Accepted Manuscript

Isolation and characterization of collagen from the outer skin of squid (Doryteuthissinghalensis)

Anguchamy Veeruraj, Muthuvel Arumugam, Thangappan Ajithkumar, ThangavelBalasubramanian

PII: S0268-005X(14)00268-9

DOI: 10.1016/j.foodhyd.2014.07.025

Reference: FOOHYD 2676

To appear in: Food Hydrocolloids

Received Date: 29 December 2013

Revised Date: 20 July 2014

Accepted Date: 29 July 2014

Please cite this article as: Veeruraj, A., Arumugam, M., Ajithkumar, T., Balasubramanian, T., Isolationand characterization of collagen from the outer skin of squid (Doryteuthis singhalensis), FoodHydrocolloids (2014), doi: 10.1016/j.foodhyd.2014.07.025.

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Graphical Abstract

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Isolation and characterization of collagen from the outer skin of squid 1

(Doryteuthis singhalensis) 2

Anguchamy Veeruraj1*., Muthuvel Arumugam1., Thangappan Ajithkumar2 3 Thangavel Balasubramanian1 4

1Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, 5

Annamalai University, Parangipettai – 608 502, India. 6

2National Bureau of Fish Genetic resources, Indian Council of Agricultural research, Canal Ring 7 road, Telibagh, P.O.Dilkusha, Lucknow, 226002, U.P., India 8

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*Corresponding Author at: Centre of Advanced Study in Marine Biology, 21

Faculty of Marine Science, Annamalai University, Parangipettai-608502, Tamilnadu, India. 22

Tel: +91 -04144-243223, +91 8015056685 (Mobile); Fax: +91 -04144-243641 23

E-mail address: anguveeruraj@gmail.com 24

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Abstract 26

Acid and Pepsin soluble collagens (ASC & PSC) were isolated from the outer skin of 27

squid (D. singhalensis) caught in the Indian waters with the yields of 56.80% for ASC and 28

24.60% for PSC, respectively. The total yield of ASC and PSC was 81.40% on the basis of 29

lyophilized dry weight, which is higher compared to other sources. ASC and PSC were 30

characterized as type I collagen, containing as α1 and α2 chains. The amino acids analysis of the 31

ASC and PSC contained glycine (332 and 328 residues/1000 residues) as the major amino acid 32

and had imino acids of 223 and 225 residues/1000 residues and the FTIR spectra confirmed that 33

limited digestion by pepsin did not disrupt the triple helical structure of collagen. Thermal 34

denaturation temperatures (Td) of the ASC and PSC measured by viscometry were 35.70 and 35

34.80ºC, respectively. The higher thermostable of squid skin collagen suggested that the 36

possibility of its utilization as a substitute for commercial collagen. Squid skin collagen has 37

potential for use as a supplementary source of collagen. Thus, collagen from squid skin could 38

serve as an alternative source of collagen for further application in food, nutraceutical and 39

pharmaceutical industries. 40

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Key words: Doryteuthis singhalensis; skin waste; Collagen; Peptides pattern; Denaturation 45

temperatures; FTIR. 46

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1. Introduction 49

Collagen is the most abundant protein in the all living animal body, comprising 50

approximately 30 % of the total protein content. Collagen is the fibrous protein, contributing to 51

unique physiological functions of tissues in skins, tendons, bones and cartilages (Jongjareonrak, 52

Benjakul, Visessanguan, & Tanaka, 2005b). Collagen has been widely used in food, cosmetic, 53

biomedical and pharmaceutical industries (Ogawa, Portier, Moody, Bell, Schexnayder, & Losso, 54

2004). Commonly isolated from by-products of land-based animals, such as cows, pigs and 55

poultry, collagen has been widely used in the food, pharmaceutical, and cosmetic industries 56

because of its excellent biocompatibility and biodegradability, and weak antigenicity (Liu, Li, 57

Miao, & Wu, 2009). 58

At present, at least 29 variants of collagen have been identified, and each differs 59

considerably in amino acid sequence, structure and function, more likely associated with specific 60

genetic variants (Liu, Liang, Regenstein, & Zhou, 2012). In addition, collagen has been utilized 61

to produce film forms, which are useful in pharmaceutical applications including wound 62

dressings and as carriers for drug delivery system. Examples are production of wound dressings, 63

vitreous implants and carriers for drug delivery, edible casings (Senaratne, Park, & Kim, 2006) 64

and production of cosmetics with good moisturizing properties (Swatschek, Schatton, 65

Kellermann, Muller, & Kreuter, 2002). However, the outbreak of bovine sponge encephalopathy 66

(BSE), transmissible spongiform encephalopathy (TSE), foot-and-mouth disease (FMD) and 67

avian influenza have resulted in anxiety among users of collagen and collagen-derived products 68

from land-based animals in recent years (Jongjareonrak, Benjakul, Visessanguan, Nagai, & 69

Tanaka, 2005a). Additionally, collagen obtained from pig can not be used as a component of 70

some foods for religious reasons (Sadowska, Kolodziejska, & Niecikowska, 2003). Hence, there 71

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is a tough need to develop alternative collagen sources and distinguish with mammalian 72

collagens, fish collagens seem to be much safer. Therefore, the global demand for collagen from 73

alternative economical sources such as aquatic animals waste has been increasing over the years. 74

Recently, marine capture fisheries contribute over 50 % of total world fish production 75

and more than 70 % of this production has been utilized for processing. As a result, large 76

quantities of protein-rich fish processing by-products, accounting for as much as 50-80 % of the 77

total weight of catch, are discarded as waste (Shahidi, Han, & Synowiecki, 1995). Therefore, 78

there is a great potential in marine bioprocess industry to convert and utilize most of these by-79

products as valuable products. With the rapid development of seafood processing industries, 80

huge quantities of by-products have been discarded which may cause pollution and emit 81

offensive odors and optimal use of these by-products is a promising way to protect the 82

environment, to produce value-added products to increase the revenue to the fish processors, and 83

to create new job/business opportunities. Fish skin is an important source for collagen, which can 84

be used as a replacement for mammalian sources. 85

Some of the researchers have found that the skin, bone, scale, fin and cartilage of 86

freshwater and marine fish, the mantle of scallops (Shen, Kurihara, & Takahashi, 2007; Veeruraj, 87

