Welcome to Scribd, the world's digital library. Read, publish, and share books and documents. See more
Download
Standard view
Full view
of .
Look up keyword
Like this
0Activity
0 of .
Results for:
No results containing your search query
P. 1
p4-

p4-

Ratings: (0)|Views: 15|Likes:
Published by Mehraj Ahmad

More info:

Published by: Mehraj Ahmad on Sep 16, 2011
Copyright:Attribution Non-commercial

Availability:

Read on Scribd mobile: iPhone, iPad and Android.
download as PDF, TXT or read online from Scribd
See more
See less

09/16/2011

pdf

text

original

 
Indigenous proteases in the skin of unicorn leatherjacket (
 Alutherus monoceros
)and their influence on characteristic and functional properties of gelatin
Mehraj Ahmad
a
, Soottawat Benjakul
a,
, Mahmoudreza Ovissipour
b
, Thummanoon Prodpran
c
a
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
b
Department of Fisheries, Gorgan Agricultural Sciences and Natural Resources, University of Gorgan, Iran
c
Department of Material Product 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 3 October 2010Receivedinrevisedform18November2010Accepted 7 January 2011Available online xxxx
Keywords:
Unicorn leatherjacket skinProteolysisSerine proteaseSBTIGelatin
a b s t r a c t
Indigenous proteases in the skin of unicorn leatherjacket (
 Alutherus monoceros
) were characterised usingautolytic study. Maximised autolysis was found at pH 7 and 50
°
C. Autolysis was markedly inhibited by0.04mM soybean trypsin inhibitor (SBTI), suggesting that heat activated serine protease was predomi-nant in the skin. The impact of indigenous proteases on the properties of gelatin extracted from unicornleatherjacket skin was investigated. Gelatin was extracted fromunicorn leatherjacket skin using distilledwater at 50
°
C for 12h in the presence and absence of 0.04mM SBTI. In the presence of SBTI, the degra-dationwasmarkedlyinhibited,butalowergelatinextractionyieldwasobtained(
<0.05).Extractedgel-atins contained
a
1
and
a
2
chains as the predominant components with some degradation peptides. FTIR spectra indicated a greater loss of molecular order of the triple helix and a higher degradation was foundingelatinextractedintheabsenceof 0.04mMSBTI. Thenetchargeof gelatinsamples extractedwithandwithout 0.04mM SBTI became zero at pHs of 8.45 and 7.31, respectively, as determined by
f
-potentialtitration. Higher gel strength (320.68±3.02g) was obtained in gelatin extracted with SBTI, comparedwith that of gelatin extracted without SBTI (288.63±1.44g). High emulsifying activity index but loweremulsifying stability index was observed in the former. Therefore, heat-activated serine protease wasinvolved in the degradation of gelatin molecules, thereby affecting the yield, proteinaceous componentsand properties of gelatin from unicorn leatherjacket skin.
Ó
2011 Elsevier Ltd. All rights reserved.
1. Introduction
Proteolysis induced by heat-activated and heat-stable indige-nous proteases associated with skin matrix can contribute to thedestabilisation as well as disintegration of collagen structure bydisrupting the intra- and intermolecular cross-links (Wu et al.,2008). Collagenases have the unique ability to degrade the majorstructuralextracellularmatrixproteins,especiallythetriplehelicalstrand of type I and type II collagens at Gly
775
-Leu (Ile)
776
. Afterthese initial and specific cleavages,
3
4
- and
1
4
-cleavage productsare formed, and collagen is spontaneously denatured (helix-to-coiltransition) to gelatin (Miller et al., 1976). Collagenases are classi-fied into two major groups, metallocollagenases and serine colla-genases (Aoki, Ahsan, Matsuo, Hagiwara, & Watabe, 2003).Furthermore, non-collagenase proteinases can cleave the collagenmolecule in the telopeptide region and contribute to hydrolysisof the collagen molecule by disrupting the regions, in which inter-molecular cross-links are formed (Bornstein & Traus, 1979).Heat-activated serine protease in bigeye snapper skin wasinvolved in the drastic degradation of the
b
- and
a
-chains of thegelatin extracted at 60
°
C(Intarasirisawat et al., 2007). These en- zymes are bound with matrix components such as collagens(Woessner, 1991). The proteolytic degradation of high molecularweightcomponentscausedbyindigenousproteasesduringextrac-tion of gelatin at high temperature resulted in adverse effects ongel-forming properties of resulting gelatin (Intarasirisawat et al.,2007). The proteolytic breakdown of collagen structure is mostlikely related to the disintegration of connective tissues, whichhas been implicated in quality deterioration of products. Gelatinstructures with large amount of high molecular weight compo-nents, like
c
-,
b
, and
a
-chains, have been known to possess themaximalfunctionalpropertiesincludinggelation(Kittiphattanaba-won, Benjakul, Visessanguan, &Shahidi, 2010), filmformingability(Hoque, Benjakul, & Prodpran, 2010), etc. Therefore, it was neces-sary to produce the gelatin with negligible hydrolysis of peptidesfor further applications in pharmacy, medicine and food industry.Hence, the use of an appropriate protease inhibitor can be aneffective means to obtain the gelatin with limited or negligiblehydrolysis of peptides. Nevertheless, no information regardingthe characteristics of indigenous proteases in the skin of unicorn
0308-8146/$ - see front matter
Ó
2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodchem.2011.01.032
Corresponding author. Tel.: +66 7428 6334; fax: +66 7455 8866.
E-mail address:
Contents lists available atScienceDirect
Food Chemistry
Please cite this article in press as: Ahmad, M., et al. Indigenous proteases in the skin of unicorn leatherjacket (
 Alutherus monoceros
) and their influence oncharacteristic and functional properties of gelatin.
Food Chemistry
 
