Food Chemistry 124 (2011) 97–105

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Food Chemistry

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Isolation and characterisation of collagen extracted from the skin of stripedcatfish (Pangasianodon hypophthalmus)

Prabjeet Singh a, Soottawat Benjakul a,*, Sajid Maqsood a, Hideki Kishimura b

a Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailandb 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

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

Keywords:Acid soluble collagen (ASC)Pepsin soluble collagen (PSC)Zeta potentialFTIRDSCStriped catfishFish skin

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.05.111

* Corresponding author. Tel.: +66 7428 6334; fax: +E-mail address: soottawat.b@psu.ac.th (S. Benjaku

a b s t r a c t

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of striped catfish (Pangasian-odon 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 PSCcomprising two different a-chains (a1 and a2) were characterised as type I and contained imino acidof 206 and 211 imino acid residues/1000 residues, respectively. Peptide maps of ASC and PSC hydrolysedby either lysyl endopeptidase or V8 protease were slightly different and totally differed from those oftype 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 hydrolysishad no marked effect on the triple-helical structure of collagen. Both ASC and PSC had the highest solu-bility at acidic pH. A loss in solubility was observed at a pH greater than 4 or when NaCl concentrationwas higher than 2% (w/v). Tmax of ASC and PSC were 39.3 and 39.6 �C, respectively, and shifted to a lowertemperature when rehydrated with 0.05 M acetic acid. Zeta potential studies indicated that ASC and PSCexhibited a net zero charge at pH 4.72 and 5.43, respectively. Thus, ASC and PSC were slightly different interms of composition and structure, leading to somewhat different properties.

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1. Introduction

Collagen is the most abundant protein in vertebrates and con-stitutes about 30% of the total proteins. Collagen, a right-handedtriple superhelical rod, is unique in its ability to form insoluble fi-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 foundin connective tissues, including tendons, bones and skins (Muyon-ga, Cole, & Duodu, 2004). All members of the collagen family arecharacterised by domains with repetitions of the proline-rich trip-eptides, Gly-X-Y, involved in the formation of the triple helix(Muyonga et al., 2004). Collagen has a wide range of applicationsin the leather and film industries, in cosmetic and biomedicalmaterials, and as food (Kittiphattanabawon, Benjakul, Visessan-guan, Nagai, & Tanaka, 2005). In pharmaceutical applications, col-lagen can be used for production of wound dressings, vitreousimplants and as carriers for drug delivery. In addition, collagenhas been used to produce edible casings for the meat processingindustries (sausages/salami/snack sticks).

ll rights reserved.

66 7421 2889.l).

Due to the outbreak of bovine spongiform encephalopathy(BSE), transmissible spongiform encephalopathy (TSE) and thefoot-and-mouth disease (FMD) crisis, the uses of collagen and col-lagen-derived products of land animal origin have become of moreconcern (Jongjareonrak, Benjakul, Visessanguan, & Tanaka, 2005).In addition, the collagens extracted from bovine sources are pro-hibited for Sikhs and Hindus, whilst porcine collagen cannot beconsumed by Muslims and Jews, both of whom require bovine tobe religiously prepared. As a consequence, the alternative sourcesof collagen, especially from aquatic animals including freshwaterand marine fish and mollusks have received increasing attention(Shen, Kurihara, & Takahashi, 2007).

Pangasianodon hypophthalmus or Pla Sawai (in Thai), a largefreshwater catfish, belongs to the order Siluriformes and is a mem-ber of Pangasidae family. It is one of the most important aquacul-ture species in Thailand (Froese & Pauly, 2007), especially in thenortheast part of Thailand. This fish is also known as Siamese sharkor sutchi catfish and is native to the Chao Phraya river in Thailandand the Mekong in Vietnam. It has become an important fish formany countries like Indonesia, Malaysia and China (Roberts &Vidthayanon, 1991). This freshwater fish normally lives in a tropi-cal climate and prefers water with a pH of 6.5–7.5 and a tempera-ture range of 22–26 �C. Adults reach up to 130 cm (4 ft) in lengthand can weigh up to a maximum of 44.0 kg (97 lb) (Roberts &

98 P. Singh et al. / Food Chemistry 124 (2011) 97–105

Vidthayanon, 1991). Its meat has been popular among the consum-ers worldwide. During processing and filleting, a huge amount ofskin from this fish is generated as a byproduct, which can be usedas a potential source for collagen extraction. The skin from this fishis thick and tough, which may be associated with the collagencross-links, especially cross-linking caused by hydroxylysine. How-ever, no information on composition and molecular properties ofcollagen from the skin of this species has been reported. Thus,the objective of the present study was to extract and characterisethe collagen, both acid soluble collagen (ASC) and pepsin solublecollagen (PSC), from the skin of striped catfish.

