Food Chemistry 124 (2011) 97105

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Isolation and characterisation of collagen extracted from the skin of striped catsh (Pangasianodon hypophthalmus)
Prabjeet Singh a, Soottawat Benjakul a,*, Sajid Maqsood a, Hideki Kishimura b
a b

Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Laboratory of Marine Products and Food Science, Research Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan

a r t i c l e

i n f o

a b s t r a c t
Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of striped catsh (Pangasianodon hypophthalmus) were isolated and characterised. The yields of ASC and PSC were 5.1% and 7.7%, based on the wet weight of skin, respectively, with the accumulated yield of 12.8%. Both ASC and PSC comprising two different a-chains (a1 and a2) were characterised as type I and contained imino acid of 206 and 211 imino acid residues/1000 residues, respectively. Peptide maps of ASC and PSC hydrolysed by either lysyl endopeptidase or V8 protease were slightly different and totally differed from those of type I calf skin collagen, suggesting some differences in amino acid sequences and collagen structure. Fourier transform infrared (FTIR) spectra of both ASC and PSC were almost similar and pepsin hydrolysis had no marked effect on the triple-helical structure of collagen. Both ASC and PSC had the highest solubility at acidic pH. A loss in solubility was observed at a pH greater than 4 or when NaCl concentration was higher than 2% (w/v). Tmax of ASC and PSC were 39.3 and 39.6 C, respectively, and shifted to a lower temperature when rehydrated with 0.05 M acetic acid. Zeta potential studies indicated that ASC and PSC exhibited a net zero charge at pH 4.72 and 5.43, respectively. Thus, ASC and PSC were slightly different in terms of composition and structure, leading to somewhat different properties. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 25 November 2009 Received in revised form 18 April 2010 Accepted 26 May 2010

Keywords: Acid soluble collagen (ASC) Pepsin soluble collagen (PSC) Zeta potential FTIR DSC Striped catsh Fish skin

1. Introduction Collagen is the most abundant protein in vertebrates and constitutes about 30% of the total proteins. Collagen, a right-handed triple superhelical rod, is unique in its ability to form insoluble bres that have high tensile strength (Gelse, Poschl, & Aigner, 2003). There are at least 27 different types of collagen, named type I XXVII (Birk & Bruckner, 2005). Type I collagen is commonly found in connective tissues, including tendons, bones and skins (Muyonga, Cole, & Duodu, 2004). All members of the collagen family are characterised by domains with repetitions of the proline-rich tripeptides, Gly-X-Y, involved in the formation of the triple helix (Muyonga et al., 2004). Collagen has a wide range of applications in the leather and lm industries, in cosmetic and biomedical materials, and as food (Kittiphattanabawon, Benjakul, Visessanguan, Nagai, & Tanaka, 2005). In pharmaceutical applications, collagen can be used for production of wound dressings, vitreous implants and as carriers for drug delivery. In addition, collagen has been used to produce edible casings for the meat processing industries (sausages/salami/snack sticks).

* Corresponding author. Tel.: +66 7428 6334; fax: +66 7421 2889. E-mail address: soottawat.b@psu.ac.th (S. Benjakul). 0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.05.111

Due to the outbreak of bovine spongiform encephalopathy (BSE), transmissible spongiform encephalopathy (TSE) and the foot-and-mouth disease (FMD) crisis, the uses of collagen and collagen-derived products of land animal origin have become of more concern (Jongjareonrak, Benjakul, Visessanguan, & Tanaka, 2005). In addition, the collagens extracted from bovine sources are prohibited for Sikhs and Hindus, whilst porcine collagen cannot be consumed by Muslims and Jews, both of whom require bovine to be religiously prepared. As a consequence, the alternative sources of collagen, especially from aquatic animals including freshwater and marine sh and mollusks have received increasing attention (Shen, Kurihara, & Takahashi, 2007). Pangasianodon hypophthalmus or Pla Sawai (in Thai), a large freshwater catsh, belongs to the order Siluriformes and is a member of Pangasidae family. It is one of the most important aquaculture species in Thailand (Froese & Pauly, 2007), especially in the northeast part of Thailand. This sh is also known as Siamese shark or sutchi catsh and is native to the Chao Phraya river in Thailand and the Mekong in Vietnam. It has become an important sh for many countries like Indonesia, Malaysia and China (Roberts & Vidthayanon, 1991). This freshwater sh normally lives in a tropical climate and prefers water with a pH of 6.57.5 and a temperature range of 2226 C. Adults reach up to 130 cm (4 ft) in length and can weigh up to a maximum of 44.0 kg (97 lb) (Roberts &

