Yu Luo, and Guo-Fang Ding

Food Sci. Biotechnol. 22(4): 909-916 (2013) DOI 10.1007/s10068-013-0163-0 RESEARCH ARTICLE Characterization of Acid-soluble Collagens from the Cartilages of Scalloped Hammerhead (Sphyrna lewini), Red Stingray (Dasyatis akajei), and Skate (Raja porosa) Chang-Feng Chi, Bin Wang, Zhong-Rui Li, Hong-Yu Luo, and Guo-Fang Ding Received: 31 October 2012 / Revised: 21 January 2013 / Accepted: 6 February 2013 / Published Online: 31 August 2013 KoSFoST and Springer 2013 Abstract Acid-soluble collagen from the cartilages of scalloped hammerhead (Sphyrna lewini, ASC-S), red stingray (Dasyatis akajei, ASC-D), and skate (Raja porosa, ASC-R) were isolated with yields of 5.64±0.41, 8.72±0.31, and 6.74±0.28% on the basis of wet weight, respectively. ASC-S, ASC-D, and ASC-R had glycine as the major amino acid with contents of 220, 231, and 228 residues/ 1,000 residues, respectively, and contained imino acid of 174, 155, and 142 residues/1,000 residues, respectively. Three kinds of collagens more likely were comprised of 2 types of collagen (type I and II) and had some differences in primary structures. The denaturation temperatures (T d s) of the ASC-S, ASC-D, and ASC-R were 23.8, 15.1, and 12.1 o C, respectively. Maximum solubility of the 3 ASCs in 0.5 M acetic acid was observed at ph 2, and a sharp decrease in solubility above 2%(w/v) was observed in the presence of NaCl. The 3 lyophilized collagens displayed loose, fibrous, and porous structures. Keywords: Sphyrna lewini, Dasyatis akajei, Raja porosa, cartilage, acid-soluble collagen (ASC) Introduction Collagen is the main protein of connective tissue in Chang-Feng Chi National Engineering Research Center of Marine Facilities Aquaculture, School of Marine Science, Zhejiang Ocean University, Qixiangtai Road 51, Zhoushan 316004, China Bin Wang ( ), Zhong-Rui Li, Hong-Yu Luo, Guo-Fang Ding School of Food and Pharmacy, Zhejiang Ocean University, Qixiangtai Road 51, Zhoushan 316004, China Tel: +86-580-2555085; Fax: +86-580-2554781 E-mail: wangbin4159@hotmail.com animals and the most abundant protein in mammals, making up approximately 30% of total protein in animal body (1). Till now, at least 29 types of collagen have been identified (named types I-XXIX) from various animal and human tissues, such as skins, bones, cartilage, and all other organs of vertebrates (2), and each type has a distinctive amino acid sequence and molecular structure to play a unique role in the tissue (3). Collagen was traditionally isolated from byproducts of land-based animals, such as cows, pigs, and poultry (4), and has been widely utilized as a material for food additives, cosmetics, biomedical materials, and experimental reagents due to its excellent biocompatibility, biodegradability, and weak antigenicity (2,5). Despite its wide range of applications, the use of collagen in food products may be a concern among consumers because of dietary restriction and concerns over bovine spongiform encephalopathy (BSE) and transmissible spongiform encephalopathy (TSE) (2,6). Furthermore, porcine collagen and other collagens from animals that are not religiously slaughtered are unacceptable to some religious and ethnic groups, such as Jews and Muslims (5). Therefore, the global demand for collagen from alternative sources such as aquatic animals has been increasing over the years due to their availability, the lack of dietary restriction or risk of disease transmission, and the possibility of high collagen yields. At present, large quantities of byproducts, accounting for 50-70% of the original raw material (7), are generated during aquatic products processing in China. Therefore, optimal use of these byproducts is a promising way to protect the environment, to produce value-added products to increase revenue for fish processors, and to create new job/business opportunities. Recently, many scientists have focused their interests on isolation and characterization of collagens

