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%
-ME. The mixtures were boiledin boiling water for 2 min. Samples (15
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
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
with automatic signalgain were collected in 32 scans at a resolution of 4 cm
and werecompared against a background spectrum recorded from the cleanempty cell at 25
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 (
) was estimated by measur-ing the area in the DSC thermogram. The maximum transitiontemperature (
) 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
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
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
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