2. Materials and methods

2.1. Fish skin preparation

Whole fresh farmed striped catfish (P. hypophthalmus) (approx-imately 2 years old) weighing 1 ± 0.5 kg/fish stored on ice wereprocured from the fish market of Hat Yai, Songhkla, Thailand. Fishwere stored in ice with a fish/ice ratio of 1:2 (w/w) and transportedwithin 1 h to the Department of Food Technology, Prince of Song-kla University, Hat Yai, Thailand. Upon arrival, fish were washedusing tap water and deskinned. The skin was washed with coldwater (5–8 �C) and cut into small pieces (0.5 � 0.5 cm2). The pre-pared skin samples were packed in polyethylene bags and keptat �20 �C until used. The storage time was not longer than1 month.

2.2. Chemicals

b-Mercaptoethanol (b-ME), V8 protease from Staphylococcusaureus (EC3.4.21.19, 800 U/mg powder); pepsin from porcinestomach mucosa (EC3.4.23.1; powderised; 750 U/mg dry matter)and type I collagen from calf skin were purchased from SigmaChemical Co. (St. Louis, MO, USA). High molecular weight markerswere obtained from GE Healthcare UK (Buckinghamshire, UK). So-dium dodecyl sulphate (SDS), trichloroacetic acid, Folin–Ciocalteu’sphenol reagent, acetic acid and tris(hydroxylmethyl) aminometh-ane were obtained from Merck (Darmstadt, Germany). Lysyl endo-peptidase from Achromobacter lyticus (EC3.4.21.50; 4.5 AU/mgprotein) were procured from Wako Pure Chemical Industries, Ltd.(Tokyo, Japan).

2.3. Pretreatment of skin

To remove non-collagenous proteins, the prepared fish skin wasmixed 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 alkalisolution was changed every 2 h. The treated skin was then washedwith cold water until a neutral or faintly basic pH of wash waterwas reached. The pH of wash water was determined using a digitalpH 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 modification. All processes were carried out at4 �C with continuous stirring. The pretreated skins were defattedwith 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 skinwas washed with cold water (5–8 �C), followed by soaking in0.5 M acetic acid with a solid/solvent ratio of 1:15 (w/v) for 24 h.The mixture was filtered through two layers of cheese cloth andthe residue was re-extracted under the same conditions. Both fil-trates were combined. The collagen was precipitated by adding

NaCl (powder) to a final concentration of 2.6 M in the presenceof 0.05 M tris(hydroxymethyl) aminomethane, pH 7.0. The resul-tant precipitate was collected by centrifuging at 20,000g for60 min, using a refrigerated centrifuge Avanti� J-E (Beckman Coul-ter, Inc., Palo Alto, CA, USA). The pellet was dissolved in a minimumvolume of 0.5 M acetic acid, and dialysed against 50 volumes of0.1 M acetic acid for 24 h, followed by the dialysis in the same vol-ume of distilled water for another 24 h. The dialysate was freezedried and was referred to as ‘‘acid soluble collagen; ASC”. The yieldof ASC was calculated from the percentage of dry weight of colla-gen extracted in comparison with the wet weight of the initial skinused.

2.5. Extraction of pepsin soluble collagen (PSC)

The undissolved residue obtained after ASC extraction was usedfor PSC extraction. The residue was soaked in 0.5 M acetic acid witha solid/solvent ratio of 1:15 (w/v) and porcine pepsin (20 U/g res-idue) was added. The mixtures were continuously stirred at 4 �Cfor 48 h, followed by filtration with two layers of cheesecloth.The filtrate was subjected to precipitation and the pellet was dia-lysed in the same manner as those for ASC previously described.The dialysate was freeze dried and was referred to as ‘‘pepsin sol-uble collagen; PSC”. The yield of PSC was also calculated in thesame manner as for ASC. Additionally, the accumulated yield ofcollagen 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 analysisThe 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 with3.5 M NaOH and diluted with 0.2 M citrate buffer (pH 2.2) (Kittip-hattanabawon, 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. SDS–polyacrylamide gel electrophoresis (SDS–PAGE)SDS–PAGE was performed following the method of Laemmli