910 Chi et al. from marine organisms and their byproducts, such as the skin of unicorn leatherjacket (8), body wall of sea cucumber (9), skin and bone of bigeye snapper (7), scale of spotted golden goatfish (3), coelomic wall of sipunculida (10), backbone of Baltic cod (11), and skin of Nile tilapia (6). Cartilaginous fishes (Chondrichthyes) are commercially important species. During processing, solid waste is generated, especially skin and cartilage, which can be used as a potential raw material for collagen extraction. For the past few years, collagens from tissue of cartilaginous fishes have been extracted, such as cartilage of milk shark (Carcharius acutus) (12), pectoral fin cartilage of skate (13), skin and bone of shark (14), skin of eagle ray, red stingray, and yantai stingray (15), and cartilages and skin of brownbanded bamboo shark and blacktip shark (16,17). However, the isolation of collagen from cartilages of scalloped hammerhead (Sphyrna lewini), red stingray (Dasyatis akajei), and skate (Raja porosa) has not been explored. To make more effective use of the wastes generated during processing, the acid-solubilized collagens from the cartilages of scalloped hammerhead, red stingray, and skate were isolated and characterize. Materials and Methods Materials The frozen scalloped hammerhead (Sphyrna lewini), red stingray (Dasyatis akajei), and skate (Raja porosa) were purchased from Nanzhen market (in Zhoushan City, Zhejiang Province of China) and placed in ice with a fish to ice ratio of 1:1 (w/w) using a polystyrene box as a container. The frozen fishes were transported to School of Food and Pharmacy, Zhejiang Ocean University and stored at 20 o C. High molecular weight markers, type I collagen from calf skin (SSC) and trypsin were used as the standards and obtained from Sigma-Aldrich (St. Louis, MO, USA). Sodium dodecyl sulphate (SDS), Coomassie Blue R-250, N,N,N',N'-tetramethylethylenediamine (TEMED), and potassium bromide (KBr) were procured from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). All other reagents used were of analytical grade. Preparation of acid-solubilized collagen ASCs from the cartilages of scalloped hammerhead, red stingray, and skate were extracted by the previous method (16,17) and referred to as ASC-S, ASC-D, and ASC-R, respectively. All the preparation procedures were carried out at 4 o C. Proximate analysis and determination of amino acid composition Moisture, ash, fat, and protein contents of ASC-S, ASC-D, and ASC-R were determined according to the methods of AOAC (2003) with the method numbers of 950.46B, 920.153, 960.39a, and 928.08, respectively. Amino acid compositions of ASC-S, ASC-D, and ASC-R were measured according to the previous method (5). Electrophoretic pattern and peptide mapping Electrophoretic patterns and peptide mapping of ASC-S, ASC-D, and ASC-R were measured according to the method of Li et al. (5). Fourier transform infrared (FTIR) spectroscopy The infrared spectra (450-4,000 1/cm) of ASCs were recorded in KBr disks with a FTIR spectrophotometer (Nicolet 6700l; Thermo Fisher Scientific Inc., Waltham, MA, USA). One mg of dry sample was mixed with 100 mg of dry KBr, and the mixture was pressed into a disk for spectrum recording. Viscosity of collagen solution The viscosity of ASC-S, ASC-D, and ASC-R solutions was determined according to the method of Li et al. (5). The denaturation temperature (T d ) was dened as the temperature causing 50% decrease in the relative viscosity of collagen solution. Solubility The solubility of ASC-S, ASC-D, and ASC-R was determined by the method of Li et al. (5). ASC-S, ASC-D, and ASC-R were dissolved in 0.5 M acetic acid to obtain a final concentration of 3 mg/ml and the mixture was stirred at 4 o C for 24 h. Thereafter, the mixture was centrifuged at 5,000 g for 15 min at 4 o C. The supernatants were used for solubility study. Ultrastructure ASC-S, ASC-D, and ASC-R were redissolved in 0.5 M acetic acid at a concentration of 5%(w/v), followed by dialyzing against distilled water, respectively. ASC-S, ASC-D, and ASC-R solutions were lyophilized in a freeze dryer (Eyela FD-1000; Tokyo Rikakikai Co., Ltd., Tokyo, Japan) and the samples were sputter coated for 90 s with gold using a Jeol JFC-1200 fine coater (Tokyo Rikakikai Co., Ltd.). The morphologies of the electrospun fibers and membrane were observed under a scanning electron microscope (Hitachi TM-1000; Hitachi High-Technologies Co., Ltd., Tokyo, Japan). Statistical analysis All experiments were performed in triplicate (n=3), and analysis of variance (ANOVA) test (using SPSS 13.0 software) was used to compare the mean values of each treatment. Significant differences between the means of parameters were determined by using Duncan s multiple range test (p<0.05).