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Vidthayanon, 1991). Its meat has been popular among the consumers worldwide. During processing and lleting, a huge amount of skin from this sh is generated as a byproduct, which can be used as a potential source for collagen extraction. The skin from this sh is thick and tough, which may be associated with the collagen cross-links, especially cross-linking caused by hydroxylysine. However, no information on composition and molecular properties of collagen from the skin of this species has been reported. Thus, the objective of the present study was to extract and characterise the collagen, both acid soluble collagen (ASC) and pepsin soluble collagen (PSC), from the skin of striped catsh. 2. Materials and methods 2.1. Fish skin preparation Whole fresh farmed striped catsh (P. hypophthalmus) (approximately 2 years old) weighing 1 0.5 kg/sh stored on ice were procured from the sh market of Hat Yai, Songhkla, Thailand. Fish were stored in ice with a sh/ice ratio of 1:2 (w/w) and transported within 1 h to the Department of Food Technology, Prince of Songkla University, Hat Yai, Thailand. Upon arrival, sh were washed using tap water and deskinned. The skin was washed with cold water (58 C) and cut into small pieces (0.5 0.5 cm2). The prepared skin samples were packed in polyethylene bags and kept at 20 C until used. The storage time was not longer than 1 month. 2.2. Chemicals b-Mercaptoethanol (b-ME), V8 protease from Staphylococcus aureus (EC3.4.21.19, 800 U/mg powder); pepsin from porcine stomach mucosa (EC3.4.23.1; powderised; 750 U/mg dry matter) and type I collagen from calf skin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). High molecular weight markers were obtained from GE Healthcare UK (Buckinghamshire, UK). Sodium dodecyl sulphate (SDS), trichloroacetic acid, FolinCiocalteus phenol reagent, acetic acid and tris(hydroxylmethyl) aminomethane were obtained from Merck (Darmstadt, Germany). Lysyl endopeptidase from Achromobacter lyticus (EC3.4.21.50; 4.5 AU/mg protein) were procured from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). 2.3. Pretreatment of skin To remove non-collagenous proteins, the prepared sh skin was mixed with 0.1 M NaOH at a skin/alkali solution ratio of 1:10 (w/v). The mixture was continuously stirred for 6 h at 4 C and the alkali solution was changed every 2 h. The treated skin was then washed with cold water until a neutral or faintly basic pH of wash water was reached. The pH of wash water was determined using a digital pH meter (Sartorious North America, Edgewood, NY, USA). 2.4. Extraction of acid soluble collagen (ASC) ASC was extracted as per the method of Nagai and Suzuki (2000) with slight modication. All processes were carried out at 4 C with continuous stirring. The pretreated skins were defatted with 10% butyl alcohol with a solid/solvent ratio of 1:10 (w/v) for 48 h and the solvent was changed every 8 h. Defatted skin was washed with cold water (58 C), followed by soaking in 0.5 M acetic acid with a solid/solvent ratio of 1:15 (w/v) for 24 h. The mixture was ltered through two layers of cheese cloth and the residue was re-extracted under the same conditions. Both ltrates were combined. The collagen was precipitated by adding