Arumugam, Ajithkumar, & Balasubramanian, 2012), and the adductor of pearl oysters (Mizuta, 88

Miyagi, Nishimiya, & Yoshinaka, 2002) can be used as new sources of collagen. Indian 89

consumes a wide range of fish species, prawns, squids, tunas, crabs daily and in particular, a 90

sliced raw fresh, food processing. The squid (Doryteuthis singhalensis) is widely distributed in 91

tropical and subtropical waters. It is one of the commercially important cephalopods (cuttlefish 92

and squid) species in India. At present, approximately 40-43 thousand tons of D. singhalensis are 93

processed per year, mainly in Madras, Cochin and Vizhinjam. Approximately 10-15 % of the 94

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leftovers are D. singhalensis skin, being considered as wastes in the fish shops and refrigerated 95

fish processing factories. If the skin is to be dumped as wastes as is the case currently, this would 96

pose a potential threat to the environment (e.g. pollution and offensive odor). Therefore, the aim 97

of this investigation was to isolate and characterize the acid and pepsin-soluble collagen from 98

outer skin waste of squid (D. singhalensis). 99

2. Materials and Methods 100

2.1. Materials and reagents 101

Acetic acid, Sodium chloride (NaCl), trichloroacetic acid, pepsin, Sodium dodecyl 102

sulphate (SDS), ammonium persulphate and Coomassie Brilliant Blue R-250 were purchased 103

from Himedia Chemicals (Hi-Media Laboratories Pvt. Ltd., Mumbai, India). The standard type I 104

collagen from human placenta and Achromopeptidase from Achromobacter lyticus were 105

purchased from SIGMA-Aldrich (EC 3.4 21.50, Mumbai, India) and Molecular weight markers 106

colourless protein were purchased from GeNei (Bangalore, India). All other chemicals and 107

reagents used were of analytical grade. 108

2.2. Sample collection and skin preparation 109

The fresh cephalopod D. singhalensis specimens were collected from a fish landing 110

centre of Mudasal odai (Lat.11°29’N long.79°46’E) in Tamilnadu, South East Coast of India 111

were brought to the laboratory and stored at -20oC until use. The outer skin was removed, cut 112

into small pieces (0.5 cm - 0.5 cm2) and the residual meat was removed manually and prepared 113

skin was used for collagen extraction. 114

2.3. Proximate composition 115

The portions of the skin were removed from different parts of squid and after blending, 116

proximate composition was determined. The amount of moisture, crude fat, ash and protein 117

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contents of skin from squid were determined according to the Association of Official Analytical 118

Chemists (AOAC, 1995) methods. 119

2.4. Preparation of collagen from the skin 120

2.4.1. Removal of non-collagenous proteins and fat from the skin 121

Skin pieces were treated with 0.1 M NaOH to remove non-collagenous proteins at a 122

sample to solution ratio of 1:10 (w/v) for 3 days and washed with distilled water. The alkali 123

solution was changed every day. Then, the sample was defatted with 10% butyl alcohol at a 124

sample to solvent ratio of 1:10 (w/v) for 24 h, washed with ample amount of distilled water and 125

freeze-dried. 126

2.4.2. Isolation of acid soluble collagen 127

The lyophilized skin was soaked in 0.5 M acetic acid with a sample to solution ratio of 128

1:10 (w/v) for 3 days at 4ºC with a gentle stirring and the mixture was centrifuged at 20,000×g 129

for 60 min at 4oC. The supernatants were collected and kept at 4ºC and the residue was re-130

extracted in 0.5 M acetic acid with a sample to solution ratio of 1:10 (w/v) for 2 days with a 131

gentle stirring, followed by centrifugation at 20,000×g for 60 min at 4ºC. The supernatants of the 132

two extracts were combined and salted-out by adding NaCl to give a final concentration of 0.9 133

M, followed by precipitation of the collagen by the addition of NaCl to the final concentration of 134

2.3 M in 0.05 M Tris-HCl (pH 7.5). The resultant precipitate was collected by centrifugation at 135

20,000×g for 30 min at 4ºC and then dissolved in 10 volumes of 0.5 M acetic acid. The solution 136

obtained was dialysed against 10 volumes of 0.1 M acetic acid in a dialysis membrane with 137

molecular weight cut-off of 30 kDa for 24 h at 4ºC with a change of solution every 8 h. 138

Subsequently, the solution was dialysed against 10 volumes of distilled water with changes of 139

water until neutral pH was obtained. The dialyzed was freeze-dried and referred to as ASC. 140

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2.4.3. Isolation of pepsin soluble collagen 141

The undissolved residues, obtained after acid extraction, was thoroughly rinsed with 142

distilled water, suspended in 5 volumes of 0.5 M acetic acid and subjected to limit hydrolysis 143

with 10% (w/v) pepsin (HiMedia, Mumbai, India) for 48 h at 4ºC with gentle stirring. The pepsin 144

solubilized collagen (PSC) was centrifuged at 20,000×g for 1h and the supernatants were 145

dialyzed to inactivate the pepsin against 0.02 M sodium phosphate buffer (Na2HPO4) (pH 7.2) 146

for 3 days with a change of solution every 8 h. The dialysate obtained was centrifuged at 147

20,000×g for 1 h. The pellet obtained was dissolved in 10 volumes of 0.5 M acetic acid. The 148

solution was further precipitated by the addition of NaCl to a final concentration of 2.3 M in 0.05 149

M Tris-HCl (pH 7.5). The resultant precipitate was collected by centrifugation at 20,000×g for 1 150

h at 4ºC and re-dissolved in 10 volumes of 0.5 M acetic acid. The solution was dialysed with 151

distilled water and freeze-dried in the same manner as for ASC preparation. The dried matter was 152

referred to as PSC. 153

2.5. Protein determination of collagen 154

The amount of protein was determined by the method of Lowry, Rosebrough, Farr, & 155