Collagens from Cartilages of Cartilaginous Fishes 911 Table 1. Proximate compositions of acid-soluble collagens (ASCs) from the cartilages of scalloped hammerhead (ASC-S), red stingray (ASC-D), skate (ASC-R), and type I collagen from calf skin (SSC) on the basis of dry weight Composition ASC-S ASC-D ASC-R SSC Moisture 00.50±0.02 d1) 4.86±0.06 a 0.85±0.05 c 4.62±0.08 b Ash 4.58±0.18 d 14.60±0.27 a 0 7.66±0.15 c 8.18±0.09 b Lipid 1.05±0.12 b 0 1.35±0.09 ab 0.66±0.17 c 1.65±0.09 a Protein 93.91±0.87 a 0 79.34±0.66 d 0 90.38±0.63 b 0 85.65±0.54 c 0 1) All values are mean±sd; (n=3); a-d Values with different letters in the same row indicate significant difference for the same composition determination (p<0.05). Results and Discussion Yield and proximate analysis The yields of ASC-S, ASC-D, and ASC-R were 5.64±0.41, 8.72±0.31, and 6.74±0.28% (based on the wet weight of cartilage), respectively, which were much higher than those found in collagen from the cartilages of brownbanded bamboo shark (0.31%), blacktip shark (0.42%) (17), and bone of bigeye snapper (1.59%) (7) and carp (1.06%, on d.w.b.) (18), but much lower than those of collagens from Baltic cod backbone (25%) (11), and bones of skipjack tuna (42.3%), yellow sea bream (40.1%), and horse mackerel (43.5%) (19). The differences in yields might be attributed to the differences in fish species, tissue structure, and preparative methods (1). As shown in Table 1, ASC-S, ASC-D, and ASC-R were similar in proximate composition. They possessed high protein content (79.34-93.91%) with the ash content ranging from 4.58 to 14.60%, and low amounts of moisture (0.50-4.86%) and lipids (0.66-1.05%). Compared with chemical compositions of SSC, ASC-S, and ASC-R were higher in protein but lower in lipid, ash, and moisture contents; but ASC-D showed the lowest content of protein and highest contents of moisture and ash. Amino acid composition As shown in Table 2, ASC-S, ASC-D, and ASC-R showed similar amino acid compositions. They had Gly as the major amino acid and were rich in Glu, Pro, and Ala, but low contents of His, Tyr, Hyl, and Cys were also observed. According to reports, Gly is the most dominant amino acid in collagen, and all members of the collagen family are characterized by domains with repetitions of the proline-rich tripeptides (Gly-X-Y) involved in the formation of the triple helix, except for the first 14 amino acid residues from the N-terminus and the first 10 amino acid residues from the C-terminus of the collagen molecules (1,5,15). On the other hand, the Gly contents (220-231 residues/1,000 residues) of ASC-S, ASC-D, and ASC-R were slightly less than that of ASC from the bone (267.7 residues/1,000 residues) of spotless smooth-hound (Mustelus griseus) (14), but significantly lower than those of ASC from the cartilages of brownbanded bamboo shark and blacktip shark (317 residues/1,000 residues) (17), and Table 2. Amino acid compositions of acid-soluble collagens (ASCs) from the cartilages of scalloped hammerhead (ASC-S), red stingray (ASC-D), skate (ASC-R), and type I collagen from calf skin (SSC) (residues/1,000 residues) Amino acid ASC-S ASC-D ASC-R SSC Hyp 69 48 50 94 Asp 59 59 55 45 Thr 30 32 37 18 Ser 32 36 51 33 Glu 114 113 113 75 Pro 105 107 92 121 Gly 220 231 228 330 Ala 94 92 91 119 Cys 3 1 1 0 Val 35 33 31 21 Met 12 14 19 6 Ile 24 21 26 11 Leu 39 39 36 23 Tyr 11 8 9 3 Phe 22 22 22 3 Hyl 8 6 7 7 Lys 36 39 35 26 His 9 12 15 5 Arg 78 87 82 50 Total 1,000 1,000 1,000 1,000 Imino acid 174 155 142 215 the bone of bigeye snapper (361 residues/1,000 residues) (7) and carp (334 residues/1,000 residues) (18). Additionally, the amounts of imino acid (Pro and Hyp) are important for the structural integrity of collagen. The imino acid contents of ASC-S, ASC-D, and ASC-R were 174, 155, and 142 residues/1,000 residues, respectively, which was similar to that of ASC from the bone of bigeye snapper (142 residues/1,000 residues) (7), but lower than those of ASC from the cartilages of brownbanded bamboo shark (203 residues/1,000 residues), blacktip shark (196 residues/1,000 residues) (17), and the bone of carp (192 residues/1,000 residues) (18). It was reported that pyrrolidine rings of Pro and Hyp imposed restrictions on the conformation of the polypeptide chain and helped to strengthen the triple helix (15,20). In particular, Hyp is believed to play a key role in the

