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Extraction and characterisation of pepsin-solubilised collagen from the skinof unicorn leatherjacket (
 Aluterus monocerous
)
Mehraj Ahmad, Soottawat Benjakul
*
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
a r t i c l e i n f o
 Article history:
Received 30 June 2009Received in revised form 19 October 2009Accepted 9 November 2009
Keywords:
PSCUnicorn leatherjacketFTIR ViscosityDenaturation temperature
a b s t r a c t
Pepsin-solubilised collagen (PSC) was extracted from the skin of unicorn leatherjacket (
 Aluterus monocer-ous
), using 0.5 M acetic acid in the presence of pepsin from albacore tuna, yellowfin tuna or porcine pep-sin at a level of 20 units/g of defatted skin. Yields of 8.48 ± 0.3%, 8.40 ± 0.3% and 7.56 ± 0.4% (wet weightbasis) were obtained for PSC extracted with the aid of albacore tuna pepsin (APSC), yellowfin tuna pepsin(YPSC) and porcine pepsin (PPSC), respectively. All PSCs were classified as Type I collagen containing two
a
1
-chains and one
a
2
-chain with no disulphide bond. The peptide maps of different PSCs hydrolysed byV8 protease and lysyl endopeptidase were different. ATR-FTIR spectra analysis revealed that PSC mole-cules had the compact triple helical structure stabilised mainly by the hydrogen bond.
max
of all PSCs(31.68–31.98
°
C) shifted to lower values (29.33–29.40
°
C) when dispersed in 0.05 M acetic acid, indicat-ing the conformational changes in the collagen structure induced by acid. Relative viscosity of 0.03% PSCin 0.1 M acetic acid solution decreased progressively as the temperature increased from 4 to 52
°
C, indi-cating thermal destabilisation or denaturation of PSC molecules. All PSCs were soluble in the pH range of 1–6 and sharply decreased at neutral pH. Decreases in solubility were observed in the presence of NaCl,especially at concentrations above 2% (w/v). Therefore, the skin of unicorn leatherjacket could serve as apotential source of collagen.
Ó
2009 Published by Elsevier Ltd.
1. Introduction
Collagen is the predominant protein of connective tissue in ani-mals, making up about 30% of the total protein content (Birk &Bruckner, 2005). It possesses a unique protein sequence and is ableto form insoluble fibres with a high tensile strength (Gelse, Poschl,& Aigner, 2003). Collagen has a wide range of applications in vari-ous branches of industry. Its pharmaceutical and biomedical usesinclude tissue engineering for implants in humans, inhibition of angiogenic diseases, treatment of hypertension, urinary inconti-nence and osteoarthritis (Lee, Singla, & Lee, 2001). It is also a veryattractive substrate for fragrance and cosmetic applications(Tzaphlidou, 2004). Its denatured form (gelatin) has been widelyused in the food industry (Kittiphattanabawon, Benjakul, Visessan-guan, Nagai, & Tanaka, 2005). All members of the collagen familyare characterised by domains with repetitions of the proline-richtripeptide Gly-X-Y, involved in the formation of trimeric collagentriple helix (Muyonga, Cole, & Duodu, 2004; Ramachandran, 1988).In general, the traditional sources of collagen are bovine andporcine skins and bones. Porcine collagen is unacceptable for somereligions, for example Judaism and Islam, while those from bovinesources are at risk of contamination with bovine spongiformencephalopathy (BSE) (Fernandez-Diaz, Montero, & Gomez-Guil-len, 2001).With the rapid development of seafood processing industries,huge quantities of by-products have been discarded, which maycause pollution and emit offensive odours. Hence, the comprehen-sive utilisation of these by-products, especially the production of value-added products, is a promising means to increase revenuefor the producers and accelerate the development of the seafoodprocessing industry. Fish skin is an important source for collagen,which can be used as a replacement for mammalian sources. Ingeneral, a low yield of collagen is obtained with traditional pro-cesses. Sincepepsin has been reported to cleavepeptides in the tel-opeptide region, the extraction of collagen partially cleaved bypepsin resulted in a higher yield ( Jongjareonrak, Benjakul, Vises-sanguan, Nagai, & Tanaka, 2005; Nalinanon, Benjakul, Visessan-guan, & Kishimura, 2007a). Due to the enormous amount of fishviscera, especially stomach, pepsin of fish origin can be recoveredand used to increase the extraction efficiency of collagen from fishskin (Nalinanon, Benjakul, Visessanguan, & Kishimura, 2007b).Unicorn leatherjacket (
 Aluterus monocerous
) belongs to the or-der Tetraodontiformes and is a member of the Monacanthidaefamily. This species has been used for fillet production, in whicha large amount of skin has been produced as by-product. Due to
0308-8146/$ - see front matter
Ó
2009 Published by Elsevier Ltd.doi:10.1016/j.foodchem.2009.11.019
*
Corresponding author. Tel.: +66 7428 6334; fax: +66 7421 2889.