(1970). The samples were dissolved in 5% SDS and the mixtureswere incubated at 85 �C for 1 h in temperature controlled waterbath (Memmert, Schwabach, Germany). The mixture was centri-fuged at 4000g for 5 min using a microcentrifuge (MIKRO20, Het-tich Zentrifugan, Germany) at room temperature to removeundissolved debris. Solubilised samples were mixed at a ratio of1:1 (v/v) with the sample buffer (0.5 M Tris HCl, pH 6.8, containing4% SDS and 20% glycerol) containing 10% b-ME. The mixtures werekept in boiling water for 2 min. Samples (15 lg protein) wereloaded onto polyacrylamide gels comprising a 7.5% running geland a 4% stacking gel and subjected to electrophoresis at a constantcurrent of 15 mA/gel for 1 h and 30 min using a Mini Protein II unit(Bio-Rad Laboratories, Inc., Richmond, CA, USA). After electropho-resis, the gel was stained with 0.05% (w/v) Coomassie blue R-250in 15% (v/v) methanol and 5% (v/v) acetic acid and destained with30% (v/v) methanol and 10% (v/v) acetic acid. High molecularweight markers were used to estimate the molecular weight ofproteins. The markers used included myosin (200 kDa), a2-macro-globulin (170 kDa), b-galactosidase (116 kDa), transferrin (76 kDa)and glutamate dehydrogenase (53 kDa). Type I calf skin collagenwas used as a standard. Quantitative analysis of protein bandintensity was performed using a Model GS-700 Imaging Densitom-eter (Bio-Rad Laboratories, Hercules, CA, USA) with Molecular Ana-lyst Software version 1.4 (image analysis system).

P. Singh et al. / Food Chemistry 124 (2011) 97–105 99

2.6.3. Peptide mapping of collagenPeptide mapping of ASC and PSC was performed according to

the method of Kittiphattanabawon et al. (2005) with a slight mod-ification. The samples (6 mg) were dissolved in 1 ml of 0.1 M so-dium phosphate, pH 7.2 containing 0.5% (w/v) SDS. Then, themixtures were preheated at 45 �C for 3 h and 300 ll of the pre-pared mixtures were transferred to test tubes for digestion. To ini-tiate the digestion, 20 ll of each enzyme solution, either S. aureusV8 protease or lysyl endopeptidase from A. lyticus, with concentra-tions of 5 and 50 ll/ml, respectively, were added to the mixtures.The reaction mixtures were then incubated at 37 �C for 1 h. Thereactions were terminated by subjecting the reaction mixture toboiling water for 3 min. Peptides generated by the protease diges-tion were separated by SDS–PAGE using a 7.5% running gel and a4% stacking gel, followed by staining and destaining as previouslydescribed. Peptide mapping of calf skin collagen type I was alsoconducted in the same manner and the peptide patterns werecompared.

2.6.4. ATR-FTIR analysisBoth ASC and PSC were subjected to attenuated total reflec-

tance-Fourier transform infrared spectroscopy (ATR-FTIR). FTIRspectrometer (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 collagensamples were placed onto the crystal cell and the cell was clampedinto the mount of the FTIR spectrometer. The spectra in the rangeof 400–4000 cm�1 with automatic signal gain were collected in 32scans at a resolution of 4 cm�1 and were ratioed against a back-ground spectrum recorded from the clean empty cell at 25 �C.

2.6.5. Differential scanning calorimetryDifferential scanning calorimetry (DSC) of ASC and PSC was run

following the method of Rochdi, Foucat, and Renou (2000) with aslight modification. The samples were rehydrated by adding deion-ised water or 0.05 M acetic acid to dried samples at a solid/solutionratio of 1:40 (w/v). The mixtures were allowed to stand for 2 daysat 4 �C. DSC was performed using a differential scanning calorime-ter (Perkin Elmer, Model DSC7, Norwalk, CA, USA). Temperaturecalibration was run using the Indium standard. The samples wereaccurately weighed into aluminium pans and sealed. The sampleswere scanned at 1 �C/min over the range of 20–50 �C using icedwater as the cooling medium. An empty pan was used as the refer-ence. Total denaturation enthalpy (DH) was estimated by measur-ing the area of the DSC thermogram. The maximum transitiontemperature (Tmax) was estimated from the thermogram.

2.6.6. Zeta potential analysisASC and PSC were dissolved in 0.5 M acetic acid to obtain a final

concentration of 0.05% (w/v). The mixtures were continuously stir-red at 4 �C using a magnetic stirrer model BIG SQUID (IKA�-WerkeGmBH & CO.KG, Stanfen, Germany) until the samples were com-pletely solubilised.