912 Chi et al. Fig. 1. SDS-PAGE patterns of acid-soluble collagens (ASCs) from the cartilages of scalloped hammerhead (ASC-S), red stingray (ASC-D), skate (ASC-R), and type I collagen from calf skin (SSC). Lane 1, SSC; lane 2, high-molecular weight marker; lane 3, ASC-S; lane 4, ASC-D; lane 5, ASC-R stabilization of the triple-stranded collagen helix due to its hydrogen bonding ability through its hydroxyl group. Furthermore, the imino acid content contributes to the thermal stability of the helix structure of collagen, due to the fact that the Pro and Hyp rich zones of the molecules are most likely to be involved in the formation of junction zones stabilized by hydrogen binding (20). Therefore, collagen helices of the 3 cartilages, especially ASC-D and ASC-R, might be less stable than those of some fishes cartilage, bone, and skin, and mammalian skin (SSC, 215 residues/1,000 residues), due to the lower imino acid content. Electrophoretic pattern Apart from amino acid composition, the properties of collagen are also influenced by the distribution of the molecular weights, structures, and composition of its subunits. As shown in Fig. 1, ASC-S, ASC-D, and ASC-R were consisted of 2 α-chains (α1 and α2), and high molecular weight components, particularly β (dimmers) and γ (trimers) components, as well as other cross-linked molecules with higher molecular weight were also observed. According to previous reports, α, β, and γ components were also found in standard collagen type I from calf skin (SSC, lane 1) and ASC from the cartilages of brownbanded bamboo shark and blacktip shark (17). However, the band intensity of α1- and α2-chain of ASC- S, ASC-D, and ASC-R was significantly different from that of SSC (lane 1), which revealed that these collagens of cartilages might contain another type of collagen except type I collagen. The result was in agreement with the previous reports (12,17), which indicated that collagens from shark cartilage were consisted of type II and type I collagen with the ratio of 2:1. Type I collagen ([α1(i)] 2 α2(i)) contained 2 of α1-chains and 1 of α2-chain, while type II collagen ([α1(ii)] 3 ) comprised 3 identical α1-chains (17). Therefore, we assumed that ASC-S, ASC-D, and ASC-R more likely comprised 2 types of collagens (type I and II) on the basis of SDS-PAGE patterns and previous reports (12,17). Peptide mapping As shown in Fig. 2, the bands of β- chains of ASC-S, ASC-D, and ASC-R as well as other high Mw components almost entirely disappeared after the digestion by the trypsin at ph of 2.5, 37 o C for 3 h. Moreover, the decreases in band intensities of the α-chains (α1 and α2) were observed with concomitant generations of lower Mw peptide fragments which were less than 130.0 kda. Fig. 2. Peptide mapping of acid-soluble collagens (ASCs) from the cartilages of scalloped hammerhead (ASC-S), red stingray (ASC-D), and skate (ASC-R) digested by trypsin using 7.5 (A) and 12% (B) gels. Lane 1 and 7, ASC-R; lane 2 and 6, ASC-D; lane 3 and 5, ASC-S; lane 4, protein marker. Lane 1-3, peptide fragments of collagens with trypsin digestion at ph 2.5; lane 5-7, peptide fragments of collagens with trypsin digestion at ph 7.8