E-mail address:
soottawat.b@psu.ac.th(S. Benjakul).Food Chemistry 120 (2010) 817–824
Contents lists available atScienceDirect
Food Chemistry
 
its thick skin, it is therefore a potential source of collagen, espe-cially when fish pepsin is implemented as the extraction aid. Theobjective of this study was to isolate and characterise the pepsin-solubilised collagen (PSC) from the skin of unicorn leatherjacket(
 A. monocerous
), with the aid of different pepsins including tunaand porcine pepsins.
2. Materials and methods
 2.1. Chemicals
Bovine haemoglobin,
b
-mercaptoethanol (
b
-ME), V8 proteasefrom
Staphylococcus aureus
(EC3.4.21.19),
-tyrosine, bovine serumalbumin, pepsin from porcine stomach mucosa (EC3.4.23.1; pow-derised, 750 U/mg dry matter), and Type I collagen from calf skinwere purchased from Sigma Chemical Co. (St. Louis, MO). Highmolecular weight markers were purchased from GE HealthcareUK (Aylesbury, UK). Sodium dodecyl sulphate (SDS), trichloroaceticacid, Folin–Ciocalteu’s phenol reagent, acetic acid and tris(hydrox-ylmethyl)aminomethane were obtained from Merck (Darmstadt,Germany). Lysyl endopeptidase from
Achromobacter lyticus
(EC3.4.21.50) was procured from Wako Pure Chemical Industries,Ltd. (Tokyo, Japan).
 2.2. Fish skin and stomach preparation
The skin of unicorn leatherjacket (
 A. monocerous
) was obtainedfrom Sea Wealth Frozen Food Co., Ltd., Songkhla, Thailand. Stom-achs of albacore tuna (
Thunnus alalunga
) and yellowfin tuna (
Thun-nus albacares
) were obtained from Tropical Canning Public Co., Ltd.,Songkhla, Thailand. The samples were packed in polythene bagsand kept in ice using a solid/ice ratio of 1:2 (w/w) and transportedto the Department of Food Technology, Prince of Songkla Univer-sity, Hat Yai within 1 h. The skin was removed manually and thecleaned skin was washed with iced tap water (0–2
°
C). Preparedskin was then cut into small pieces (0.5
Â
0.5 cm
2
). Both preparedskin and stomachs were placed in polyethylene bags and stored at
À
20
°
C until use. The storage time was less than 2 months.