NaCl (powder) to a nal concentration of 2.6 M in the presence of 0.05 M tris(hydroxymethyl) aminomethane, pH 7.0. The resultant precipitate was collected by centrifuging at 20,000g for 60 min, using a refrigerated centrifuge Avanti J-E (Beckman Coulter, Inc., Palo Alto, CA, USA). The pellet was dissolved in a minimum volume of 0.5 M acetic acid, and dialysed against 50 volumes of 0.1 M acetic acid for 24 h, followed by the dialysis in the same volume of distilled water for another 24 h. The dialysate was freeze dried and was referred to as acid soluble collagen; ASC. The yield of ASC was calculated from the percentage of dry weight of collagen extracted in comparison with the wet weight of the initial skin used. 2.5. Extraction of pepsin soluble collagen (PSC) The undissolved residue obtained after ASC extraction was used for PSC extraction. The residue was soaked in 0.5 M acetic acid with a solid/solvent ratio of 1:15 (w/v) and porcine pepsin (20 U/g residue) was added. The mixtures were continuously stirred at 4 C for 48 h, followed by ltration with two layers of cheesecloth. The ltrate was subjected to precipitation and the pellet was dialysed in the same manner as those for ASC previously described. The dialysate was freeze dried and was referred to as pepsin soluble collagen; PSC. The yield of PSC was also calculated in the same manner as for ASC. Additionally, the accumulated yield of collagen was calculated from the yields of both ASC and PSC. 2.6. Analyses Both ASC and PSC were subjected to the following analyses. 2.6.1. Amino acid analysis The samples were hydrolysed under reduced pressure in 4 M methane sulphonic acid containing 0.2% (v/v) 3-2(2-aminoethyl) indole at 115 C for 24 h. The hydrolysates were neutralised with 3.5 M NaOH and diluted with 0.2 M citrate buffer (pH 2.2) (Kittiphattanabawon, Benjakul, Visessanguan, Kishimura, & Shahidi, 2010). An aliquot of 0.4 ml was applied to an amino acid analyser (MLC-703; Atto Co., Tokyo, Japan). 2.6.2. SDSpolyacrylamide gel electrophoresis (SDSPAGE) SDSPAGE was performed following the method of Laemmli (1970). The samples were dissolved in 5% SDS and the mixtures were incubated at 85 C for 1 h in temperature controlled water bath (Memmert, Schwabach, Germany). The mixture was centrifuged at 4000g for 5 min using a microcentrifuge (MIKRO20, Hettich Zentrifugan, Germany) at room temperature to remove undissolved debris. Solubilised samples were mixed at a ratio of 1:1 (v/v) with the sample buffer (0.5 M Tris HCl, pH 6.8, containing 4% SDS and 20% glycerol) containing 10% b-ME. The mixtures were kept in boiling water for 2 min. Samples (15 lg protein) were loaded onto polyacrylamide gels comprising a 7.5% running gel and a 4% stacking gel and subjected to electrophoresis at a constant current of 15 mA/gel for 1 h and 30 min using a Mini Protein II unit (Bio-Rad Laboratories, Inc., Richmond, CA, USA). After electrophoresis, the gel was stained with 0.05% (w/v) Coomassie blue R-250 in 15% (v/v) methanol and 5% (v/v) acetic acid and destained with 30% (v/v) methanol and 10% (v/v) acetic acid. High molecular weight markers were used to estimate the molecular weight of proteins. The markers used included myosin (200 kDa), a2-macroglobulin (170 kDa), b-galactosidase (116 kDa), transferrin (76 kDa) and glutamate dehydrogenase (53 kDa). Type I calf skin collagen was used as a standard. Quantitative analysis of protein band intensity was performed using a Model GS-700 Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA, USA) with Molecular Analyst Software version 1.4 (image analysis system).

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2.6.3. Peptide mapping of collagen Peptide mapping of ASC and PSC was performed according to the method of Kittiphattanabawon et al. (2005) with a slight modication. The samples (6 mg) were dissolved in 1 ml of 0.1 M sodium phosphate, pH 7.2 containing 0.5% (w/v) SDS. Then, the mixtures were preheated at 45 C for 3 h and 300 ll of the prepared mixtures were transferred to test tubes for digestion. To initiate the digestion, 20 ll of each enzyme solution, either S. aureus V8 protease or lysyl endopeptidase from A. lyticus, with concentrations of 5 and 50 ll/ml, respectively, were added to the mixtures. The reaction mixtures were then incubated at 37 C for 1 h. The reactions were terminated by subjecting the reaction mixture to boiling water for 3 min. Peptides generated by the protease digestion were separated by SDSPAGE using a 7.5% running gel and a 4% stacking gel, followed by staining and destaining as previously described. Peptide mapping of calf skin collagen type I was also conducted in the same manner and the peptide patterns were compared. 2.6.4. ATR-FTIR analysis Both ASC and PSC were subjected to attenuated total reectance-Fourier transform infrared spectroscopy (ATR-FTIR). FTIR spectrometer (Model Equinox 55, Bruker, Ettlingen, Germany) equipped with a horizontal ATR trough plate crystal cell (45 ZnSe; 80 mm long, 10 mm wide and 4 mm thick) (PIKE Technology Inc., Madison, WI, USA) was used. For spectra analysis, the collagen samples were placed onto the crystal cell and the cell was clamped into the mount of the FTIR spectrometer. The spectra in the range of 4004000 cm1 with automatic signal gain were collected in 32 scans at a resolution of 4 cm1 and were ratioed against a background spectrum recorded from the clean empty cell at 25 C. 2.6.5. Differential scanning calorimetry Differential scanning calorimetry (DSC) of ASC and PSC was run following the method of Rochdi, Foucat, and Renou (2000) with a slight modication. The samples were rehydrated by adding deionised water or 0.05 M acetic acid to dried samples at a solid/solution ratio of 1:40 (w/v). The mixtures were allowed to stand for 2 days at 4 C. DSC was performed using a differential scanning calorimeter (Perkin Elmer, Model DSC7, Norwalk, CA, USA). Temperature calibration was run using the Indium standard. The samples were accurately weighed into aluminium pans and sealed. The samples were scanned at 1 C/min over the range of 2050 C using iced water as the cooling medium. An empty pan was used as the reference. Total denaturation enthalpy (DH) was estimated by measuring the area of the DSC thermogram. The maximum transition temperature (Tmax) was estimated from the thermogram. 2.6.6. Zeta potential analysis ASC and PSC were dissolved in 0.5 M acetic acid to obtain a nal concentration of 0.05% (w/v). The mixtures were continuously stirred at 4 C using a magnetic stirrer model BIG SQUID (IKA-Werke GmBH & CO.KG, Stanfen, Germany) until the samples were completely solubilised. The zeta (f) potential of collagen solutions was measured with a zeta potential analyser (model ZetaPALs; Brookhaven Instruments Co., Holtsville, NY, USA). Solutions (20 ml) were transferred to an autotitrator (model BI-ZTU, Brookhaven Instruments Co.), in which the pH of the solutions were adjusted to 26 using either 1.0 M nitric acid or 1.0 M KOH. The zeta potential for each solution was recorded. 2.6.7. Solubility The solubility of ASC and PSC was determined by the method of Montero, Jimennez-Colmenero, and Borderias (1991) with a slight modication. Collagens were dissolved in 0.5 M acetic acid to ob-