Randall (1951) using bovine serum albumin as a standard. 156

2.6. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis (SDS-PAGE) 157

The Protein patterns of ASC and PSC were analysed using sodium dodecyl sulfate-158

polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (1970). 159

Collagen samples (1 mg/mL) (ASC and PSC) were dissolved in 0.02 M sodium phosphate buffer 160

(pH 7.2) containing 1% (w/v) SDS and 3.5 M urea. The sample mixtures were gently stirred at 161

4ºC for 12 h to dissolve total proteins. Supernatants were collected after centrifuging at 10,000×g 162

for 3 min at 4ºC. Solubilized ASC and PSC were mixed with the sample buffer (0.5 M Tris-HCl, 163

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pH 6.8 containing 4% (w/v) SDS, 20% (v/v) glycerol) with 10% (v/v) β-mercaptoethanol (β-164

ME), using the sample to sample buffer ratio of 1:1(30:30 µL) (v/v). Samples were loaded onto a 165

polyacrylamide gel made of 10% running gel and 4% stacking gel and subjected to 166

electrophoresis at a constant current of 20 mA per gel, using a Mini Protein II unit (Bio-Rad 167

Laboratories Inc., Richmond, CA, USA). After electrophoresis, the gel was stained with 0.05% 168

(w/v) Coomassie brilliant blue R-250 in 15% (v/v) methanol and 5% (v/v) acetic acid and 169

destained with 30% (v/v) methanol and 10% (v/v) acetic acid. High-molecular-weight markers 170

(29-205 kDa, GeNei, Bangalore) were used to estimate the molecular weights of proteins. Acid-171

soluble type I collagen from human placenta (Sigma Chemical Co., St. Louis, Mo, USA) were 172

used as standard collagens. 173

2.7. Determination of subunit composition 174

The denatured squid skin collagen subunits were fractionated by CM-Toyopearl 650 M 175

column chromatography, essentially according to the method of Piez, Eigner, & Lewis (1963) 176

with a slight modification. One hundred milligrams of collagen samples (ASC and PSC) were 177

dissolved in 50 mL of 0.02 M sodium acetate buffer, pH 4.8, containing 3 M urea, and denatured 178

by heating to 45 oC, respectively. The mixture was centrifuged at 12,000×g for room temperature 179

for 30 min. The supernatant was applied onto a CM-Toyopearl 650 M column (1.8×20 cm) 180

previously equilibrated with 10 volumes of the starting buffer at a flow rate of 60 mL per hr. 181

After loading, the unbound proteins were washed by the same buffer until the A230 was less than 182

0.05. Elution was achieved with a linear gradient of 0-0.15 M NaCl in the same buffer, at a flow 183

rate of 60 mL per hr, with a total volume of over 250 mL. The eluant was monitored at 230 nm 184

(spectrophotometer, UV-1800, Shimadzu, Kyoto, Japan) and fractions (3 mL) were collected. 185

The selected fractions were subject to SDS-PAGE as described previously. 186

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2.8. Peptide mapping 187

Peptide mapping of ASC and PSC were performed according to the method mentioned 188

by Saito, Kunisaki, Urano, & Kimura (2002) with some modifications. Collagen samples (ASC 189

and PSC) (0.5 mg/mL) were dissolved in 0.1 M sodium phosphate buffer (pH 7.2) containing 190

0.5% (w/v) SDS and heated at 100ºC for 5 min. After cooling in ice, the digestion was carried 191

out at 37 ºC for 30 min using 5 µL of achromopeptidase from Achromobacter lyticus (EC 192

3.421.50, SIGMA-Aldrich, Mumbai, India) and the proteolysis was stopped by boiling for 5 min 193

after adding SDS to a final concentration of 2%. Electrophoretic patterns of samples were 194

checked according to the SDS-PAGE of Laemmli (1970) using 12% gel. Molecular weight 195

markers (colourless protein molecular weight markers, High molecular weight range (29-205 196

kDa, GeNei, Bangalore, India), were used as marker proteins. 197

2.9. UV absorption spectrum 198

UV absorption spectrum of ASC and PSC was measured using a Shimadzu-UV-199

Spectrophotometer. The ASC and PSC (1 mg) was dissolved in 100 mL 0.02 M sodium acetate 200

buffer, pH 4.8 containing 2 M urea. The solution was placed into a quartz cell with a path length 201

of 1 mm. UV spectrum was measured at wavelength between 190-400 nm at a scan speed of 2 202

nm per second with an interval of 1 nm. 203

2.10. Determination of denaturation temperature (Td) 204

Determination of denaturation temperature was based on the method described by 205

Kimura, Ohno, Miyauchi, & Uchida (1987). Briefly, the Ostwald’s viscometer was filled with 206

0.1% (m/v) collagen solution in 0.1 M acetic acid. After immersing viscometer in the water bath 207

at 15°C, it was kept for 30 min to allow the collagen solution to equilibrate to the water bath 208

temperature. The temperature was increased stepwise up to 50°C and maintained at each 209

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temperature for 30 min. The viscosities of the collagen solution were measured at temperature 210

intervals of about 2°C from 15°C up to 50°C. Then, the fractional viscosities were calculated for 211

each temperature by the following equation: 212

Fractional viscosity 213

= (maximum viscosity - measured viscosity)/ (maximum viscosity - minimum viscosity). 214

2.11. Amino acid analysis 215

Twenty milligrams of lyophilized ASC and PSC were dissolved in 2 mL of 6 N HCl and 216

the mixture was evacuated, vacuum-sealed and hydrolysed at 110oC for 24 h. The hydrolysate 217

was analysed on amino acid auto-analyser (Hitachi 835-50, Shimadzu Seisakusho Co. Ltd., 218

Kyoto, Japan). Amino acids were determined by derivatization with ninhydrin and measurement 219

of absorbance at 570 nm except for proline and hydroxyproline, for which absorbance at 440 nm 220

was measured. The amino acid content was expressed by the number of residues per 1000 221

residues. 222

2.12. Fourier transform infrared spectroscopy 223

Fourier transform infrared spectroscopy (FTIR) spectra were obtained from discs 224

containing 2 mg collagen samples (ASC and PSC) in approximately 50 mg potassium bromide 225