The zeta (f) potential of collagen solutions was measured with azeta potential analyser (model ZetaPALs; Brookhaven InstrumentsCo., Holtsville, NY, USA). Solutions (20 ml) were transferred to anautotitrator (model BI-ZTU, Brookhaven Instruments Co.), in whichthe pH of the solutions were adjusted to 2–6 using either 1.0 M ni-tric acid or 1.0 M KOH. The zeta potential for each solution wasrecorded.

2.6.7. SolubilityThe solubility of ASC and PSC was determined by the method of

Montero, Jimennez-Colmenero, and Borderias (1991) with a slightmodification. Collagens were dissolved in 0.5 M acetic acid to ob-

tain a final concentration of 3 mg/ml and the mixture was stirredat 4 �C for 24 h. Thereafter, the mixture was centrifuged at 5000gfor 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 adjustedwith either 6 N NaOH or 6 N HCl to obtain the final pH rangingfrom 1 to 10. The volume of solution was made up to 10 ml bydeionised water (Hydrochem group-Harlte pool, Cleveland, UK)previously adjusted to the same pH as the collagen solution. Thesolution was centrifuged at 20,000g for 30 min at 4 �C. Protein con-tent in the supernatant was determined by the Lowry method(Lowry, Rosebrough, Farr, & Randall, 1951) using bovine serumalbumin as a standard. Relative solubility was calculated in com-parison 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 variousconcentrations to give the final concentrations of 0%, 1%, 2%, 3%,4%, 5% and 6%. The mixture was stirred continuously at 4 �C for30 min, followed by centrifuging at 20,000g for 30 min at 4 �C. Pro-tein content in the supernatant was measured and the relative sol-ubility was calculated as previously described.

2.6.8. Statistical analysesAll 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 Duncan’smultiple range tests. For comparison, the T-test was used (Steel& Torrie, 1980). Analysis was performed using a SPSS statisticalpackage (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 catfish

ASC and PSC were isolated from the skin of striped catfish withyields of 5.1% and 7.7% (based on the wet weight of skin), respec-tively. The skin was not completely solubilised by 0.5 M acetic acid,as shown by the low yield of ASC. This result was in agreementwith Jongjareonrak, Benjakul, Visessanguan, and Tanaka (2005)who reported the incomplete solubilisation of bigeye snapper skinin 0.5 M acetic acid. The present result was explained by the factthat collagen molecules in striped catfish skin were most likelycross-linked by covalent bonds through the condensation of alde-hyde groups at the telopeptide region as well as the inter-molecu-lar cross-linking, leading to a decrease in the solubility of collagen(Foegeding, Lanier, & Hultin, 1996; Zhang et al., 2007). The yields ofASC and PSC from brownbanded bamboo shark (Chiloscylliumpunctatum) were 9.38% and 8.86% (wet weight basis), respectively(Kittiphattanabawon et al., 2010) and those from bigeye snapperskin were 6.4% and 1.1% (wet weight basis), respectively (Jongjar-eonrak, Benjakul, Visessanguan, Nagai, & Tanaka, 2005), whereasfor brownstripe red snapper skin, the yields of ASC and PSC were9% and 4.7% (wet weight basis), respectively (Jongjareonrak, Benja-kul, Visessanguan, & Tanaka, 2005). With further limited pepsindigestion, the cross-linked molecules at the telopeptide regionwere cleaved, resulting in further extraction. Pepsin was able tocleave specifically at the telopeptide region of collagen from theskin 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 extractionyield of collagen from the skin of striped catfish.

