research article characteristics of pepsin-solubilised...

Research ArticleCharacteristics of Pepsin-Solubilised Collagen fromthe Skin of Splendid Squid (Loligo formosana)

Phanat Kittiphattanabawon1 Sitthipong Nalinanon2

Soottawat Benjakul3 and Hideki Kishimura4

1Department of Food Science and Technology Faculty of Technology and Community Development Thaksin UniversityPhatthalung Campus Phatthalung 93210 Thailand2Faculty of Agro-Industry King Mongkutrsquos Institute of Technology Ladkrabang Ladkrabang Bangkok 10520 Thailand3Department of Food Technology Faculty of Agro-Industry Prince of Songkla University Hat Yai Songkhla 90112 Thailand4Laboratory of Marine Products and Food Science Research Faculty of Fisheries Sciences Hokkaido UniversityHakodate Hokkaido 041-8611 Japan

Correspondence should be addressed to Sitthipong Nalinanon sitthipongnakmitlacth

Received 12 October 2015 Accepted 7 December 2015

Academic Editor Murat Senturk

Copyright copy 2015 Phanat Kittiphattanabawon et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Pepsin-solubilised collagen from the skin of splendid squid (SC) was isolated partially purified by salt precipitation and dialysisprior to characterisation The yield of SC was 753 (dry weight basis) SC with high purity was obtained as shown by the distinctUV absorption peak at 232 nm and high hydroxyproline content Total sugar content of SC was 470 (dry weight basis) whichwas higher than that of collagen from calf skin (CC) (145 dry weight basis) (119875 lt 005) Based on SDS-PAGE and elution profileSC might contain the mixed types of collagen (type SQ-I and type SQ-II) in which 120572- and 120573-chains were the major componentsSC was rich in glycine and had high content of imino acids (189 residues1000 residues)The degradation induced by chymotrypsinand lysyl endopeptidase was more pronounced in CC compared with SC The maximum transition temperature (119879max) of SC was341∘C which was about 7∘C lower than that of CC Fourier transform infrared spectra revealed that the triple-helical structure ofSC was predominant with the copresence of carbohydrate moieties Therefore the skin of splendid squid a byproduct from squidprocessing can be an alternative source for collagen production

1 Introduction

Collagen is the fibrous protein that contributes to the uniquephysiological functions of connective tissues in skins ten-dons bones cartilages and so forth and is associated withtoughness of mammalian muscle [1ndash3]The structural unit ofcollagen is tropocollagen a rod-shaped protein consisting ofthree polypeptides units (called120572 chains) intertwined to forma triple-helical structure [4] Each polypeptide chain forms aleft-handed helix and consists of repeating triplets (Gly-119883-119884)n where 119883 and 119884 are with a high possibility proline orhydroxyproline [5]

Generally pig and cow skins and bones are the mainsources of collagen and gelatin The outbreak of bovinespongiform encephalopathy (BSE) has resulted in anxiety

among users of cattle gelatin Additionally the collagen andgelatin obtained from pig skin and bones cannot be useddue to the religious constraint [6] As a consequence theincreasing attention of alternative sources for replacementof mammalian collagen has been paid Seafood processingbyproducts particularly the skins are alternative sourcesfor collagen preparation Collagens from several fish andcephalopods such as unicorn leatherjacket [7] arabesquegreenling [8] brownbanded bamboo shark [2] cod [9] deep-sea redfish [10] bigeye snapper [11 12] black drum [5]sheephead seabream [5] octopus [13] and cuttlefish [14] havebeen isolated and characterised

Squids have become an important fishery product inThailand as well as other Southeast Asian countries and aremainly exported worldwide [15] From the squid processing

Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 482354 8 pageshttpdxdoiorg1011552015482354

2 Journal of Chemistry

the skin byproduct is generated in large quantity It has beenmainly used for fish meal or fertilizer with a low marketvalue Nagai et al [14] reported that skin waste materialfrom cuttlefish contained high amount of collagen Hencesquid skin can be served as an alternative source of collagenHowever no information regarding the collagen from theskin of splendid squid (Loligo formosana) has been reportedTherefore the objective of this study was to isolate andcharacterise the collagen from the skin of splendid squid (Lformosana) a byproduct from squid processing