(KBr). All spectra were obtained using an infrared spectrophotometer (Bruker Tensor 27 226

Instruments, Billerica, MA, Germany) from 4000 to 400 cm-1 at a data acquisition rate of 2 cm-1 227

per point. Background was subtracted using the Opus software (Bruker Instruments, Billerica, 228

MA). 229

2.13. Statistical analysis 230

All methods of extraction of collagen and analysis were replicated three times. The 231

results were presented with mean ± standard deviations. 232

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3. Results and Discussion 233

3.1. Proximate compositions of squid skin 234

The proximate compositions of raw materials were given in Table 1. According to the 235

proximate composition results, the squid skin was found to have a fairly high moisture content 236

(60.34 ± 0.09%), which was similar to the results obtained for the balloon fish skin (62.23%) 237

(Huang, Shiau, Chen, & Huang, 2011), and skin of brownbanded bamboo shark (Chiloscyllium 238

punctatum) (61.96%) (Kittiphattanabawon, Benjakul, Visessanguan, Kishimura, & Shahidi, 239

2010a), but was lower than moisture content obtained for skin of blacktip shark (Carcharhinus 240

limbatus) (67.62%) and brown backed toadfish (73.4%) (Kittiphattanabawon, Benjakul, 241

Visessanguan, & Shahidi, 2010b; Senaratne, Park, & Kim, 2006). The crude protein, lipid and 242

ash content of the squid skin on wet weight basis were obtained as 31.79 ± 0.50%, 1.10 ± 0.50% 243

and 07.26 ± 0.10%, respectively. These result were slightly different that the moisture, protein, 244

fat and ash contents of brownbanded bamboo shark (Chiloscyllium punctatum) skin (61.96%, 245

24.75%, 0.19% & 12.12%, respectively) and brown backed toadfish (Lagocephalus gloveri) skin 246

(73.4 %, 90.3 %, 1.3 % & 8.4 %, respectively) (Kittiphattanabawon, Benjakul, Visessanguan, 247

Kishimura, & Shahidi, 2010a; Senaratne, Park, & Kim, 2006). In addition, Nile perch skin 248

contained moisture (68.4%), protein (21.6%), fat (6.8%) and ash (6.0%), and bigeye snapper 249

(Priacanthus tayenus) skin contained moisture (64.08%), protein (31.99%), fat (0.98%), and ash 250

(3.23%) (Muyonga, Cole, & Duodu, 2004; Kittiphattanabawon, Benjakul, Visessanguan, Nagai, 251

& Tanaka, 2005). However, the crude fat content on wet weight basis of squid skin was little 252

lower than those contained in Nile perch skin (5-6 %), but higher than bigeye snapper 253

(Priacanthus tayenus) skin (0.98 %), balloon fish skin (0.73 %) and shark skin (0.19 %) 254

(Kittiphattanabawon, Benjakul, Visessanguan, Nagai, & Tanaka, 2005; Huang, Shiau, Chen, & 255

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Huang, 2011; Kittiphattanabawon, Benjakul, Visessanguan, Kishimura, & Shahidi, 2010a). The 256

ash content of squid skin was lower than those contained in balloon fish (15.87 %) and brown 257

backed toadfish (8.4 %) (Huang, Shiau, Chen, & Huang, 2011; Senaratne, Park, & Kim, 2006), 258

probably because the skin was covered without scals and small spine comparatively that both 259

fishes. After demineralization, ash content of 7.15 % was obtained, in which approximately 93 % 260

of inorganic matters were removed. Almost complete demineralization might cause the looser 261

matrix of skin, which could be easier for collagen extraction. 262

3.2. Yield of ASC and PSC from the skin of squid 263

The present study, the yield of ASC and PSC from the skin of squid was about 56.80 % 264

and 24.60 % (on a dry weight basis). Defatted squid skin was not soluble entirely in 0.5 M acetic 265

acid, but after adding 0.5 M acetic acid with 10% (w/v) pepsin, squid skin pieces were 266

completely solubilized by making a viscous solution. Cross-link mediated by covalent bonds 267

through the condensation of aldehyde groups at the telopeptide region as well as the 268

intermolecular cross links might lead to a decrease in the solubility of collagen in the acidic 269

solution used for extraction (Foegeding, Lanier, & Hultin, 1996; Zhang, Liu, Li, Shi, Miao, & 270

Wu, 2007; Jongjareonrak, Benjakul, Visessanguan, & Tanaka, 2005b). With further limited 271

pepsin digestion, the cross-links at the telopeptide region were cleaved without damaging the 272

integrity of the triple helix. Therefore, a high solubility of collagen in acid was obtained after 273

adding pepsin. Pepsin was able to cleave specifically at the telopeptide region of collagen from 274

snakehead fish scale (Liu, Li, Miao, & Wu, 2009). This result was in agreement with Zhang, 275

Duan, Ye, & Konno (2010) who reported that the limited amount of pepsin (0.1%) effectively 276

solubilized collagen from silver carp fish scale. The difference in efficacy of pepsin in extracting 277

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collagen might be governed by fish species, collagen composition and configuration, amount of 278

pepsin used, etc. 279

The extractable yield (sum of yield of ASC and PSC) of squid skin collagens (81.40% 280

(dry weight basis) was much higher than that of squid skin (52.6 %), brownstripe red snapper 281

(13.7%) and bigeye snapper (7.5%) (Mingyan, Bafang, & Xue, 2009; Jongjareonrak, Benjakul, 282

Visessanguan, Nagai, & Tanaka, 2005a; Jongjareonrak, Benjakul, Visessanguan, & Tanaka, 283

2005b). As a consequence, similar and higher yield of ASC and PSC based on dry weight from 284

other fish skin has been reported for marine eel fish (Evenchelys macrura) (80 % and 7.1 %, 285

respectively), black drum (2.3 % and 15.8 %, respectively), largefin longbarbel catfish (16.8 % 286

and 28.0 %, respectively), and brown backed toadfish (ASC: 54.3) (Veeruraj, Arumugam, 287

Ajithkumar, & Balasubramanian, 2012; Ogawa, Portier, Moody, Bell, Schexnayder, & Losso, 288