100 P. Singh et al. / Food Chemistry 124 (2011) 97–105

3.2. Amino acid composition

ASC and PSC from the skin of striped catfish showed similaramino acid compositions as shown in Table 1. Both collagens had(309–317 glycine residues/1000 residues) as the major amino acid,followed by proline (120–126 residues/1000 residues), alanine(116–114 residues/1000 residues) and hydroxyproline (86–91 res-idues/1000 residues). Generally, glycine in collagen representsnearly one third of the total residues and occurs as every third res-idue in collagen except for the first 14 amino acid residues fromthe N-terminus and the first 10 residues from the C-terminus (Foe-geding et al., 1996). The imino acid content (proline + hydroxypro-line) of ASC and PSC was 206 and 217/1000 residues, which washigher than those of most fish collagens such as grass carp skin col-lagen (186 residues/1000 residues) and bigeye snapper skin colla-gen (193 residues/1000 residues) (Kittiphattanabawon et al., 2005;Zhang et al., 2007). The variation in imino acid content amongstdifferent species is mostly due to different living environments ofhabitat, particularly temperature. Additionally, the imino acid con-tent was reported to have a major influence on the thermal stabil-ity of collagen (Muyonga et al., 2004). Pro + Hyp rich zones ofcollagen molecules are most likely involved in the formation ofjunction zones that are stabilised by hydrogen bonding (John-ston-Banks, 1990). The pyrrolidine rings of proline and hydroxy-proline impose restrictions on the conformation of thepolypeptide chain and help to strengthen the triple helix (Wong,1989). Thus, collagens from striped catfish are expected to be morestable than those from the other fish species. Hydroxylysine (5–6residues/1000 residues) was found in both ASC and PSC fromstriped catfish skin. Hydroxylysine might undergo cross-linking,leading to the compact structure of collagen from striped catfishskin (Balian & Bowes, 1977). When comparing the imino acid con-tent between ASC and PSC, it was found that the former containeda slightly lower content than the latter. It was suggested that theteleopeptides removed by pepsin digestion contained fewer iminoacids. As a result, the PSC obtained had a higher proportion of imi-no acids. No cysteine and tryptophan were found in either ASC orPSC. Generally, type I collagen has low amounts of cysteine(�0.2%) and methionine (�1.24–1.33%) (Owusu-Apenten, 2002).

The amino acid composition of collagen from striped catfishskin was found to be almost similar to those of collagen from other

Table 1Amino acid composition of acid soluble collagen and pepsin soluble collagen from theskin of striped catfish (Pangasianodon hypophthalmus) (expressed as residues per 1000total amino acid residues).

Amino acids ASC PSC

Alanine 116 114Arginine 54 54Aspartic acid/asparagine 46 44Cysteine 0 0Glutamic acid/glutamine 80 77Glycine 309 317Histidine 4 4Isoleucine 14 12Leucine 26 24Lysine 27 26Hydroxylysine 5 6Methionine 10 10Phenylalanine 13 12Hydroxylproline 86 91Proline 120 126Serine 37 34Threonine 24 24Tyrosine 5 3Tryptophan 0 0Valine 23 22Total 1000 1000Imino acid 206 211

freshwater fish including common carp, channel catfish and silvercarp (Duan, Zhang, Du, Yao, & Konno, 2009).

3.3. Gel electrophoresis

The electrophoretic patterns of ASC and PSC from the skin ofstriped catfish are shown in Fig. 1. Both ASC and PSC consisted ofa1- and a2-chains at a ratio of approximately 2:1. Therefore bothcollagens should most likely be classified as type I collagen. Similarelectrophoretic patterns of type I collagen from the skin of brownstripe red snapper were reported by Jongjareonrak, Benjakul, Vis-essanguan, Nagai, et al. (2005). Apart from a-chains, both ASCand PSC also contained high molecular weight (MW) components,including b- and c-components as well as their cross-linked mole-cules. Generally, starving fish have more cross-linked collagen thanthose fed well (Love, Yamaguchi, Creach, & Lavety, 1976). However,the cross-linking of collagen in fish skins is extremely low and thehighly cross-linked molecules are rarely found (Cohen-Solal, Louis,Allian, & Meunier, 1981). When comparing the proportion of highMW components between ASC and PSC, the former contained thehigher band intensity of b- and c-chains as well as more cross-linked components than the latter. The result suggested that theintra- and inter-molecular cross-links of collagens were richer inASC than in PSC. After digestion by pepsin, some b- and c-compo-nents of ASC might be cleaved into a-components, as evidenced bythe increased band intensity of the a-chains. Similar electropho-retic protein patterns were found in ASC and PSC from the skinof large fin long barbel catfish (Mystus macropterus) (Zhang, Liu,& Li, 2009). Pepsin cleaves the cross-link containing the telopep-tide, 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 (Monteroet 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 catfish skin di-gested by lysyl endopeptidase and V8 protease with type 1 calfskin collagen as a control are shown in Fig. 2. For peptide mapsof all collagens digested with lysyl endopeptidase, all components