tain a nal concentration of 3 mg/ml and the mixture was stirred at 4 C for 24 h. Thereafter, the mixture was centrifuged at 5000g for 15 min at 4 C. The supernatant was used for solubility study.

2.6.7.1. Effect of pH on solubility. Collagen solution (3 mg/ml; 8 ml) was transferred to a 50 ml centrifuge tube and the pH was adjusted with either 6 N NaOH or 6 N HCl to obtain the nal pH ranging from 1 to 10. The volume of solution was made up to 10 ml by deionised water (Hydrochem group-Harlte pool, Cleveland, UK) previously adjusted to the same pH as the collagen solution. The solution was centrifuged at 20,000g for 30 min at 4 C. Protein content in the supernatant was determined by the Lowry method (Lowry, Rosebrough, Farr, & Randall, 1951) using bovine serum albumin as a standard. Relative solubility was calculated in comparison with that obtained at the pH giving the highest solubility.

2.6.7.2. Effect of NaCl on solubility. Collagen solution (6 mg/ml; 5 ml) was mixed with 5 ml of NaCl in 0.5 M acetic acid at various concentrations to give the nal concentrations of 0%, 1%, 2%, 3%, 4%, 5% and 6%. The mixture was stirred continuously at 4 C for 30 min, followed by centrifuging at 20,000g for 30 min at 4 C. Protein content in the supernatant was measured and the relative solubility was calculated as previously described.

2.6.8. Statistical analyses All experiments were performed in triplicate and a completely randomised design (CRD) was used. Analysis of variance (ANOVA) was performed and means comparison were done by Duncans multiple range tests. For comparison, the T-test was used (Steel & Torrie, 1980). Analysis was performed using a SPSS statistical package (SPSS 11.0 for Windows, SPSS Inc, Chicago, IL, USA).

3. Results and discussion 3.1. Yield of ASC and PSC from the skin of striped catsh ASC and PSC were isolated from the skin of striped catsh with yields of 5.1% and 7.7% (based on the wet weight of skin), respectively. The skin was not completely solubilised by 0.5 M acetic acid, as shown by the low yield of ASC. This result was in agreement with Jongjareonrak, Benjakul, Visessanguan, and Tanaka (2005) who reported the incomplete solubilisation of bigeye snapper skin in 0.5 M acetic acid. The present result was explained by the fact that collagen molecules in striped catsh skin were most likely cross-linked by covalent bonds through the condensation of aldehyde groups at the telopeptide region as well as the inter-molecular cross-linking, leading to a decrease in the solubility of collagen (Foegeding, Lanier, & Hultin, 1996; Zhang et al., 2007). The yields of ASC and PSC from brownbanded bamboo shark (Chiloscyllium punctatum) were 9.38% and 8.86% (wet weight basis), respectively (Kittiphattanabawon et al., 2010) and those from bigeye snapper skin were 6.4% and 1.1% (wet weight basis), respectively (Jongjareonrak, Benjakul, Visessanguan, Nagai, & Tanaka, 2005), whereas for brownstripe red snapper skin, the yields of ASC and PSC were 9% and 4.7% (wet weight basis), respectively (Jongjareonrak, Benjakul, Visessanguan, & Tanaka, 2005). With further limited pepsin digestion, the cross-linked molecules at the telopeptide region were cleaved, resulting in further extraction. Pepsin was able to cleave specically at the telopeptide region of collagen from the skin of bigeye snapper (Nalinanon, Benjakul, Visessanguan, & Kishimura, 2007). The combined yield of ASC and PSC was 12.8%. Thus pepsin could be used as an aid for increasing the extraction yield of collagen from the skin of striped catsh.