2004; Zhang, Liu, & Li, 2009; Senaratne, Park, & Kim, 2006). 289

The ASC was 2.3-fold higher than the PSC, and it could be invented that this was 290

because there were many inter-chain cross-links at the telopeptide region, leading to the partial 291

solubility of collagen in acid (Foegeding, Lanier, & Hultin, 1996; Zhang, Liu, Li, Shi, Miao, & 292

Wu, 2007; Zhang, Liu, & Li, 2009). This result supported that of Kittiphattanabawon, Benjakul, 293

Visessanguan, & Shahidi (2010b), who point out that squid skin, may have a loosened matrix, 294

via swelling mechanism in acidic solution, leading to the ease of pepsin to cleave the telopeptide 295

region. Thus, it could be supposed that the degree of cross-linking at the telopeptide region of 296

collagen from squid skin was in accordance with those of collagen from bigeye snapper 297

(Priacanthus marcracanthus) skin, brownstripe red snapper (Lutjanus vitta) skin was ASC 298

(Jongjareonrak, Benjakul, Visessanguan, & Tanaka, 2005b; Jongjareonrak, Benjakul, 299

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Visessanguan, Nagai, & Tanaka, 2005a), but was lower than those of collagen from many other 300

fish species. 301

3.3. Electrophoretic characterization of collagen from the skin of squid 302

The protein patterns of isolated ASC and PSC and type I collagen from human placenta 303

were revealed that both ASC and PSC consisted of two α-chains (α1 and α2) as the major 304

constituents. High molecular weight components, including γ-components, as well as their cross-305

linked molecules, were also observed in both products (Fig. 1.) and the α1 and α2 chains of ASC 306

with molecular weight of 107 and 91 kDa, respectively, were found at a ratio of approximately 307

2:1. The result suggested that the isolated collagen from squid skin was characterized by Type I 308

collagen. The electrophoretic patterns of isolated ASC and PSC were similar to that of the type I 309

collagen from human placenta (lane 2), and also in accordance with the collagens of marine eel 310

fish (Veeruraj, Arumugam, Ajithkumar, & Balasubramanian, 2012), sardine, red seabream, 311

Japanese seabass (Nagai, 2004), black drum, sheephead seabream (Ogawa, Portier, Moody, Bell, 312

Schexnayder, & Losso, 2004). 313

The α1 and α2 chains of PSC had slightly higher molecular weight (110 and 95 kDa, 314

respectively), compared with those of ASC. However, Nalinanon, Benjakul, & Kishimura (2010) 315

suggested that the α1 and α2 chains of PSC from the skin of arabesque greenling had slightly 316

lower molecular weight than that of ASC. Pepsin is able to cleave the peptides localised at the 317

telopeptide region. As a result, some part of peptide was removed. In addition, β-chain and γ-318

chain, representing dimer and trimer, respectively, were also observed in ASC and PSC. 319

Nevertheless, band intensity of β and γ -chains from ASC was higher than that of PSC. 320

Accordingly, it could be concluded that the intra and inter- molecular cross links of collagens 321

were richer in ASC than in PSC. This was explained by conversions of some β- and γ-322

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components in the PSC matrix to α-components by the treatment with pepsin. Pepsin cleaves the 323

crosslink containing teleopeptide, and the β-chain is converted to two α-chains (Sato, Ebihara, 324

Adachi, Kawashima, Hattori, & Irie, 2000; Kittiphattanabawon, Benjakul, Visessanguan, Nagai, 325

& Tanaka, 2005). However, it cannot be determined whether α3-chain exists in the collagens, 326

since α3-chain has a migration similar to that of α1-chain and it can not be separated from α1-327

chain under the electrophoretic conditions employed. It was reported that the heterotrimer (α1 α2 328

α3) was found as a major component in ASC from the scale of sheep head and black drum 329

(Ogawa, Portier, Moody, Bell, Schexnayder, & Losso, 2004). α3 chain, which was able to 330

migrate to the same mobility with α1 chain, might be present in ASC and PSC. In addition, no 331

differences in the electrophoretic patterns of ASC and PSC analysed in the presence and absence 332

of β-ME were observed. The result suggested that no disulphide bonds were present in both 333

collagens from skin of squid. 334

3.4. Subunit compositions of collagen from the skin of squid 335

The elution profiles of ASC and PSC on the CM-Toyopearl 650M column 336

chromatography after being dissociated with heat treatment are shown in Figure 2. The fractions 337

of ASC (Fig. 2A) and PSC (Fig. 2B) were eluted as 2 major peaks. The α1-chain was found in 338

the first peak (fraction Nos. 16-22 and 17-21 for ASC and PSC, respectively) whilst the α2- 339

chains was found in the second peak (fraction Nos. 16-22 and 17-21 for ASC and PSC, 340

respectively). Additionally, a small amount of this kind of β- chain (dimer) was observed in the 341

ASC (fraction Nos. 16-21). The present results revealed that the both collagens might be 342

classified type I collagen, although the band intensity of α1-chain was not 2-fold higher than that 343

of α 2-chain. As a result, much lower band intensity of α2-chain was detected on SDS-PAGE. 344

Similar results have been reported for collagen type I from other elasmobranches and other 345

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squids (Bae, Osatomi, Yoshida, Osako, Yamaguchi, & Hara, 2008; Mingyan, Bafang, & Xue, 346

2009; Veeruraj, Arumugam, Ajithkumar, & Balasubramanian, 2012). 347

3.5. Peptide mapping of collagen from the skin of squid 348

Peptide maps of isolated collagens (ASC and PSC) digested by Achromopeptidase with 349

type I collagen from human placenta, were analysed by SDS– PAGE (12 % gel) as shown in 350

Figure 3. Generally, band intensity of α1- and α2-chains, as well as high MW cross-link, β- and 351

γ-components of ASC, PSC and type I collagen, decreased after limited digestion by 352

Achromopeptidase. The standard type I collagen from human placenta was digested by 353

Achromopeptidase (Lane 1) underwent slightly decrease in band intensity of α, β-chain 354

components and high MW cross-linked molecules with molecular weight of 139, 82, 67, 52 and 355