220 kDa 170 kDa

116 kDa

76 kDa 70 kDa

53 kDa

γ

β

α1 α2

M I ASC PSC

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

Lysyl endopeptidase V8 protease

220 kDa 170 kDa

116 kDa

76 kDa 70 kDa

53 kDa

M I ASC PSC I ASC PSC

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

P. Singh et al. / Food Chemistry 124 (2011) 97–105 101

including a-1, a-2, b- and c-chains were markedly hydrolysed andvarying degradation peptides with different molecular weightswere obtained for all collagens. Peptides with MW of 178, 162,138, 123, 87, 76, 66, 65 and 54 kDa were the dominant productsof ASC and PSC after being hydrolysed with lysyl endopeptidase.When comparing between ASC and PSC, the former was more sus-ceptible to hydrolysis by lysyl endopeptidase than the latter. a-Chains were more retained in the latter. For ASC and PSC digestedwith V8 protease, b- and c-components were almost completelyhydrolysed. V8 protease shows a high specific preference for glu-tamic acid and aspartic acid residues of proteins (Vercaigne-Marko,Kosciarz, Nedjar-Arroume, & Guillochon, 2000). Due to the slightlylower contents of glutamic acid and aspartic acid (75 and 45 resi-dues/1000 residues) in calf skin collagen (Herbage, Bouillet, &Bernengo, 1977), ASC (80 and 46 residues/1000 residues) andPSC (77 and 44 residues/1000 residues) might be more susceptibleto hydrolysis by V8 protease. After hydrolysis with V8 protease,peptides with MW of 95.4, 66 and 25.7 kDa were obtained forASC. For PSC, peptides with MW of 71.1, 61.6 and 26.3 kDa wereobserved. However, type I calf skin collagen was resistant to thehydrolysis by V8 protease. When comparing the peptide maps be-tween ASC and PSC hydrolysed by the V8 protease, PSC was moreresistant to hydrolysis than ASC, as indicated by a greater bandintensity of the a-1 chain.

It was found that all collagens were more susceptible to hydro-lysis by lysyl endopeptidase than by V8 protease. The differences inthe peptide maps between the different collagens generated by ly-syl endopeptidase and V8 protease digestion suggested some dif-ferences in their primary structure (Nagai & Suzuki, 2002). ForPSC, some portions of the telopeptides were presumably removedby pepsin. As a result, chain lengths as well as amino acids at boththe C- and N-termini were different. This might determine theaccessibility of collagen molecules to proteinases, leading to vary-ing degrees of hydrolysis between ASC and PSC. Peptide maps ofcollagens were reported to differ amongst sources and species(Mizuta, Yamasa, Miyagi, & Yoshinaka, 1999). Thus, ASC and PSCfrom the skin of striped catfish might be different in terms of do-main or cross-links and totally different from type I calf skin colla-gen 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 stripedcatfish are depicted in the Fig. 3. FTIR spectra for both ASC andPSC were similar to those of collagens from other fish species(Muyonga et al., 2004). The amide A band of ASC and PSC wasfound at a wavenumber of 3321 cm�1. According to Doyle, Bendit,and Blout (1975), a free N–H stretching vibration occurs in therange of 3400–3440 cm�1 and when the NH group of a peptide isinvolved in a hydrogen bond, the position is shifted to lower fre-quencies, usually around 3300 cm�1. The result indicated that theNH groups of this collagen were involved in hydrogen bonding,probably with a carbonyl group of the peptide chain. The amideB band positions of ASC and PSC were found at wavenumbers of2926 and 2928 cm�1, respectively, representing the asymmetricalstretch of CH2 (Muyonga et al., 2004). The amide I band of ASCand PSC were found at wavenumbers of 1651 and 1649 cm�1,respectively. Due to the greater non-helical portion of the telopep-tides in ASC, intramolecular H-bond between C@O of the peptidebackbone and the adjacent hydrogen donor should be lower inASC, in comparison with PSC. The amide I band with characteristicfrequencies in the range from 1600 to 1700 cm�1 was mainly asso-ciated with the stretching vibrations of the carbonyl group (C@Obond) along the polypeptide backbone (Payne & Veis, 1988), andwas a sensitive marker of the peptide secondary structure (Sure-wicz & Mantsch, 1988). The amide II band of ASC and PSC was sit-uated at a wavenumber of 1551 cm�1, whilst the amide III band ofASC and PSC was located at wavenumbers of 1242 and 1244 cm�1,respectively. The amide II and amide III bands represent N–H bend-ing vibrations and C–H stretching, respectively (Payne & Veis,1988). Furthermore, a strong C–H stretching also occurred at wave-number of 1747 cm�1 for both ASC and PSC, which was in agree-ment with the findings of Duan et al. (2009) who found a similarstretching pattern of collagen from the skin of common carp, afreshwater fish.