 2.3. Preparation of stomach extract 
The frozen stomachs of both types of tuna were thawed usingrunning water (26–28
°
C) until the core temperature reached
À
2to0
°
C.Thesamplewascutintopieces(1
Â
1 cm
2
)andfinelygroundinliquidnitrogenusingaNationalModelMX-T2GNblender(Taipei,Taiwan),accordingtothemethodof Klomklao,Benjakul,andVises-sanguan(2004).Stomachpowderwassuspendedin50 mMsodiumphosphate buffer (pH 7.2) at a ratio of 1:10 (w/v). The mixture wasstirred continuously at 4
°
C for 30 min. The suspension was centri-fuged at 7700
 g 
for 30 min at 4
°
C using a Beckman Model Avanti J-Ecentrifuge(BeckmanCoulter,Inc.,Fullerton,CA)toremovethetis-sue debris. The supernatant was collected and referred to as ‘stom-ach extract’. The protein content in the stomach extract wasmeasuredasperthemethodof Lowry,Rosebrough,Farr,andRandall(1951), using bovine serum albumin as a standard.
 2.4. Assay of proteolytic activity
Prior to assay, the stomach extracts from albacore tuna or yel-lowfin tuna or porcine pepsin were adjusted to pH 2 with 1 MHCl and the mixtures were allowed to stand at 4
°
C for 10 min,as described byNalinanon et al. (2007a). The treated stomach ex-tracts were centrifuged at 5000
 g 
for 10 min at 4
°
C, using a refrig-erated centrifuge. The acidified supernatants were collected andused as the sources of activated pepsin.Proteolytic activity of acidified stomach extract from both tunaand pepsin powder from porcine stomach mucosa was determinedusing haemoglobin as a substrate, as per the method of Nalinanonet al. (2007a), with a slight modification. To initiate the reaction,200
l
l of stomach extract were added into the assay mixture con-taining 200
l
l of 2% haemoglobin, 200
l
l of distilled water and625
l
l of McIlvaine’s buffer (0.2 M Na-phosphate and 0.1 M Na cit-rate, pH 2.0). Appropriate dilution was made to ensure that theamount of enzyme was not excessive for the available substratein the assay system. The reaction was conducted at 50
°
C for20 min. To terminate the enzymatic reaction, 200
l
l of 50% (w/v)trichloroacetic acid (TCA) were added. Unhydrolysed protein sub-strate was allowed to precipitate for 15 min at 4
°
C, followed bycentrifuging at 4725
 g 
for 10 min at room temperature (26–28
°
C)using a MIKRO 20 centrifuge (Hettich Zentrifugen Tuttlingen, Ger-many). The oligopeptide content in the supernatant was measuredby the Lowry method (Lowry et al., 1951), using tyrosine as a stan-dard. One unit of activity was defined as the amount releasing1
l
mol of tyrosine per min. A blank was performed in the samemanner, except that the acidified stomach extract was added intothe reaction mixture after the addition of 50% TCA (w/v).
 2.5. Extraction of pepsin-solubilised collagen (PSC)
All procedures were carried out at 4
°
C with gentle stirring. Thecollagen from the skin of unicorn leatherjacket was extracted fol-lowing the method of Nalinanon et al. (2007b)with slight modifi-cation. Skin was soaked in 0.1 M NaOH with a skin/solution ratio of 1:20 (w/v), with continuous stirring to remove non-collagenousproteins for 6 h. The solution was changed every 2 h. The alka-line-treated skin was then washed with cold water until the pHof the wash water became neutral or faintly basic. Residual fat inthe skin was removed using 10% (v/v) butyl alcohol with a sam-ple/solution ratio of 1:10 for 18 h with a change of solution every6 h. Defatted skin was thoroughly washed with 15 volumes of coldwater (4–5
°
C).To extract PSC, the prepared skin was soaked in 0.5 M aceticacid with a solid/solution ratio of 1:15 (w/v) for 48 h in the pres-ence of acidified stomach extracts or acidified porcine pepsin at alevel of 20 units/g of defatted skin. The mixture was filtered withtwo layers of cheesecloth to remove undissolved debris. The fil-trate was added with NaCl to obtain a final concentration of 2.6 M in the presence of 0.05 M tris(hydroxymethyl) aminometh-ane (pH 7.0). The resultant precipitate was collected by centrifug-ing at 20,000
 g 
for 60 min at 4
°
C. The pellets were dissolved in 10volumes of 0.5 M acetic acid and dialysed against 9 volumes of 0.1 M acetic acid and distilled water, respectively. The dialysatewas finally freeze-dried using a Model Coolsafe 55, Scanvac, (Cool-safe, Lynge, Denmark). The yield of PSC was calculated and ex-pressed as dry matter/wet weight of skin. PSC was thensubjected to analysis.