leatherjacket (
 Alutherus monoceros
) 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
 2.1. Chemicals
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),
trans
-epoxysuccinyl-
-leucyl-amido (4-guanidino) butane (E-64), iodoacetic acid, pepstatin A,phenylmethanesulfonyl fluoride (PMSF), soybean trypsin inhibitor(SBTI),
b
-mercaptoethanol(
b
-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),
,
,
0
,
0
-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 (
 A. monoceros
) 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
Â
0.5cm
2
), placed in polyethylene bags and stored at
À
20
°
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
Ò
-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
À
20
°
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
 g 
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
2
HPO
4
–Na
2
B
4
O
7
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 
Free amino group content was determined following the meth-od of Benjakul and Morrissey (1997). Properly diluted samples(125
l
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 
-leucine.
 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
l
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.
2
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 (
 Alutherus monoceros
) and their influence oncharacteristic and functional properties of gelatin.
Food Chemistry
 
 2.5. Study on the impact of indigenous proteases on extraction andcharacteristics of gelatin from unicorn leatherjacket skin
To inhibit indigenous serine proteases, the pretreated skin wasmixed with 0.04mM SBTI solution at a pretreated skin/SBTI solu-tion ratio of 1:5 (w/v). The mixture was stirred for 24h at 4
°
Cusing an overhead mechanical stirrer at a speed of 150rpm. Then,the mixtures in the absence and presence of 0.04mM SBTI wereincubated at 50
°
C in a temperature-controlled water bath for12h with continuous stirring to extract the gelatin. The mixtureswere then filtered using two layers of cheesecloth. The filtrateswere further filtered using a Whatman No. 4 filter paper (What-man International, Ltd., Maidstone, England) with the aid of anelectric aspirator, (Model VE-11, JEIO TECH, Korea). The resultantfiltrate was freeze-dried using a Scanvac Model Coolsafe 55 freezedryer (Coolsafe, Lynge, Denmark). Gelatins extracted in the ab-sence and presence of 0.04mM SBTI were referred to as GAI andGPI, respectively. Both gelatin samples were subjected to SDSPAGE as previously described and further analyses were also con-ducted as follows:
 2.5.1. Fourier transform infrared spectroscopy (FTIR)
FTIR analysis was performed as per the method of Ahmad,Benjakul, and Nalinanon (2010). A Bruker Model EQUINOX 55 FTIR spectrometer (Bruker, Ettlingen, Germany) equipped with a deu-terated
-alanine triglycine sulphate (DLATGS) detector was used.The horizontal attenuated total reflectance (HATR) accessory wasmounted into the sample compartment. The internal reflectioncrystal (Pike Technologies, Madison, WI, USA), made of zinc sele-nide, had a 45
°
angle of incidence to the IR beam. Spectra were ac-quired at a resolution of 4cm
À
1
and the measurement range was4000–650cm
À
1
(mid-IR region) at room temperature. Automaticsignals were collected in 32 scans at a resolution of 4cm
À
1
andwere normalised against a background spectrum recorded fromthe clean, empty cell at 25
°
C. Analysis of spectral data was carriedout using the OPUS 3.0 data collection software programme (Bru-ker, Ettlingen, Germany).
 2.5.2.
f
-Potential analysis
Gelatin samples were dissolved in distilled water at a concen-tration of 0.5mg/ml. The mixtures were stirred at room tempera-ture for 6h. The
f
-potential of each sample (20ml) wasmeasured using a
f
-potential analyser (ZetaPALS, BrookhavenInstruements Co., Holtsville, NY, USA).
f
-Potential of samples, ad- justed to different pHs with 1.0M nitric acid or 1.0M KOH usingan autotitrator (BI-ZTU, Brookhaven Instruments Co., Holtsville,New York, USA), was determined. The pI was estimated from pHrendering
f
-potential of zero.
 2.5.3. Determination of gel strength
Gels of gelatin were prepared by the method of Fernandez-Daz,Montero, and Go
9
mez-Guille˙n (2001), with a slight modification.Gelatin samples were dissolved in distilled water (60
°