2 Materials and Methods

21 Chemicals Bovine haemoglobin 120573-mercaptoethanol(120573ME) pepsin from porcine stomach mucosa (EC 34231)(750 unitsmg dry matter) and type I collagen from calf skinwere purchased from Sigma Chemical Co (St Louis MOUSA) Coomassie Blue R-250 and NNN1015840N1015840-tetramethylethylene diamine (TEMED) were procured from Bio-RadLaboratories (Hercules CA USA) Bovine serum albu-min was obtained from Fluka (Buchs Switzerland) High-molecular-weight protein marker was purchased from GEHealthcare UK Limited (Buckinghamshire UK) TOY-OPEARL CM-650M was purchased from Tosoh Corpo-ration (Tokyo Japan) 120572-Chymotrypsin from bovine pan-creas (EC 34211) lysyl endopeptidase (EC 342150) fromAchromobacter lyticus and type II III and V collagensfrom porcine cartilage skin and placenta respectively wereobtained from Wako Pure Chemical Industries Ltd (TokyoJapan)

22 Preparation of Splendid Squid Skin Splendid squid(Loligo formosana) were obtained from the dock in Samut-sakhonThailandThe squidwere packed in polyethylene bagkept in ice with a solidice ratio of 1 2 (wv) and transportedto the Faculty of Agro-Industry King Mongkutrsquos Instituteof Technology Ladkrabang Bangkok Thailand within 1 hAll procedures were performed at 0ndash4∘C Upon the arrivalthe squid skin was removed and washed with cold waterdrained and cut into small pieces (05 times 05 cm2) using thescissor To remove noncollagenous proteins the skin wasmixed with 01M NaOH using a samplealkaline solutionratio of 1 10 (wv) The mixture was stirred continuously for6 h The alkaline solution was changed every 2 h Then thealkaline treated skin was washed with cold water until neutralor faintly basic pH of wash water was obtained The treatedskin was then defatted with 10 (vv) butyl alcohol with asolidsolvent ratio of 1 10 (wv) for 18 h and the solvent waschanged every 6 h Defatted skin was then washed with 10volumes of cold water for 3 times prior to lyophilisation

23 Isolation of Collagen from the Skin of Splendid SquidPepsin-solubilised collagen from the skin of splendid squidwas isolated following the method of Nalinanon et al [12]with some modifications All procedures were performedat 4∘C To extract the collagen the lyophilised skin wassoaked in 05M acetic acid with a samplesolution ratio of1 250 (wv) in the presence of porcine pepsin (10 g100 g

lyophilised skin) The mixture was gently stirred for 72 hfollowed by centrifugation at 20000timesg for 1 h using thecentrifuge (Sorvall Legend Mach 16R Thermo Fisher Sci-entific Germany) Then the supernatant was immediatelyprecipitated by the addition of NaCl to a final concentrationof 26M in 005M Tris-HCl (pH 75) The mixture wasallowed to stand for 1 h for pepsin inactivation The resultantprecipitate was collected by centrifugation at 20000timesg for1 h and dissolved in 10 volumes of 05M acetic acid Thesolution obtained was dialysed against 10 volumes of 01Macetic acid in a dialysis bag with a molecular weight cutoff of14 kDa for 48 h with the change of dialysis solution every 6 hSubsequently the solution was dialysed with 60 volumes ofdistilled water The changes of dialysis water were performeduntil neutral pH of dialysate was obtained The dialysate waslyophilised and referred to as ldquopepsin-solubilised collagenfrom the skin of splendid squid SCrdquo SC was subjected toanalyses The yield of collagen was calculated based on theweight of SC in comparison with that of dry defatted skin