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3.2. Amino acid composition ASC and PSC from the skin of striped catsh showed similar amino acid compositions as shown in Table 1. Both collagens had (309317 glycine residues/1000 residues) as the major amino acid, followed by proline (120126 residues/1000 residues), alanine (116114 residues/1000 residues) and hydroxyproline (8691 residues/1000 residues). Generally, glycine in collagen represents nearly one third of the total residues and occurs as every third residue in collagen except for the rst 14 amino acid residues from the N-terminus and the rst 10 residues from the C-terminus (Foegeding et al., 1996). The imino acid content (proline + hydroxyproline) of ASC and PSC was 206 and 217/1000 residues, which was higher than those of most sh collagens such as grass carp skin collagen (186 residues/1000 residues) and bigeye snapper skin collagen (193 residues/1000 residues) (Kittiphattanabawon et al., 2005; Zhang et al., 2007). The variation in imino acid content amongst different species is mostly due to different living environments of habitat, particularly temperature. Additionally, the imino acid content was reported to have a major inuence on the thermal stability of collagen (Muyonga et al., 2004). Pro + Hyp rich zones of collagen molecules are most likely involved in the formation of junction zones that are stabilised by hydrogen bonding (Johnston-Banks, 1990). The pyrrolidine rings of proline and hydroxyproline impose restrictions on the conformation of the polypeptide chain and help to strengthen the triple helix (Wong, 1989). Thus, collagens from striped catsh are expected to be more stable than those from the other sh species. Hydroxylysine (56 residues/1000 residues) was found in both ASC and PSC from striped catsh skin. Hydroxylysine might undergo cross-linking, leading to the compact structure of collagen from striped catsh skin (Balian & Bowes, 1977). When comparing the imino acid content between ASC and PSC, it was found that the former contained a slightly lower content than the latter. It was suggested that the teleopeptides removed by pepsin digestion contained fewer imino acids. As a result, the PSC obtained had a higher proportion of imino acids. No cysteine and tryptophan were found in either ASC or PSC. Generally, type I collagen has low amounts of cysteine ($0.2%) and methionine ($1.241.33%) (Owusu-Apenten, 2002). The amino acid composition of collagen from striped catsh skin was found to be almost similar to those of collagen from other
Table 1 Amino acid composition of acid soluble collagen and pepsin soluble collagen from the skin of striped catsh (Pangasianodon hypophthalmus) (expressed as residues per 1000 total amino acid residues). Amino acids Alanine Arginine Aspartic acid/asparagine Cysteine Glutamic acid/glutamine Glycine Histidine Isoleucine Leucine Lysine Hydroxylysine Methionine Phenylalanine Hydroxylproline Proline Serine Threonine Tyrosine Tryptophan Valine Total Imino acid ASC 116 54 46 0 80 309 4 14 26 27 5 10 13 86 120 37 24 5 0 23 1000 206 PSC 114 54 44 0 77 317 4 12 24 26 6 10 12 91 126 34 24 3 0 22 1000 211

freshwater sh including common carp, channel catsh and silver carp (Duan, Zhang, Du, Yao, & Konno, 2009). 3.3. Gel electrophoresis The electrophoretic patterns of ASC and PSC from the skin of striped catsh are shown in Fig. 1. Both ASC and PSC consisted of a1- and a2-chains at a ratio of approximately 2:1. Therefore both collagens should most likely be classied as type I collagen. Similar electrophoretic patterns of type I collagen from the skin of brown stripe red snapper were reported by Jongjareonrak, Benjakul, Visessanguan, Nagai, et al. (2005). Apart from a-chains, both ASC and PSC also contained high molecular weight (MW) components, including b- and c-components as well as their cross-linked molecules. Generally, starving sh have more cross-linked collagen than those fed well (Love, Yamaguchi, Creach, & Lavety, 1976). However, the cross-linking of collagen in sh skins is extremely low and the highly cross-linked molecules are rarely found (Cohen-Solal, Louis, Allian, & Meunier, 1981). When comparing the proportion of high MW components between ASC and PSC, the former contained the higher band intensity of b- and c-chains as well as more crosslinked components than the latter. The result suggested that the intra- and inter-molecular cross-links of collagens were richer in ASC than in PSC. After digestion by pepsin, some b- and c-components of ASC might be cleaved into a-components, as evidenced by the increased band intensity of the a-chains. Similar electrophoretic protein patterns were found in ASC and PSC from the skin of large n long barbel catsh (Mystus macropterus) (Zhang, Liu, & Li, 2009). Pepsin cleaves the cross-link containing the telopeptide, and the b-chain is concomitantly converted to two a-chains (Sato et al., 2000). Type I collagen was found in the skins of hake (Montero, Borderias, Turnay, & Leyzarbe, 1990), trout (Montero et al., 1990), Nile perch (Muyonga et al., 2004) and bigeye snapper (Kittiphattanabawon et al., 2005). 3.4. Peptide mapping The peptide maps of ASC and PSC from striped catsh skin digested by lysyl endopeptidase and V8 protease with type 1 calf skin collagen as a control are shown in Fig. 2. For peptide maps of all collagens digested with lysyl endopeptidase, all components

ASC

PSC

220 kDa 170 kDa 116 kDa 76 kDa 70 kDa

1 2

53 kDa

Fig. 1. SDSPAGE of acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of striped catsh. M, high molecular weight markers; I: type I calf skin collagen.