43 kDa. 356

The present results suggested that the α-chain component and high MW cross-linked 357

molecules from human placenta collagen type I are less tolerant to hydrolysis by 358

Achromopeptidase than are ASC and PSC from squid skin. Moreover, these patterns was also 359

similar to that of type I collagen. Achromopeptidase shows a high specific preference for 360

glutamic acid and aspartic acid residues of proteins. Due to the lower contents of glutamic acid 361

and aspartic acid residues in ASC (56 and 44 residues per 1000 residues) and PSC (52 and 47 362

residues per 1000 residues), which had the greater glutamic acid and aspartic acid contents, 363

might be more susceptible to hydrolysis by Achromopeptidase. From this result, ASC was more 364

prone to hydrolysis than PSC. However, the present results of ASC and PSC was similar patterns 365

of peptide fragments were observed. After hydrolysis, the α- component and high MW cross-366

linked molecules of ASC and PSC from the skin of squid were degraded into small MW peptides 367

which ranged between 103 and 39 kDa and 108 and 35 kDa, respectively. 368

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The peptide fragments of high MW of ASC and PSC were ranging from 143 to 127 kDa 369

and 147 to 131 kDa respectively, were also observed. Compared to the skin collagens of bigeye 370

snapper and Brownstripe red snapper digested by V8 protease, the peptide maps were different 371

(Kittiphattanabawon, Benjakul, Visessanguan, Nagai, & Tanaka, 2005; Jongjareonrak, Benjakul, 372

Visessanguan, Nagai, & Tanaka, 2005a). The peptide maps of isolated ASC and PSC were 373

reported to differ among sources and species (Mizuta, Miyagi, Nishimiya, & Yoshinaka, 2002). 374

As a result, the pattern of the peptide fragment of squid skin collagen might be similar to that of 375

mammalian collagen. 376

3.6. UV absorption spectrum 377

The UV absorption spectrums of ASC and PSC at the wavelengths between 190–400 nm 378

were presented in Figure 4. Most proteins have a maximum ultraviolet absorption at 356 nm. The 379

numbers of tyrosine and tryptophane residues contribute to the ultraviolet absorption at 280 nm. 380

The present results of the amount of tyrosine in ASC and PSC from squid were 3 and 3 residues 381

per 1000 residues, respectively and the both extracted collagens showed as a maximum 382

absorption at 230 and 222 nm. Results of the present study revealed that intensity of UV 383

absorption spectrum of collagen with a maximum at 275 nm due to the aromatic residues 384

(tyrosine and phenylalanine) increases with the increasing dose of UV radiation (Metreveli, 385

Jariashvili, Namicheishvili, Syintradze, Chikvaidze, Sionkowska, & Skopinska, 2010). Edwards, 386

Farwell, Holder, & Lawson (1997) suggested that the groups of C=O, -COOH, CONH2 was 387

accessible in polypeptides chains of collagen. 388

3.7. Thermal denaturation temperature of collagen from the skin of squid 389

Thermal denaturation profiles of isolated collagen provided useful clues to the thermal 390

stability of collagen in relation to environment and amino acids content. To determine the Td of 391

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ASC and PSC from squid, the changes in viscosity and the Td were calculated from thermal 392

denaturation curve (Fig. 5). On the contrary, the Td of from squid (ASC and PSC) was found to 393

be at 35.80 ºC (ASC) and 34.80 ºC (PSC) which was comparatively higher than the control 394

(standard type I collagen) (33.70 ºC). This is quite similar to that of collagen from other 395

elasmobranches such as eel fish (39 and 35 ºC), eagle ray (34.1ºC), red stingray (33.2 ºC) and 396

yantai stingray (32.2ºC) (Veeruraj, Arumugam, & Balasubramanian, 2013; Bae, Osatomi, 397

Yoshida, Osako, Yamaguchi, & Hara, 2008). Since, Td of both ASC and PSC was not different; 398

pepsin digestion might not affect collagen structure, especially triple helical structure. 399

The thermal denaturation temperature of porcine skin collagen was 37ºC (Nagai, & 400

Suzuki, 2000). When comparing with Td of porcine skin collagen, Td of squid skin collagen was 401

about 1.5ºC lower than that of porcine skin collagen because Td of collagen is correlated with 402

their body temperature and the environmental temperature that they are living (Rigby, 1968). 403

Squid commonly lives in seawater and distributed in the tropics, but are relatively uncommon in 404

temperate regions and completely absent from cold water. Thermal denaturation temperature of 405

collagen from animal species is to be correlated with the imino acid content; proline and 406

hydroxyproline. Moreover, cold-water fish collagen has a low Td since their imino acid contents 407

are very low. ASC and PSC from the skin of squid contained a higher amount of imino acids, 408

compared with those from the skin of brownbanded bamboo shark (204 and 207 residues per 409

1000 residues, respectively), sailfish (213 and 221 per 1000 residues, respectively) 410

(Kittiphattanabawon, Benjakul, Visessanguan, Kishimura, & Shahidi, 2010a; Tamilmozhi, 411

Veeruraj, & Arumugam, 2013). The cleavage of telopeptide region by pepsin or removal of some 412

of those peptides might facilitate the denaturation of PSC induced by heat. Similar results have 413

been obtained for ASC and PSC from Brownstripe red snapper (36.5ºC) and brownbanded 414

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bamboo shark (34.5ºC) (Jongjareonrak, Benjakul, Visessanguan, Nagai, & Tanaka, 2005a; 415

Kittiphattanabawon, Benjakul, Visessanguan, Kishimura, & Shahidi, 2010a). In contrast, it was 416

much higher than that of cold-water fish skin, including cod skin (15ºC) (Sadowska, 417

Kolodziejska, & Niecikowska, 2003) and that of other tropical fish, such as brownstripe red 418

snapper (31.5ºC), and chub mackerel, 25.6ºC, bullhead shark, 25ºC (Jongjareonrak, Benjakul, 419

Visessanguan, Nagai, & Tanaka, 2005a; Nagai, & Suzuki, 2000). 420

3.8. Amino acid composition of collagen from skin of squid 421

The amino acid compositions of ASC and PSC from the skin of squid were presented in 422