Collagens from Cartilages of Cartilaginous Fishes 913 Based on the peptide mapping of ASC-S, ASC-D, and ASC-R digested by trypsin at ph 2.5 (lane 1-3), it could be found that ASC-D was more susceptible to hydrolysis than ASC-S and ASC-R as evidenced by the lower band intensity of each component retained and new peptide fragments ranged from 43 to 130 kda. However, ASC-R was more tolerant to digestion by trypsin than ASC-S and ASC-D as indicated by a greater band intensity of higher Mw peptide fragments ranging from 43 to 130 kda. When comparing with peptide hydrolysis patterns of ASC-D and ASC-R, peptide mapping of ASC-S showed the maximum band number of peptide fragments, especially the peptide fragments ranging from 14.4 to 43 kda. According to the peptide mapping results, ASC-S, ASC-D, and ASC-R might be different in primary structure, in terms of composition of amino acids. FTIR spectroscopy As shown in Fig. 3, FTIR spectra for ASC-S, ASC-D, and ASC-R were similar to those of collagens from channel catfish (21), Nile perch (1), and swim bladders (4). The amide A band is associated with the N-H stretching frequency. It was reported that a free N-H stretching vibration occurs in the range 3,400-3,440 1/cm, and when the NH group of a peptide is involved in a hydrogen bond, the position is shifted to lower frequency (22). The amide A bands of ASC-S, ASC-D, and ASC-R were at 3,430, 3,430, and 3,434 1/cm, respectively, which indicated that less NH groups of ASC-S, ASC-D, and ASC-R were involved in hydrogen bonding. The result was in accordance with the low amount of imino acids, especially Hyp (Table 2), which was believed to play a key role in the formation of hydrogen bond through its hydroxyl group. The amide B band positions of ASC-S, ASC-D, and ASC-R were found at wave number of 2,925 1/cm, representing the asymmetrical stretch of CH 2. The wavenumbers of the amide I, amide II, and amide III bands are directly associated with the configuration of collagen (23,24). The amide I band with the characteristic strong absorbance in the range of 1,600-1,700 1/cm was mainly related to the C=O stretching vibrations coupled to N-H bending vibrations, CN stretch and CCN deformation along the polypeptide backbone, and it could be a sensitive marker of the peptide s secondary structure (1,24). The amide I band of ASC-S, ASC-D, and ASC-R were found at the wavenumber of 1,641, 1,642, and 1,639 1/cm, respectively. Furthermore, the amide II band, which is caused by the N-H bending vibration coupled with a C-N stretching vibration, normally occurs at 1,550-1,600 1/cm, and the shift to lower wavenumbers suggests the existence of hydrogen bonds (8,18). The amide II band of ASC-S, ASC-D, and ASC-R were found at the wavenumber of 1,548, 1,549, and 1,548 1/cm, respectively, which further Fig. 3. FTIR spectra of acid-soluble collagens (ASCs) from the cartilages of scalloped hammerhead (ASC-S), red stingray (ASC-D), skate (ASC-R). confirmed that less hydrogen bonding existed in ASC-S, ASC-D, and ASC-R. In addition, the amide III band (1,220-1,320 1/cm) represented the combination peaks between C-N stretching vibrations and N-H deformation from amide linkages as well as absorptions arising from wagging vibrations from CH 2 groups from the glycine backbone and proline side-chains (24). The amide III bands of ASC-S, ASC-D, and ASC-R were found at the wavenumber of 1,242, 1,243, and 1,240 1/cm, respectively. The triple helical structures of ASC-S, ASC-D, and ASC- R were confirmed from the absorption ratios between amide III and 1,452 1/cm bands, which were approximately equal to 1.0 (23). Viscosity of ASC-S, ASC-D, and ASC-R solution It is known that the hydrogen bonds of collagen are gradually broken with increasing temperature. Consequently, the triple helix structure of collagen organized by hydrogen bonds is converted into the random coil configuration of collagen by the process of thermal depolymerisation, which is accompanied by changes in physical properties, such as viscosity, sedimentation, diffusion, light scattering, and optical activity (25). Hence, viscosity measurement is usually used to investigate the thermal stability of collagen and learn about the loss of viscosity with heating, attributed to denaturation of collagen. The changes in viscosity and T d s of ASC-S, ASC-D, and ASC-R solution at the concentration of 0.6%(w/v) were shown in Fig. 4. ASC-S (Fig. 4A) exhibited a slow loss of