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Lysyl endopeptidase

V8 protease

220 kDa 170 kDa 116 kDa

76 kDa 70 kDa 53 kDa

ASC

PSC

ASC

PSC

Fig. 2. Peptide maps of ASC and PSC from the skin of striped catsh digested by lysyl endopeptidase and V8 protease. M, high MW markers; I, type I calf skin collagen.

including a-1, a-2, b- and c-chains were markedly hydrolysed and varying degradation peptides with different molecular weights were obtained for all collagens. Peptides with MW of 178, 162, 138, 123, 87, 76, 66, 65 and 54 kDa were the dominant products of ASC and PSC after being hydrolysed with lysyl endopeptidase. When comparing between ASC and PSC, the former was more susceptible to hydrolysis by lysyl endopeptidase than the latter. aChains were more retained in the latter. For ASC and PSC digested with V8 protease, b- and c-components were almost completely hydrolysed. V8 protease shows a high specic preference for glutamic acid and aspartic acid residues of proteins (Vercaigne-Marko, Kosciarz, Nedjar-Arroume, & Guillochon, 2000). Due to the slightly lower contents of glutamic acid and aspartic acid (75 and 45 residues/1000 residues) in calf skin collagen (Herbage, Bouillet, & Bernengo, 1977), ASC (80 and 46 residues/1000 residues) and PSC (77 and 44 residues/1000 residues) might be more susceptible to hydrolysis by V8 protease. After hydrolysis with V8 protease, peptides with MW of 95.4, 66 and 25.7 kDa were obtained for ASC. For PSC, peptides with MW of 71.1, 61.6 and 26.3 kDa were observed. However, type I calf skin collagen was resistant to the hydrolysis by V8 protease. When comparing the peptide maps between ASC and PSC hydrolysed by the V8 protease, PSC was more resistant to hydrolysis than ASC, as indicated by a greater band intensity of the a-1 chain. It was found that all collagens were more susceptible to hydrolysis by lysyl endopeptidase than by V8 protease. The differences in the peptide maps between the different collagens generated by lysyl endopeptidase and V8 protease digestion suggested some differences in their primary structure (Nagai & Suzuki, 2002). For PSC, some portions of the telopeptides were presumably removed by pepsin. As a result, chain lengths as well as amino acids at both the C- and N-termini were different. This might determine the accessibility of collagen molecules to proteinases, leading to varying degrees of hydrolysis between ASC and PSC. Peptide maps of collagens were reported to differ amongst sources and species (Mizuta, Yamasa, Miyagi, & Yoshinaka, 1999). Thus, ASC and PSC from the skin of striped catsh might be different in terms of domain or cross-links and totally different from type I calf skin collagen in term of sequence and composition of amino acids.

3.5. ATR-FTIR spectra ATR-FTIR spectra of both ASC and PSC from the skin of striped catsh are depicted in the Fig. 3. FTIR spectra for both ASC and PSC were similar to those of collagens from other sh species (Muyonga et al., 2004). The amide A band of ASC and PSC was found at a wavenumber of 3321 cm1. According to Doyle, Bendit, and Blout (1975), a free NH stretching vibration occurs in the range of 34003440 cm1 and when the NH group of a peptide is involved in a hydrogen bond, the position is shifted to lower frequencies, usually around 3300 cm1. The result indicated that the NH groups of this collagen were involved in hydrogen bonding, probably with a carbonyl group of the peptide chain. The amide B band positions of ASC and PSC were found at wavenumbers of 2926 and 2928 cm1, respectively, representing the asymmetrical stretch of CH2 (Muyonga et al., 2004). The amide I band of ASC and PSC were found at wavenumbers of 1651 and 1649 cm1, respectively. Due to the greater non-helical portion of the telopeptides in ASC, intramolecular H-bond between C@O of the peptide backbone and the adjacent hydrogen donor should be lower in ASC, in comparison with PSC. The amide I band with characteristic frequencies in the range from 1600 to 1700 cm1 was mainly associated with the stretching vibrations of the carbonyl group (C@O bond) along the polypeptide backbone (Payne & Veis, 1988), and was a sensitive marker of the peptide secondary structure (Surewicz & Mantsch, 1988). The amide II band of ASC and PSC was situated at a wavenumber of 1551 cm1, whilst the amide III band of ASC and PSC was located at wavenumbers of 1242 and 1244 cm1, respectively. The amide II and amide III bands represent NH bending vibrations and CH stretching, respectively (Payne & Veis, 1988). Furthermore, a strong CH stretching also occurred at wavenumber of 1747 cm1 for both ASC and PSC, which was in agreement with the ndings of Duan et al. (2009) who found a similar stretching pattern of collagen from the skin of common carp, a freshwater sh. The IR ratios between the amide III and 1454 cm1 of ASC and PSC were 1.17 and 1.16, respectively. An IR ratio of approximately 1 indicates the presence of helical structure (Plepis, Goissis, & Das-Gupta, 1996). Due to the similarity of the IR ratio between

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ASC Amide A Amide I

PSC Amide II

Amide III Amide B

4000

3600

3200

2800

2400

2000
-1

1600

1200

800

400

Wavenumber (cm )
Fig. 3. Fourier transform infrared spectra of ASC and PSC from the skin of striped catsh.