 2.6. Amino acid analysis
PSC samples were hydrolysed under reduced pressure in 4 Mmethanesulfonic acid containing 0.2% (v/v) 3-2(2-amino-ethyl)indole at 115
°
C for 24 h. The hydrolysates were neutralisedwith 3.5 M NaOH and diluted with 0.2 M citrate buffer (pH 2.2).An aliquot of 0.4 ml was applied to an amino acid analyser (MLC-703; Atto Co., Tokyo, Japan).
 2.7. SDS–polyacrylamide gel electrophoresis (SDS–PAGE)
SDS–PAGE was performed by the method of Laemmli (1970).The PSC samples were dissolved in 5% SDS and the mixtures wereincubated at 85
°
C for 1 h. The mixture was centrifuged at 4000
 g 
818
M. Ahmad, S. Benjakul/Food Chemistry 120 (2010) 817–824
 
for 5 min at room temperature to remove undissolved debris. Sol-ubilised samples were mixed at 1:1 (v/v) ratio with the samplebuffer (0.5 M Tris–HCl, pH 6.8, containing 4% SDS and 20% glycerol)in the presence or absence of 10%
b
-ME. The mixtures were boiledin boiling water for 2 min. Samples (15
l
g protein) were loadedonto polyacrylamide gels comprising a 7.5% running gel and a 4%stacking gel and subjected to electrophoresis at a constant currentof 15 mA/gel, using a Mini Protein II unit (Bio-Rad Laboratories,Inc., Hercules, CA). After electrophoresis, gel was stained with0.05% (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 esti-mate the molecular weight of proteins. Type I calf skin collagenwas used as a standard.
 2.8. Peptide mapping of collagen
PeptidemappingofPSCwasperformedaccordingtothemethodof Saito, Kunisaki, Urano, and Kimura (2002)with slight modifica-tion. The sample (0.2 mg) were dissolved in 0.1 ml of 0.1 M sodiumphosphate (pH 7.2) containing 0.5% (w/v) SDS. After the addition of 10
l
lofthesamplebuffercontaining5
l
gofV8proteasefrom
S.aur-eus
or0.05
l
goflysylendopeptidasefrom
 A.lyticus
tocollagensolu-tions, the reaction mixture was incubated at 37
°
C for 25 and 5 minfor V8 protease and lysyl endopeptidase, respectively. The reactionwasterminatedbysubmergingthereactionmixtureinboilingwaterfor 3 min.Peptidesgeneratedbytheproteasedigestionweredeter-mined by SDS–PAGE using 7.5% separating gel and 4% stacking gel.High molecular weight markers (GE Healthcare UK) were used toestimate the molecular weight of peptides.
 2.9. ATR-FTIR analysis
PSC samples from the skin of unicorn leatherjacket were sub- jected to attenuate total reflectance/Fourier-transform infraredspectroscopy (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 and4 mm thick) (PIKE Technologies Inc., Madison, WI) was used. Forspectral analysis, the PSC samples were placed onto the crystal celland the cell was clamped into the mount of the FTIR spectrometer.The spectra in the range of 600–4000 cm
À
1
with automatic signalgain were collected in 32 scans at a resolution of 4 cm
À
1
and werecompared against a background spectrum recorded from the cleanempty cell at 25
°
C.