914 Chi et al. Fig. 4. Thermal behaviors of acid-soluble collagens (ASCs) from the cartilages of scalloped hammerhead (ASC-S) (A), red stingray (ASC-D) (B), and skate (ASC-R) (C) measured by viscosity change in deionized water. All values are mean±sd (n=3); Values with differrent letters indicate significant difference (p<0.05). viscosity with heating from 4 (7,649.87±90.69 mpa s) to 16 o C (6,673.83±133.54 mpa s) and a rapid loss of viscosity with heating from 16 (6,673.83±133.54 mpa s) to 36 o C (300.17±54.95 mpa s), and remained low viscosity above 36 o C; ASC-D (Fig. 4B) showed a rapid loss of viscosity with heating from 4 (3,733.33±89.76 mpa s) to 20 o C (883.40±76.15 mpa s) and a slow loss of viscosity with heating from 20 (883.40±76.15 mpa s) to 40 o C (163.53±3.05 mpa s); ASC-R (Fig. 4C) exhibited a rapid loss of viscosity with heating from 4 (19,954.33±489.81 mpa s) to 36 o C (429.27±41.31 mpa s) and a slow loss of viscosity with heating from 36 (429.27±41.31 mpa s) to 40 o C (123.00±9.29 mpa s). The T d s of ASC-S, ASC-D, and ASC-R were 23.8, 15.1, and 12.1 o C, respectively, which were lower than the T d s of calf and pig skin collagen (37 o C) (20,26). These results indicate that the helices of ASC-S, ASC-D, and ASC-R are less stable than those of mammalian collagens. T d s of ASC-S, ASC-D, and ASC-R were similar to those of coldwater fish, such as Argentine hake (10.0 o C), Baltic cod (15.0 o C), and Alaska pollack (16.8 o C) (27), and were much Fig. 5. Solubility of acid-soluble collagens from the cartilages (ASCs) of scalloped hammerhead (ASC-S), red stingray (ASC- D), and skate (ASC-R) in 0.5 M acetic acid at different ph (A) and NaCl concentrations (B). All values are mean±sd (n=3); Values with different letters indicate significant difference (p<0.05). lower than those of tropical fish species, such as common mackerel (26.1 o C), Japanese seabass (26.5 o C), paper nautilus (27 o C), ocellate puffer (28 o C), eel (29.3 o C), skipjack tuna (29.7 o C), ayu (29.7 o C), and Nile perch (36.5 o C) (1,26,28). This finding was in agreement with Rigby s report that the thermal stability of collagen was correlated with environmental and body temperatures (29). T d may be affected by the content of imino acid in fish collagens, and the increasing imino acid content resulted in increased denaturation temperature of the collagen. Based on the analysis of amino acid composition, the contents of imino acid were listed in a decreasing order as followed: ASC-S>ASC-D> ASC-R (Table 2). The T d results of the 3 cartilages collagens also brought into correspondence with their imino acid content order. Solubility Effect of ph on ASCs solubility: The effect of ph on the solubilities of ASC-S, ASC-D, and ASC-R in 0.5 M acetic acid was depicted in Fig. 5A. The solubility of ASC-S, ASC-D, and ASC-R reached maxima at ph 2. In general, the 3 cartilage collagens were solubilised to a greater extent in acidic ph ranging from 1 to 4 and a significant decrease

Collagens from Cartilages of Cartilaginous Fishes 915 Fig. 6. SEM images of acid-soluble collagens (ASCs) from the cartilages of scalloped hammerhead (ASC-S) (A), red stingray (ASC-D) (B), and skate (ASC-R) (C) (A1-C1, 100 ; A2-C2, 300 ; A3-C3, 800, magnification). in solubility was observed when ph ranged from 4 to 7 and the solubility reached the minimums (ASC-S, 24.5±0.7%; ASC-D 17.8±0.7%; ASC-R 23.2±1.0%) at ph 7.0. However, the solubility of ASC-S, ASC-D, and ASC-R appeared to increase slightly when ph increased from 7 to 11. It is known that the net charge residues of protein molecules are greater and the solubility is increased by the repulsion forces between chains as the ph is lower or higher than pi. In contrast, total net charges of protein molecules are zero and hydrophobic-hydrophobic interaction increases, thereby leading to the precipitation and aggregation at pi. The pi of ASC-S, ASC-D, and ASC-R was obtained at ph around 7 which was consistent with the report that collagen has isoelectric points at ph 