ASC and PSC, pepsin hydrolysis apparently had no pronounced effect on the triple-helical structure of PSC. However, there might be the slight differences between ASC and PSC, especially at the telopeptide region, which was cleaved by pepsin. Nagai, Suzuki, and Nagashima (2008) reported that there were some differences between the secondary structural components such as a-helix, bsheet, b-turn and other random coils between ASC and PSC from the skin of the common mink whale (Balaenoptera acutorostrata). 3.6. Thermal stability DSC thermograms of ASC and PSC from the skin of striped catsh rehydrated in 0.05 M acetic acid and deionised water are shown in Fig. 4. The endothermic peaks, with maximum temperatures (Tmax) of 39.66 and 39.31 C, were observed for ASC and PSC rehydrated in deionised water, respectively. When ASC and PSC were rehydrated in acetic acid, it was noted that Tmax shifted to lower temperatures, 35.35 and 35.38 C for ASC and PSC, respectively. The result suggested that the intramolecular hydrogen bonds stabilising the triple helix structure of collagen might be disrupted to some levels in the presence of acetic acid, mainly due to the repulsion of collagen molecules in acidic solution (Ahmad & Benjakul, 2010). Furthermore, a higher cross-linkage of striped catsh collagen more likely contributed to the higher Tmax of both ASC and PSC. Tmax of ASC was similar to that of PSC in both media, suggesting no differences in the denaturation temperature between both collagens. The triple helix structure was predominant in PSC (Hickman et al., 2000). Tmax of collagen from the skin of striped catsh was slightly higher than that of pig skin collagen (37 C) (Ikoma, Kobayashi, Tanaka, Walsh, & Mann, 2003) but was close to that of calf skin collagen (40.8 C) (Komsa-Penkova, Koyonava, Kostov, & Tenchov, 1999). In contrast, Tmax of collagen from striped catsh was much higher than that of collagen from cold-water sh skin including cod skin (15 C) and that of other tropical sh such as brownstripe red snapper (31.5 C) and bigeye snapper (30.4 C) (Jongjareonrak, Benjakul, Visessanguan, & Tanaka, 2005). The difference in Tmax amongst collagens from different species was correlated with the different imino acid contents (proline and hydroxyproline), body temperature and environmental temperature (Kittiphattanabawon et al., 2005; Nagai et al., 2008). The water temperature from where the striped catsh were caught ranged from 27 to 32 C. The higher content of imino acids is associated with increasing thermal denaturation

(Wong, 1989). The denaturation temperature in sh species is also correlated with the environment, in which they are living. The higher imino acid content of ASC and PSC (206 and 217 residues/1000 residues) was noticeable, compared with those of collagen from the skin of cod (154 residues/1000 residues) (Duan et al., 2009), bigeye snapper (193 residues/1000 residues) (Kittiphattanabawon et al., 2005), carp (190 residues/1000 residues) (Duan et al., 2009) and brown banded bamboo shark (207/1000 residues) (Kittiphattanabawon et al., 2010). When the transition enthalpy (DH) of both ASC and PSC was determined, PSC had the higher DH than ASC when rehydrated in the same medium. The higher DH of PSC might be associated with the higher imino acid content (Table 1) after the removal of telopeptides. The lower DH was found when acetic acid was used for rehydration, in comparison with that found in collagen rehydrated with deionised water. Acetic acid can cleave hydrogen bonds, which stabilise the triple-helical structure of collagen (Xiong, 1997). Thus, the collagen structure was destabilised, leading to decreased thermal stability of collagens, as shown by the lowered Tmax and enthalpy. 3.7. Zeta potentials The zeta potentials of ASC and PSC solutions at different pHs are shown in Fig. 5. The zero surface net charge of ASC and PSC was observed at pH 4.72 and 5.43, respectively. Vojdani (1996) reported that a protein in an aqueous system has a zero net charge at its isoelectric point (pI), when the positive charges are balanced out by the negative charges (Bonner, 2007). Difference in surface charge of ASC and PSC might be ascribed to the difference in acidic and basic amino acid residues, which was more likely governed by the removal of telopeptides by pepsin. ASC and PSC from the skin of brown branded bamboo shark had a net charge of zero at pH 6.21 and 6.56, respectively (Kittiphattanabawon et al., 2010). 3.8. Collagen solubility 3.8.1. Effect of pH The effect of pH on the solubility of ASC and PSC from the skin of striped catsh is shown in Fig. 6a. Both ASC and PSC showed a maximum solubility at pH 2 (P < 0.05). In general, both collagens were solubilised in the acidic pH range (14) (Jongjareonrak,