C) to obtainthe final concentrationof 6.67%(w/v). The solutionwas stirred un-til the gelatin was solubilised completely, followed by incubatingthe samples in a refrigerator at 10
°
C for 18h for gel maturation.The dimensions of the sample were 3cm in diameter and 2.5cmin height. The gel strength of the samples prepared at 10
°
C wasdetermined using a Model TA-XT2 Texture Analyser (Stable MicroSystem, Surrey, UK) with a load cell of 5kN and equipped with a1.27cm diameter flat-faced cylindrical Teflon
Ò
plunger. The maxi-mumforce(ingrams)wasrecordedwhenthepenetrationdistancereached 4mm. The speed of the plunger was 0.5mm/s.
 2.5.4. Determination of gel microstructure
Microstructures of gelatingelswerevisualisedusinga scanningelectron microscope (SEM). Gelatin gels having a thickness of 2–3mm were fixed with 2.5% (v/v) glutaraldehyde in 0.2Mphosphate buffer (pH 7.2) for 12h. The samples were then rinsedwithdistilledwater for 1handdehydratedin ethanol witha serialconcentration of 50%, 70%, 80%, 90% and 100% (v/v). Dried sampleswere mounted on a bronze stub and sputter-coated with gold(SputtercoaterSPI-Module,WestChester,PA,USA).Thespecimenswere observed with a scanning electron microscope (JEOL JSM-5800 LV, Tokyo, Japan) at an acceleration voltage of 15kV.
 2.5.5. Determination of emulsifying properties
Emulsion activity index (EAI) and emulsion stability index (ESI)of gelatin samples were determined according to the method of Pearce and Kinsella (1978)as modified byAewsiri, Benjakul, andVisessanguan (2009). Soy bean oil (2ml) and gelatin solution (1%gelatin, 6ml) were homogenised using a homogeniser at a speedof 20,000rpm for 1min. Emulsions were pipetted out at 0 and10minand100-folddilutedwith0.1%SDS.Themixturewasmixedthoroughly for 10s using a vortex mixer (Scientific Industries, Inc.,Bohemia, NY, USA).
A
500
of the resulting dispersion was measuredusing a spectrophotometer. EAI and ESI were calculated by the fol-lowing equation:
EAI
ð
m
2
=
g
Þ¼ð
2
Â
2
:
303
Â
 A
Â
DF
Þ
=
l
ø
where
A
=
 A
500
, DF=dilution factor (100),
l
=path length of cuvette(m), ø=oil volume fraction and
=protein concentration in aque-ous phase (g/m
3
)
ESI
ð
min
Þ¼
 A
0
=
ð
 A
0
À
 A
10
ÞÂ
D
where
A
0
=
 A
500
at time of 0min;
A
10
=
 A
500
at time of 10min and
D
=10min.
 2.6. Statistical analyses
All experiments were performed in triplicate and a completelyrandomised design (CRD) was used. Data were presented asmeans±standarddeviationanda probabilityvalue <0.05was con-sidered significant. Analysis of variance (ANOVA) was performedand the mean comparisons were done by
-test. Analysis was per-formed using SPSS 11.0 for Windows (SPSS Inc., Chicago, IL).
3. Results and discussion
 3.1. Effect of temperature and pH on autolysis of pretreated skin
The autolytic degradation of pretreated skin at different tem-peratures, as monitored by free amino group content, is shown inFig. 1a. The maximal autolytic activity of pretreated skin was ob-served at 50
°
C as indicated by the highest free amino group con-tent (
P <
0.05). However, a decrease in autolytic activity wasfoundwhenthetemperaturewasabove50
°
C, suggestingthether-mal denaturation of the indigenous proteases. When the proteinpatterns of pretreated skin, autolysed at different temperatures,were compared, the highest degradation of 
b
-chain and
a
-chainswas obtained at 50
°
C(Fig. 1c). This was concomitant with the for- mation of degradation products with low molecular weightappearingatthedyefront.Furthermore,thehighmolecularweightcross-linked proteins were also degraded at 50–70
°
C.
b
- and
a
-chains constituted as the major proteins in the collagen from uni-corn leatherjacket skin (Ahmad & Benjakul, 2010a). The resultssuggested that the proteases still remained within the skin afterpretreatment with alkali and acid for non-collagenous protein re-moval and swelling processes, respectively. Those heat-activated
M. Ahmad et al./Food Chemistry xxx (2011) xxx–xxx
3
Please cite this article in press as: Ahmad, M., et al. Indigenous proteases in the skin of unicorn leatherjacket (
 Alutherus monoceros
) and their influence oncharacteristic and functional properties of gelatin.
Food Chemistry

You're Reading a Free Preview

Download
scribd
/*********** DO NOT ALTER ANYTHING BELOW THIS LINE ! ************/ var s_code=s.t();if(s_code)document.write(s_code)//-->