Table 2. Both collagens had glycine (332–328 per 1000 residues) as their major amino acid, 423

followed by proline (122–126 per 1000 residues), alanine (119–112 per 1000 residues) and the 424

hydroxyproline (101–99 per 1000 residues) contents. About one-third of the total amino acid 425

residues of isolated collagen were glycine with about 12 % proline, 12 % alanine and 10 % 426

hydroxyproline. Additionally, the amounts of imino acid (proline and hydroxyproline) contents 427

are important for the structural integrity of collagen. 428

The imino acid contents of the ASC and PSC from the squid skin was found 223 and 225 429

per 1000 residues, respectively, which was slightly higher than that of type I collagen from calf 430

skin (215 per 1000 residues), young and adult Nile perch (193–200 per 1000 residues), bigeye 431

snapper (193 per 1000 residues), eel fish (190 and 200 per 1000 residues) and red stingray 432

collagen (216 per 1000 residues) (Ogawa, Portier, Moody, Bell, Schexnayder, & Losso, 2004; 433

Muyonga, Cole, & Duodu, 2004; Kittiphattanabawon, Benjakul, Visessanguan, Nagai, & 434

Tanaka, 2005; Veeruraj, Arumugam, Ajithkumar, & Balasubramanian, 2012; Bae, Osatomi, 435

Yoshida, Osako, Yamaguchi, & Hara, 2008), but lower than mammalian collagens (Foegeding, 436

Lanier, & Hultin, 1996). By the way, the imino acid content of the collagen from the squid skin 437

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was lower than that of edible jellyfish, dusky spine foot, cod, ocellate puffer and brown backed 438

toadfish, which contained imino acids ranging from 122 to 187 per 1000 residues (Bae, Osatomi, 439

Yoshida, Osako, Yamaguchi, & Hara, 2008; Senaratne, Park, & Kim, 2006). Supporting to the 440

above fact, difference in the imino acid content of animals was associated with the differences in 441

their habitats (Love, Yamaguchi, Creach, & Lavety, 1976) and the stability of collagen was 442

proportional to the total content of imino acids (Kittiphattanabawon, Benjakul, Visessanguan, & 443

Shahidi, 2010b). It is known that the pyrrolidine rings of proline and hydroxyproline impose 444

restrictions on the conformation of a polypeptide chain and help to strengthen the triple helix. 445

The hydroxyproline plays an important role in stabilization of the helix structure by preventing 446

rotation of the N-C bond (Foegeding, Lanier, & Hultin, 1996). The amino acid composition 447

indicated that ASC and PSC from squid skin might be classified by type І collagen. 448

3.9. Fourier transform infrared (FTIR) spectra of collagen from the skin of squid 449

FTIR spectra of ASC and PSC from the skin of squid are depicted in the Figure 6 and 450

Table 3. FTIR spectra of both ASC and PSC were similar to those of collagens from other fish 451

species (Muyonga, Cole, & Duodu, 2004; Veeruraj, Arumugam, Ajithkumar, & 452

Balasubramanian, 2012; Nagai, Suzuki, & Nagashima, 2008). Similar FTIR spectra were 453

observed between ASC and PSC and the amide A bands were found at wavenumber of 3307 and 454

3428 cm-1, respectively. According to Doyle, Bendit, & Blout (1975), a free N-H stretching 455

vibration occurs in the range of 3400–3440 cm-1 and when the NH group of a peptide is involved 456

in a hydrogen bond, the position is shifted to lower frequencies, usually around 3300 cm-1. The 457

result indicated that the NH groups of this collagen were involved in hydrogen bonding, 458

probably with a carbonyl group of the peptide chain. The amide B band positions of ASC and 459

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PSC were found at wavenumbers of 2928 and 2927 cm-1, respectively, representing the 460

asymmetrical stretching of CH2 (Muyonga, Cole, & Duodu, 2004). 461

The amide I band with characteristic frequencies in the ranges from 1600 to 1700 cm-1 462

was mainly associated with the stretching vibrations of the carbonyl group (C=O bond) along the 463

polypeptide backbone (Payne, & Veis, 1988), and was a sensitive marker of the peptide 464

secondary structure (Surewicz, & Mantsch, 1988). The amide I band of ASC and PSC were 465

found at wavenumbers of 1654 and 1647 cm-1, respectively. This observation confirmed that the 466

formation of hydrogen bond between N–H stretch (X position) and C=O (Gly) of the fourth 467

residue is responsible for introducing into triple helix (Zanaboni, Rossi, Onana, & Tenni, 2000). 468

Due to the greater non-helical portion of the telopeptides in ASC, intramolecular H-bond 469

between C=O of the peptide backbone and the adjacent hydrogen donor should be lower in ASC, 470

in comparison with PSC (Muyonga, Cole, & Duodu, 2004; Singh, Benjakul, Maqsood, & 471

Kishimura, 2011). The amide II band of ASC and PSC was situated at a wavenumber of 1541 472

and 1544 cm-1, respectively, whilst the amide III band of ASC and PSC was located at 473

wavenumbers of 1236 and 1239 cm-1, respectively. The amide II and amide III bands represent 474

N-H bending vibrations coupled with C-N stretching vibration and C-H stretching, respectively 475

(Barth, & Zscherp, 2002; Payne, & Veis, 1988). 476

The amide I peak underwent a decrease in absorbance, followed by a broadening 477

accompanied by the appearance of additional shoulders when collagen was heated at higher 478

temperature (Bryan, Brauner, Anderle, Flach, Brodsky, & Mendelsohn, 2007). Due to the 479

similarity in the amplitude, both collagens were most likely not denatured during the extraction. 480

The IR ratios between the amide III and 1454 cm-1 of ASC and PSC were 1.176 and 1.17, 481

respectively. The IR values were approximately 1.0, confirming that the triple helix of both ASC 482

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and PSC still existed and a high extent of intermolecular structure was still maintained (Plepis, 483

Goissis, & Das Gupta, 1996). However, a slight change in structure of PSC was noticed, more 484

likely due to the removal of telopeptide region by pepsin, but the triple-helical structure was still 485

predominant with the stronger bond. Nagai, Suzuki, & Nagashima (2008) suggested that the 486

some differences between the secondary structural components such as α-helix, β sheet, β-turn 487

and other random coils between ASC and PSC from the skin of the common mink whale 488