6-9 (5). In addition, the result that ASC-S, ASC-D, and ASC-R had lowest solubility in the neutral ph range was in accordance with the solubility of collagens from the skin of the balloon fish (20), Nile tilapia (6), and bigeye snapper (7). Effect of NaCl concentration on ASCs solubility: As shown in Fig. 5B, the solubility of ASC-S, ASC-D, and ASC-R showed similar pattern with slight differences when NaCl concentrations ranged from 0 to 6%(w/v). The solubility of ASC-S, ASC-D, and ASC-R remained high (more than 90%) in the presence of NaCl up to 2%(w/v), and sharply decreased when the NaCl concentration was between 2 and 4%(w/v), after which solubility of ASC-S, ASC-D, and ASC-R slowly reduced when the NaCl concentration was from 4 and 6%(w/v). At the NaCl concentration of 6%(w/v), the solubilities of ASC-S, ASC- D, and ASC-R were 25.5±1.7, 18.8±0.7, and 23.7±1.2%, respectively. The result was in accordance with the reports that the solubility of collagens from the skin of yellowfin tuna, dusky spinefoot, eagle ray, red stingray, yantai stingray, brownstripe red snapper, bigeye snapper, and striped catfish in acetic acid solution generally decreased with an increase in NaCl concentration (5,7,15). The decrease in solubility of ASC-S, ASC-D, and ASC-R could be described as being due to a salting out effect, which occurred at relatively high NaCl concentrations. An increase in ionic strength causes a reduction in protein solubility by enhancing hydrophobic-hydrophobic interactions between protein chains, and increasing the competition for water with the ionic salts, thereby leading to the induced protein precipitation (15). Ultrastructures As shown in Fig. 6, the lyophilized ASC-S, ASC-D, and ASC-R were loose, fibrous, and porous structures because of the evaporation of fluid, just like collagens from the skin of grass carp (27), coelomic wall of Sipunculida (10), and scales of barramundi (Lates calcarifer) (30). However, the structures of ASC-S, ASC- D, and ASC-R were not endowed with uniform and regular alveolate pores, which might be attributed to the different collagen concentrations and quantities before lyophilizing (27). Taking the structure into account, ASC-S, ASC-D, and ASC-R might also have good properties for distribution of the drugs as carriers.

916 Chi et al. Acknowledgments This work was financed by National Natural Science Foundation of China (No. 31001109) and the Special Program for the Science and Technology Plan of Zhejiang Province, China (2011C02003). References 1. Muyonga JH, Cole CGB, Duodu KG. Characterisation of acid soluble collagen from skins of young and adult Nile perch (Lates niloticus). Food Chem. 85: 81-89 (2004) 2. Liu Z, Oliveira ACM, Su YC. Purification and characterization of pepsin-solubilised collagen from skin and connective tissue of giant red sea cucumber (Parastichopus californicus). J. Agr. Food Chem. 58: 1270-1274 (2010) 3. Matmaroh K, Benjakul S, Prodpran T, Encarnacion AB, Kishimura H. Characteristics of acid soluble collagen and pepsin soluble collagen from scale of spotted golden goatfish (Parupeneus heptacanthus). Food Chem. 129: 1179-1186 (2011) 4. Liu D, Liang L, Regenstein JM, Zhou P. Extraction and characterisation of pepsin-solubilised collagen from fins, scales, skins, bones, and swim bladders of bighead carp (Hypophthalmichthys nobilis). Food Chem. 133: 1441-1448 (2012) 5. Li Z, Wang B, Chi C, Zhang Q, Gong Y, Tang J, Luo H, Ding G. Isolation and characterization of acid soluble collagens and pepsin soluble collagens from the skin and bone of Spanish mackerel (Scomberomorous niphonius). Food Hydrocolloid. 