P. Singh et al. / Food Chemistry 124 (2011) 97105

103

(a)

Tmax = 35.3C
H = 0.578 j/g

Heat flow (w/g)

T max=35.3C
H= 0.764 j/g

ASC PSC

25

30

35

40

45

50

Temperature (C)

(b)
Tmax=39.6C
H= 1.589 j/g

Heat flow (w/g)

T max=39.6C
H= 3.766 j/g

ASC PSC

25

30

35

40

45

50

Temperature (C)
Fig. 4. DSC thermogram of ASC and PSC from the skin of striped catsh dispersed in 0.05 M acetic acid (a) and in deionised water (b).

Benjakul, Visessanguan, Nagai, et al., 2005b). There was a sharp decrease in solubility of both ASC and PSC at a pH higher than 4
25

ASC PSC

20 15 10 5 0 1 -5 2 3 4

(P < 0.05). Marked decreases in solubility were observed in the neutral and alkaline pH ranges. When the pH is lower or higher than pI, the net charge of protein molecules are greater and the solubility is increased by the repulsion forces between chains (Vojdani, 1996). In contrast, when the total net charges of protein molecules are zero, the hydrophobichydrophobic interaction increases, thereby leading to the precipitation and aggregation at the pI. It has been reported that collagen has isoelectric points ranging from pH 6 to 9 (Foegeding et al., 1996). The lowest solubility of ASC and PSC was obtained at around pH 7. This result was in accordance with the solubility of collagen from trout muscle and skin, which was lowest at pH 7 (Montero et al., 1991). From the result, ASC had a solubility prole similar to PSC.

Zeta potential (mV)

pH

Fig. 5. Zeta (f) potential of ASC and PSC from the skin of striped catsh at different pH.

3.8.2. Effect of salt concentration The effect of NaCl on the solubility of ASC and PSC is shown in Fig. 6b. Solubility of ASC and PSC in 0.5 M acetic acid remained constant in the presence of NaCl at concentrations of 02% (P > 0.05).

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P. Singh et al. / Food Chemistry 124 (2011) 97105

(a) 100
Relative collagen solubility(%)
90 80 70 60 50 40 30 20 10 0

PSC ASC

pH ranges. However, the solubility decreased in the presence of NaCl at concentrations above 2%. Pepsin aided extraction can serve as a tool for obtaining the greater yield without a marked effect on the triple-helical structure. Therefore, the skin of striped catsh could be an alternative source of collagen for further applications.

Acknowledgements The authors would like to express their sincere thanks to the Graduate School, Prince of Songkla University, TRF Senior Research Scholar and National Research Council of Thailand for the nancial support.
0 1 2 3 4 5 pH 6 7 8 9 10

References
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(b)100
Relative Collagen solubility(%)
90 80 70 60 50 40 30 20 10 0

PSC ASC

Concentration NaCl (% (w/v))

Fig. 6. Relative solubility (%) of ASC and PSC from the skin of striped catsh as affected by different pH (a) and NaCl (b) concentrations.

However a slight decrease in solubility was observed at 3% NaCl. A drastic decrease was noticeable when the concentration was increased to 4% and above (P < 0.05). The solubility of collagens from the skin of trout, hake, bigeye snapper (Priacanthus tayenus), and bigeye snapper (Priacanthus marcracanthus) in acetic acid solution generally decreased with increasing NaCl concentration (Jongjareonrak, Benjakul, Visessanguan, & Tanaka, 2005a; Kittiphattanabawon et al., 2005; Montero et al., 1991). The decrease in solubility of collagens could be described as being due to a salting out effect, which occurred at relatively high NaCl concentrations (Asghar & Henrickson, 1982). An increase in ionic strength causes a reduction in protein solubility by enhancing hydrophobichydrophobic interactions between protein chains, and increasing the competition for water with the ionic salts, leading to the induced protein precipitation (Vojdani, 1996). Similar behaviours were found for ASC and PSC. However, PSC showed a greater solubility than ASC at NaCl concentrations greater than 2%. The result was in accordance with that of Jongjareonrak, Benjakul, Visessanguan, Nagai, et al. (2005). A greater solubility of PSC could be due to the partial hydrolysis of high MW cross-linked molecules by pepsin. In addition, the differences in amino acid compositions and structure between ASC and PSC might result in such differences.

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