The IR ratios between the amide III and 1454 cm�1 of ASC andPSC were 1.17 and 1.16, respectively. An IR ratio of approximately1 indicates the presence of helical structure (Plepis, Goissis, &Das-Gupta, 1996). Due to the similarity of the IR ratio between

40080012001600200024002800320036004000

Wavenumber (cm -1)

ASC PSC

Amide I Amide A

Amide B

Amide II

Amide III

Fig. 3. Fourier transform infrared spectra of ASC and PSC from the skin of striped catfish.

102 P. Singh et al. / Food Chemistry 124 (2011) 97–105

ASC and PSC, pepsin hydrolysis apparently had no pronounced ef-fect on the triple-helical structure of PSC. However, there might bethe slight differences between ASC and PSC, especially at the telo-peptide region, which was cleaved by pepsin. Nagai, Suzuki, andNagashima (2008) reported that there were some differences be-tween the secondary structural components such as a-helix, b-sheet, b-turn and other random coils between ASC and PSC fromthe skin of the common mink whale (Balaenoptera acutorostrata).

3.6. Thermal stability

DSC thermograms of ASC and PSC from the skin of striped catfishrehydrated in 0.05 M acetic acid and deionised water are shown inFig. 4. The endothermic peaks, with maximum temperatures (Tmax)of 39.66 and 39.31 �C, were observed for ASC and PSC rehydrated indeionised water, respectively. When ASC and PSC were rehydratedin acetic acid, it was noted that Tmax shifted to lower temperatures,35.35 and 35.38 �C for ASC and PSC, respectively. The result sug-gested that the intramolecular hydrogen bonds stabilising the triplehelix structure of collagen might be disrupted to some levels in thepresence of acetic acid, mainly due to the repulsion of collagen mol-ecules in acidic solution (Ahmad & Benjakul, 2010). Furthermore, ahigher cross-linkage of striped catfish collagen more likely contrib-uted to the higher Tmax of both ASC and PSC. Tmax of ASC was similarto that of PSC in both media, suggesting no differences in the dena-turation temperature between both collagens. The triple helixstructure was predominant in PSC (Hickman et al., 2000). Tmax ofcollagen from the skin of striped catfish was slightly higher thanthat 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 catfish was much higher than that ofcollagen from cold-water fish skin including cod skin (15 �C) andthat of other tropical fish such as brownstripe red snapper(31.5 �C) and bigeye snapper (30.4 �C) (Jongjareonrak, Benjakul,Visessanguan, & Tanaka, 2005). The difference in Tmax amongst col-lagens from different species was correlated with the different imi-no acid contents (proline and hydroxyproline), body temperatureand environmental temperature (Kittiphattanabawon et al., 2005;Nagai et al., 2008). The water temperature from where the stripedcatfish were caught ranged from 27 to 32 �C. The higher contentof imino acids is associated with increasing thermal denaturation

(Wong, 1989). The denaturation temperature in fish species is alsocorrelated with the environment, in which they are living. The high-er imino acid content of ASC and PSC (206 and 217 residues/1000residues) was noticeable, compared with those of collagen fromthe skin of cod (154 residues/1000 residues) (Duan et al., 2009), big-eye snapper (193 residues/1000 residues) (Kittiphattanabawonet al., 2005), carp (190 residues/1000 residues) (Duan et al., 2009)and brown banded bamboo shark (207/1000 residues) (Kittiphat-tanabawon et al., 2010).

When the transition enthalpy (DH) of both ASC and PSC wasdetermined, PSC had the higher DH than ASC when rehydrated inthe same medium. The higher DH of PSC might be associated withthe higher imino acid content (Table 1) after the removal oftelopeptides. The lower DH was found when acetic acid was usedfor rehydration, in comparison with that found in collagen rehy-drated with deionised water. Acetic acid can cleave hydrogenbonds, which stabilise the triple-helical structure of collagen(Xiong, 1997). Thus, the collagen structure was destabilised, lead-ing to decreased thermal stability of collagens, as shown by thelowered Tmax and enthalpy.

3.7. Zeta potentials

The zeta potentials of ASC and PSC solutions at different pHs areshown in Fig. 5. The zero surface net charge of ASC and PSC was ob-served at pH 4.72 and 5.43, respectively. Vojdani (1996) reportedthat a protein in an aqueous system has a zero net charge at its iso-electric point (pI), when the positive charges are balanced out bythe negative charges (Bonner, 2007). Difference in surface chargeof ASC and PSC might be ascribed to the difference in acidic and ba-sic amino acid residues, which was more likely governed by the re-moval of telopeptides by pepsin. ASC and PSC from the skin ofbrown branded bamboo shark had a net charge of zero at pH6.21 and 6.56, respectively (Kittiphattanabawon et al., 2010).