(Balaenoptera acutorostrata). From these result both ASC and PSC showed a similar secondary 489

structure of the protein. 490

4. Conclusion 491

The higher quantity of collagen (ASC and PSC) could be extracted from skin of squid 492

which consist of two α-chains (α1 and α2) were characterized as type I collagen. Further, acetic 493

acid digestion could increase the yield of collagen by 2.3-fold. The denaturation temperature and 494

the imino acid content of extracted collagen from the skin of squid were found to be higher than 495

those reported for most other fish species and closer to those of mammalian collagens. The FTIR 496

spectra of ASC and PSC were observed, there were still some differences in protein patterns. 497

Therefore, the collagen from squid skin could be a considerable potential as a substitute for 498

mammalian collagen. Thus, collagen from squid skin waste could serve as an alternative source 499

of cattle and porcine collagen for further application in food and nutraceutical industries 500

purposes. 501

Acknowledgement 502

This work was supported in parts by the grant from the Centre of Advanced Study in 503

Marine Biology, Faculty of marine sciences, Annamalai University, Parangipettai, Tamilnadu, 504

India. We would like to express our heartfelt gratitude to the donors. 505

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1343–1354. 634

635

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Figure caption

Fig. 1. SDS-polyacrylamide gel electrophoresis pattern of squid outer skin collagen. Lanes: (1)

Pepsin soluble collagen, (2) Acid soluble collagen, (3) Standard molecular weight markers, (4)

Type I collagen from human placenta.

Fig. 2(A). CM- TOYOPEARL® 650 M column chromatography of denatured acid soluble outer

skin collagen from squid. The CM- TOYOPEARL® 650 M column was equilibrated with 0.02 M

sodium acetate buffer (pH 4.8) containing 2 M urea. The soluble collagen (100 mg) was

dissolved in 50 mL of the same buffer, denatured at 45°C for 30 min, and then eluted from the

column with a linear gradient of 0-0.15 M NaCl at a flow rate of 1.0 mL per min. The fractions

indicated by the numbers were examined by SDS-PAGE.

Fig. 2(B). CM- TOYOPEARL® 650 M column chromatography of denatured pepsin soluble

outer skin collagen from squid. The CM- TOYOPEARL® 650 M column was equilibrated with

0.02 M sodium acetate buffer (pH 4.8) containing 2 M urea. The soluble collagen (100 mg) was

dissolved in 50 mL of the same buffer, denatured at 45°C for 30 min, and then eluted from the

column with a linear gradient of 0–0.15 M NaCl at a flow rate of 1.0 mL per min. The fractions

indicated by the numbers were examined by SDS-PAGE.

Fig. 3. Peptide mapping of Achromopeptidase digests from collagen samples at 12 % gel, Lanes:

(1) Type I collagen from human placenta, (2) ASC, (3) PSC, (4) Molecular weight marker.

Fig. 4. UV absorption spectrum of ASC and PSC from squid skin.

Fig. 5. Thermal denaturation curves of collagens (ASC, PSC) from squid skin and human

placenta type I collagen as a standard.

Fig. 6. Fourier transforms infrared spectra of standard type I collagen from human placenta, acid

soluble collagen (ASC) and pepsin soluble collagen (PSC) from the outer skin of squid.

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Tables

Table 1. Proximate analyses of skins of Doryteuthis singhalensis

Sample

Proximate compositions a (% of wet weight)

Moisture Protein Fat Ash

Squid skin 60.34±0.09 31.79±0.50 1.10±0.50 07.26±0.10

a Average ± SD from triplicate determinations.

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Table 2. Amino acid composition of Acid soluble collagen and Pepsin soluble collagen from the skin of D. singhalensis (expressed as

residues per 1000 total amino acid residues)

Amino Acids ASC PSC

Alanine 119 112

Arginine 52 53

Aspartic acid /Asparagine 44 47

Cysteine 6 5

Glutamic acid/glutamine 56 52

Glycine 332 328

Histidine 3 4

Isoleucine 11 11

Leucine 17 15

Lysine 32 35

Hydroxylysine 5 7

Methionine 13 12

Phenylalanine 8 8

Hydroxyproline 101 99

Proline 122 126

Serine 29 28

Threonine 21 29

Tryptophan 0 1

Tyrosine 12 10

Valine 17 18

Total 1000 1000

Imino acids 223 225

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Table 3. General peak assignment of the FTIR spectra consist of ASC and PSC from D. singhalensis skin

Peak Wavenumber (cm-1) Peak Assignments

Type I Collagen ASC PSC

3420 3307 3428 Amid A : mainly N-H stretching coupled with hydrogen bond

2928 2928 2927 Amid B : CH2-asymmetric stretching

2853 2853 2853 CH3-asymmetric stretching mainly protein

1646 1654 1647 Amide I : C=O stretching/ hydrogen bond coupled with COO-

1536 1541 1544 Amide II : N-H Bend coupled with C-N stretching

1436 1425 1436 CH2 bending vibration

1319 1318 1319 CH2 wagging of proline

1236 1236 1239 Amide III : N-H Bend coupled with C-N stretching

1160 1163 - COO-C asymmetric stretching

1079 1082 1080 PO2-symmetric stretching

1032 1037 1024 C-O stretching/C-O band

- 928 - C-O stretching

779,667 899,831,669 778,668 Skeletal stretching

473 467 465 Out of plane band

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List of Figures

Figure 1

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Figure 2 (A)

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Figure 2 (B)

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Figure 3

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Figure 4

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0 20 25 30 35 40 45 50 55 60

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ASC PSC Type I collagen

Figure 5

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Figure 6

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Highlights

• Isolated collagen was characterized by type I and also higher imino acid content.

• D. singhalensis skin waste as a potential and rich sources of type I collagen.

• Isolated collagen was higher thermal stability comparison to other fish skin collagens.

• D. singhalensis collagen could be used as alternative source of mammalian collagen.

• Isolated collagen could be used in foods, cosmetics and pharmaceutical industries.