 2.10. Differential scanning calorimetry
Differential scanning calorimetry (DSC) of the PSC samples wasrunfollowingthemethodof Rochdi,Foucat,and Renou(2000)withslight 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). Temperature calibra-tion was run using the Indium thermogram. 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 (
D
) was estimated by measur-ing the area in the DSC thermogram. The maximum transitiontemperature (
max
) was estimated from the thermogram.
 2.11. Viscosity of collagen solution
Collagen was dissolved in 0.1 M acetic acid to obtain a concen-tration of 0.03% (w/v). The solution (500 ml) was subjected to vis-cosity measurement using a Brookfield Synchrolectic viscometer(model DVII+, Brookfield Eng. Labs Inc., Stoughton, MA) with spin-dle No. 1 and speed of 100 rpm. Collagen solution was heated from4 to 52
°
C with a heating rate of 4
°
C/min. At the designated tem-perature, the solution was held for 30 min prior to viscosity deter-mination. The relative viscosity was calculated in comparison tothat obtained at 4
°
C.
 2.12. Collagen solubility
The collagen solubility was determined by the method of Mon-tero, Jimenez-Colmenero, and Borderias (1991)with slight modifi-cation. Collagen was dissolved in 0.5 M acetic acid to obtain a finalconcentration of 3 mg/ml and the mixture was stirred at 4
°
C untilcollagen was completely solubilised.
 2.12.1. Effect of pH on collagen solubility
Collagensolution(8 ml)wastransferredtoacentrifugetubeandthepHwasadjustedwitheither6 NNaOHor6 NHCltoobtainafinalpH ranging from 1 to 10. The volume of solutions was made up to10 ml by deionised water previously adjusted to the same pH asthe collagen solution. The solution was centrifuged at 20,000
 g 
for30 min at 4
°
C. Protein content in the supernatant was measuredby the Lowry method(Lowryet al., 1951) using bovine serumalbu-min as a standard. Relative solubility was calculated in comparisonto that obtained at the pH rendering the highest solubility.
 2.12.2. Effect of NaCl on collagen solubility
Collagen (6 mg/ml) was dissolved in 0.5 M acetic acid. Solution(5 ml) was added with 5 ml of NaCl in 0.5 M acetic acid at variousconcentrations (0%, 2%, 4%, 6%, 8%, 10% and 12% (w/v)), in whichthe final concentrations of 0%, 1%, 2%, 3%, 4%, 5% and 6% were ob-tained. The mixture was stirred continuously at 4
°
C for 30 min,followed by centrifuging at 20,000
 g 
for 30 min at 4
°
C. Protein con-tent in the supernatant was measured and the relative solubilitywas calculated as previously described.
 2.13. Statistical analyses
All experiments were performed in triplicate and a completelyrandomised design (CRD) was used. Data were presented asmeans ± standard deviation and a probability value of <0.05 wasconsidered significant. Analysis of variance (ANOVA) was per-formed and mean comparisons were done by Duncan’s multiplerange tests. Analysis was performed using SPSS 11.0 for Windows(SPSS Inc., Chicago, IL).
3. Results and discussion
 3.1. Yields of PSC from the skin of unicorn leatherjacket 
The yields of APSC and YPSC extracted with the aid of albacoretuna and yellowfin tuna stomach extract were 8.48 ± 0.3% and8.40 ± 0.3% (wet weight basis), respectively. A lower yield(7.56 ± 0.4%) was obtained for PPSC extracted with the aid of por-cine pepsin. Without pepsin, much lower yield (4.19 ± 0.4%) wasobtained. The skin was not completely solubilised in 0.5 M aceticacid. The dense covalent cross-linking through the condensationof aldehyde groups at the telopeptide regions of collagen chainsand the inter-molecular cross-linked collagen were not readily sol-ubilised by acetic acid ( Jongjareonrak et al., 2005). The results sug-
M. Ahmad, S. Benjakul/Food Chemistry 120 (2010) 817–824
819

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