3.8. Collagen solubility

3.8.1. Effect of pHThe effect of pH on the solubility of ASC and PSC from the skin of

striped catfish is shown in Fig. 6a. Both ASC and PSC showed amaximum solubility at pH 2 (P < 0.05). In general, both collagenswere solubilised in the acidic pH range (1–4) (Jongjareonrak,

25 30 35 40 45 50

Temperature (°C)

Heat

flow

(w/g

)ASCPSC

25 30 35 40 45 50

Temperature (°C)

Heat

flow

(w/g

)

ASCPSC

Tmax=35.3°C

T

T

T

Δ H= 0.764 j/g

(b)

max = 35.3°C

Δ H = 0.578 j/g

(a)

max=39.6°C

Δ H= 1.589 j/g

max=39.6°C

Δ H= 3.766 j/g

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

P. Singh et al. / Food Chemistry 124 (2011) 97–105 103

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

-5

0

5

10

15

20

25

1 2 3 4 5 6 7

pH

Zeta

pot

entia

l (m

V)

ASC

PSC

Fig. 5. Zeta (f) potential of ASC and PSC from the skin of striped catfish at differentpH.

(P < 0.05). Marked decreases in solubility were observed in theneutral and alkaline pH ranges. When the pH is lower or higherthan pI, the net charge of protein molecules are greater and the sol-ubility is increased by the repulsion forces between chains(Vojdani, 1996). In contrast, when the total net charges of proteinmolecules are zero, the hydrophobic–hydrophobic interaction in-creases, thereby leading to the precipitation and aggregation atthe pI. It has been reported that collagen has isoelectric pointsranging from pH 6 to 9 (Foegeding et al., 1996). The lowest solubil-ity of ASC and PSC was obtained at around pH 7. This result was inaccordance with the solubility of collagen from trout muscle andskin, which was lowest at pH 7 (Montero et al., 1991). From the re-sult, ASC had a solubility profile similar to PSC.

3.8.2. Effect of salt concentrationThe 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 con-stant in the presence of NaCl at concentrations of 0–2% (P > 0.05).

(a)

0

1020

3040

50

6070

8090

100

0 1 2 3 4 5 6 7 8 9 10

pH

Rel

ativ

e co

llage

n so

lubi

lity(

%)

PSCASC

0

1020

3040

50

6070

8090

100

0 1 2 3 4 5 6

Concentration NaCl (% (w/v))

Rel

ativ

e C

olla

gen

solu

bilit

y(%

)

PSCASC

(b)

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

104 P. Singh et al. / Food Chemistry 124 (2011) 97–105

However a slight decrease in solubility was observed at 3% NaCl. Adrastic decrease was noticeable when the concentration was in-creased to 4% and above (P < 0.05). The solubility of collagens fromthe skin of trout, hake, bigeye snapper (Priacanthus tayenus), andbigeye snapper (Priacanthus marcracanthus) in acetic acid solutiongenerally decreased with increasing NaCl concentration (Jongjar-eonrak, Benjakul, Visessanguan, & Tanaka, 2005a; Kittiphattanaba-won et al., 2005; Montero et al., 1991). The decrease in solubility ofcollagens 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 reductionin protein solubility by enhancing hydrophobic–hydrophobicinteractions between protein chains, and increasing the competi-tion for water with the ionic salts, leading to the induced proteinprecipitation (Vojdani, 1996). Similar behaviours were found forASC and PSC. However, PSC showed a greater solubility than ASCat NaCl concentrations greater than 2%. The result was in accor-dance with that of Jongjareonrak, Benjakul, Visessanguan, Nagai,et al. (2005). A greater solubility of PSC could be due to the partialhydrolysis of high MW cross-linked molecules by pepsin. In addi-tion, the differences in amino acid compositions and structure be-tween ASC and PSC might result in such differences.

4. Conclusion

Both ASC and PSC were successfully isolated from the skin ofstriped catfish and classified as type I collagen. Telopeptides ofASC were cleaved by pepsin, resulting in the increased yield ofPSC. Both ASC and PSC had a much higher Tmax, as compared tothose from temperate and cold-water fish. ASC and PSC with a pIof 6.21 and 6.56, respectively, showed high solubility in the acidic

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

Acknowledgements

The authors would like to express their sincere thanks to theGraduate School, Prince of Songkla University, TRF Senior ResearchScholar and National Research Council of Thailand for the financialsupport.

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