31: 103-113 (2013) 6. Zeng SK, Zhang CH, Lin H, Yang P, Hong PZ, Jiang ZH. Isolation and characterisation of acid-solubilised collagen from the skin of Nile tilapia (Oreochromis niloticus). Food Chem. 116: 879-883 (2009) 7. Kittiphattanabawon P, Benjakul S, Visessanguan W, Nagai T, Tanaka M. Characterisation of acid-soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus). Food Chem. 89: 363-372 (2005) 8. Ahmad M, Benjakul S. Extraction and characterisation of pepsinsolubilised collagen from the skin of unicorn leatherjacket (Aluterus monocerous). Food Chem. 120: 817-824 (2010) 9. Cui FX, Xue CH, Li ZJ, Zhang YQ, Dong P, Fu XY, Gao X. Characterization and subunit composition of collagen from the body wall of sea cucumber Stichopus japonicus. Food Chem. 100: 1120-1125 (2007) 10. Su XR, Sun B, Li YY, Hu QH. Characterization of acid-soluble collagen from the coelomic wall of Sipunculida. Food Hydrocolloid. 23: 2190-2194 (2009) 11. Z elechowska E, Sadowska M, Turk M. Isolation and some properties of collagen from the backbone of Baltic cod (Gadus morhua). Food Hydrocolloid. 24: 325-329 (2010) 12. Rama S, Chandrakasan G. Distribution of different molecular species of collagen in the vertebral cartilage of shark (Carcharius acutus). Connect. Tissue Res. 12: 111-118 (1984) 13. Mizuta S, Hwang JH, Yoshinaka R. Molecular species of collagen in pectoral fin cartilage of skate (Raja kenojei). Food Chem. 80: 1-7 (2003) 14. Chen S, Cai Y, Zhou Q, Zhang Q, Cao M. Study on collagen from shark skin and bone. J. Chin. Inst. Food Sci. Technol. 6: 173-178 (2006) 15. Bae I, Osatomi K, Yoshida A, Osako K, Yamaguchi A, Hara K. Biochemical properties of acid-solubilized collagens extracted from the skins of under-utilised fishes. Food Chem. 108: 49-54 (2008) 16. Kittiphattanabawon P, Benjakul S, Visessanguan W, Kishimura H, Shahidi F. Isolation and characterisation of collagen from the skin of brownbanded bamboo shark (Chiloscyllium punctatum). Food Chem. 119: 1519-1526 (2010) 17. Kittiphattanabawon P, Benjakul S, Visessanguan W, Shahidi F. Isolation and characterization of collagen from the cartilages of brownbanded bamboo shark (Chiloscyllium punctatum) and blacktip shark (Carcharhinus limbatus). LWT-Food Sci. Technol. 43: 792-800 (2010b) 18. Duan R, Zhang J, Du X, Yao X, Konno K. Properties of collagen from skin, scale, and bone of carp (Cyprinus carpio). Food Chem. 112: 702-706 (2009) 19. Nagai T, Suzuki N. Preparation and characterization of several fish bone collagens. J. Food Biochem. 24: 427-436 (2000) 20. Huang YR, Shiau CY, Chen HH, Huang BC. Isolation and characterization of acid and pepsin-solubilized collagens from the skin of balloon fish (Diodon holocanthus). Food Hydrocolloid. 25: 1507-1513 (2011) 21. Liu HY, Li D, Guo SD. Studies on collagen from the skin of channel catfish (Ictalurus punctaus). Food Chem. 101: 621-625 (2007) 22. Doyle BB, Bendit EG, Blout ER. Infrared spectroscopy of collagen and collagen-like polypeptides. Biopolymers 14: 937-957 (1975) 23. Heu MS, Lee JH, Kim HJ, Jee SJ, Lee JS, Jeon YJ, Shahidi F, Kim J-S. Characterization of acid- and pepsin-soluble collagens from flatfish skin. Food Sci. Biotechnol. 10: 27-33 (2010) 24. Ahmad M, Benjakul S, Nalinanon S. Compositional and physicochemical characteristics of acid solubilize d collagen extracted from the skin of unicorn leatherjacket (Aluterus monoceros). Food Hydrocolloid. 24: 588-594 (2010) 25. Engel J, Bächinger HP. Structure, stability, and folding of the collagen triple helix. Top. Curr. Chem. 247: 7-33 (2005) 26. Nagai T, Araki Y, Suzuki N. Collagen of the skin of ocellate puffer fish (Takifugu rubripes). Food Chem. 78: 173-177 (2002) 27. Zhang Y, Liu WT, Li GY, Shi B, Miao YQ, Wu XH. Isolation and partial characterization of pepsin-soluble collagen from the skin of grass carp (Ctenopharyngodon idella). Food Chem. 103: 906-912 (2007) 28. Nagai T, Yamashita E, Taniguchi K, Kanamori N, Suzuki N. Isolation and characterisation of collagen from the outer skin waste material of cuttlefish (Sepia lycidas). Food Chem. 72: 425-429 (2001) 29. Rigby BJ. Amino-acid composition and thermal stability of the skin collagen of the Antarctic ice-fish. Nature 219: 166-167 (1968) 30. Sankar S, Sekar S, Mohan R, Rani S, Sundaraseelan J, Sastry TP. Preparation and partial characterization of collagen sheet from fish (Lates calcarifer) scales. Int. J. Biol. Macromol. 42: 6-9 (2008)