) and their role in properties of gelatin extracted has been reported. Unicorn leatherjacket is aneconomically important species commonly used for frozen ﬁlletproduction. Thickskin of this species, generated during deskinningprocess,canserveasthepromisingrawmaterialforgelatinextrac-tion. Therefore, the objectives of this investigation were to studythe autolysis of skin from unicorn leatherjacket and to elucidatetheir impact on gelatin extraction and characteristics of resultinggelatin.
2. Materials and methods
2,4,6-Trinitrobenzenesulfonic acid (TNBS), sodium sulphite,
-leucine, bovine serum albumin, ethylenediaminetetraacetic acid(EDTA), 1,10-phenanthroline monohydrate, ethylene-bis (oxyethy-lenenitrilo) tetraacetic acid (EGTA),
-leucyl-amido (4-guanidino) butane (E-64), iodoacetic acid, pepstatin A,phenylmethanesulfonyl ﬂuoride (PMSF), soybean trypsin inhibitor(SBTI),
-ME)andtypeIcollagenfromcalfskinwere obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fo-lin–Ciocalteu’s phenol reagent and phosphoric acid were pur-chased from Merck (Darmstadt, Germany). Sodium dodecylsulphate (SDS),
-tetramethyl ethylene diamine (TEMED)and Coomassie blue R-250 were procured from Bio-Rad Laborato-ries (Hercules, CA, USA). All chemicals were of analytical grade.
2.2. Collection and preparation of ﬁsh skin
The skin of unicorn leatherjacket (
) was obtainedfrom Sea Wealth Frozen Food Co., Ltd., Songkhla, Thailand. Uponarrival to the Department of Food Technology, Prince of SongklaUniversity, Hat Yai, the skin was cleaned and washed with icedtap water (0–2
C). Prepared skin was then cut into small pieces(0.5
), placed in polyethylene bags and stored at
Cuntil use. The storage time was less than 2months.
2.3. Pretreatment of unicorn leatherjacket skin
Prepared skin was pretreated following the method of Ahmadand Benjakul (2010a), with a slight modiﬁcation. To remove non-collagenous proteins, the prepared skin was mixed with 0.1MNaOH at a skin/alkali solution ratio of 1:20 (w/v). The mixturewas stirred for 6h at 4
C using an overhead mechanical stirrer(W20.n,IKA
-WerkeGmbH&CO.KG,Stanfen,Germany)ataspeedof300rpm.Thealkalisolutionwaschangedevery2h.Thesampleswerethenwashedwithicedtapwateruntilneutral orfaintlybasicpH was obtained. Thereafter, the prepared skin was swollen bymixing the skins with 0.2M phosphoric acid at a ratio of 1:10(w/v). The mixture was stirred for 24h at 4
C. Finally, the swollenskin was washed thoroughly with iced tap water until neutral orfaintly acidic pH of wash water was obtained. The pretreated skinwas stored at
C until use.
2.4. Autolysis study of pretreated unicorn leatherjacket skin
Prior to study, the frozen pretreated skin was powderised in li-quid nitrogen using a blender (Model MX-T2GN, National, Taipei,Taiwan). The powder obtained was used for autolysis study.
2.4.1. Temperature proﬁle
Autolytic activity was assayed according to the method of Morrissey, Wu, Lin, and An (1993),with a slight modiﬁcation. Pre-treated skin powder (3g) was added to 9ml of McIlvaine’s buffer(0.2M Na-phosphate and 0.1M Na-citrate), pH 7.0. The mixturewas homogenised for 2min at a speed of 11,000rpm using ahomogeniser(ModelT25basic,IKA,Labortechnik,Selangor,Malay-sia). The resulting homogenate was incubated at different temper-atures (40, 45, 50, 55, 60, 70, 80 and 90
C) for 30min in atemperature-controlled water bath (Memmert, Schwabach, Ger-many). Autolytic reaction was terminated by addition of 18ml of 10% SDS (85
C). The reaction mixture was further incubated at85
C for 1h in a temperature-controlled water bath. The superna-tant was obtained by centrifuging at 8500
for 15min using amicrocentrifuge (Model MIKRO20, Hettich Zentrifugen, Germany)and was subjected to sodium dodecyl sulphate–polyacrylamidegel electrophoresis (SDS–PAGE) analysis. Samples were also deter-mined for free amino group content.
2.4.2. pH proﬁle
Autolytic activity of pretreated skin was determined at variouspHs using different buffers at 50
C for 30min. Buffers used in-cluded 50mM maleate buffer for pH 1; McIlvaine’s buffer (0.2MNa-phosphate and 0.1M Na-citrate) for pHs 2–6, 50mM Tris–HClbuffer for pHs 7–9 and 0.1M Na
buffer for pH 10.After the incubation at 50
C for 30min, the autolysis was termi-nated and was monitored in the same manner as the temperatureproﬁle study.
2.4.3. Inhibitor study
Pretreated skin powder (0.5g) was homogenised with 1.5ml of McIlvaine’sbuffer(pH7)ataspeedof11,000rpmfor2minusingahomogeniser. The homogenate was mixed with 2ml of variousprotease inhibitors to obtain the different ﬁnal concentrations.Those inhibitors included EDTA (10mM), EGTA (10mM), iodoace-tic acid (10mM), E-64 (1mM), pepstatin A (1mM) and 1,10-phe-nanthroline (10mM), PMSF (1mM) and SBTI (0.04mM). Themixtures were allowed to stand in ice for 2h, followed by incuba-tion at optimal temperature (50
C) for 30min. The reaction wasterminatedbyadditionof2mlof10%(w/v)SDS(85
C).Aftercom-plete solubilisation, the autolytic pattern was then analysed byusing SDS–PAGE. The control was performed in the same manner,but deionised water was added instead of protease inhibitorsolution.
2.4.4. Determination of free amino group content
l) were mixed thoroughly with 2.0ml of 0.2125M phos-phate buffer, pH 8.2, followed by the addition of 1.0ml of 0.01%TNBS solution. The mixtures were then placed in a temperaturecontrolled water bath at 50
C for 30min in the dark. The reactionwas terminated by adding 2.0ml of 0.1M sodium sulphite. Themixtures were cooled down at room temperature for 15min. Theabsorbancewasmeasuredat420nmusingadoublebeamspectro-photometer (Model UV-1800, Shimadzu, Kyoto, Japan) and freeamino group content was expressed in terms of
2.4.5. SDS–polyacrylamide gel electrophoresis (SDS–PAGE)
ProteinpatternsweredeterminedusingSDS–PAGEaccordingtothe method of Laemmli (1970)using a 4% stacking gel and a 7.5%separatinggel.Samplesweremixedat1:1(v/v)ratiowiththesam-plebuffer(0.5MTris–HCl,pH6.8,containing4%SDS,20%glycerol).Samples (15
g) were loaded onto the gel. After electrophoresisusing 15mA/gel (Mini Protein II, Bio-Rad, Hercules, CA, USA), thegel 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. Type I collagen was usedas the standard.
M. Ahmad et al./Food Chemistry xxx (2011) xxx–xxx
Please cite this article in press as: Ahmad, M., et al. Indigenous proteases in the skin of unicorn leatherjacket (
) and their inﬂuence oncharacteristic and functional properties of gelatin.