24 Characterisation of Collagen fromthe Skin of Splendid Squid

241 Hydroxyproline Content Collagenwas dehydratedwithacetone and then hydrolysed in 6M HCl at 110∘C for 24 hprior to the determination of hydroxyproline content usingthe colorimetricmethod as described byNalinanon et al [12]The hydroxyproline content was calculated and expressed asmgg collagen

242 Total Sugar Content Total sugar content of collagenwas determined according to the phenol-sulfuric methodas described by Fournier [16] with a slight modificationCollagen was dissolved in 05M acetic acid to obtain afinal concentration of 6mgmL and further stirred untilcompletely solubilised To determine total sugar content col-lagen sample (250120583L) was mixed with 500120583L of 4 phenoland 25mL of 96 sulfuric acid The mixture was allowedto stand at room temperature for 30min The absorbanceof the reaction mixture was then measured at 490 nm Tocalculate the concentration of sugar the calibration curvewas performedusingD-glucose (0ndash04mgmL) as a standardTotal sugar content was calculated and expressed as (dryweight basis)

243 UV Absorption Measurement Collagen was dissolvedin 05M acetic acid to obtain a concentration of 1mgmLThe solution was placed into a quartz cell with a pathlength of 1 cm UV absorption spectrum of collagen wasmeasured using a spectrophotometer (UV-1800 ShimadzuKyoto Japan) Prior to measurement the base line wasset with 05M acetic acid The spectrum was obtained byscanning the wavelength in the range of 190ndash350 nm with ascan speed of 50 nmmin at room temperature

244 Differential Scanning Calorimetry (DSC) DSC analysisof collagen samples was carried out following the methods ofNalinanon et al [12] with a slight modification The samples

Journal of Chemistry 3

were rehydrated by adding 005M acetic acid to driedsamples at a solidsolution ratio of 1 40 (wv) The mixturewas allowed to stand for 2 days at 4∘C DSC analysis wasperformed using a differential scanning calorimeter (ModelDSC 7 Norwalk CT USA) Temperature calibration wasdone using the indium thermogram The collagen solutions(5ndash10mg) were accurately weighed into aluminium pansand sealed The samples were scanned at 1∘Cmin over therange of 20ndash50∘C using iced water as the cooling mediumAn empty pan was used as the reference The maximumtransition temperature (119879max) was estimated from the DSCthermogram

245 Amino Acid Analysis Collagen was hydrolysed underreduced pressure in 40M methanesulfonic acid containing02 (vv) 3-2(2-aminoethyl)indole at 115∘C for 24 h Thehydrolysates were neutralised with 35M NaOH and dilutedwith 02M citrate buffer (pH 22) An aliquot of 04mL wasapplied to an amino acid analyser (MLC-703 AttoCo TokyoJapan)

246 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)SDS-PAGE was performed by the method of Laemmli [17]The samples were mixed with 5 (wv) SDS and heatedat 85∘C for 1 h The mixtures were then centrifuged at8500timesg for 5min to remove undissolved debris Solubilisedsamples were mixed at 1 1 (vv) ratio with the sample buffer(05M Tris-HCl pH 68 containing 4 (wv) SDS 20 (vv)glycerol) in the presence of 10 (vv) 120573ME Samples (15 120583gprotein) were loaded onto a polyacrylamide gel made of 75or 5 separating gel and 4 stacking gel and subjected toelectrophoresis at a constant current of 15mAgel using aMini-PROTEAN II unit (Bio-Rad Laboratories Inc Rich-mond CA USA) After electrophoresis gels were fixed witha mixture of 50 (vv) methanol and 10 (vv) acetic acidfor 45min followed by staining with 005 (wv) CoomassieBlue R-250 in 15 (vv) methanol and 5 (vv) acetic acidfor 3 h Finally gels were destained with the mixture of30 (vv) methanol and 10 (vv) acetic acid for 45minHigh-molecular-weight protein markers (GE Healthcare UKLimited Buckinghamshire UK) were used to estimate themolecular weight of proteins Gels were imaged using aCanon image scanner (CanoScan LiDE100 Canon IncTokyo Japan)

247 TOYOPEARLCM-650 ColumnChromatography TOY-OPEARL CM-650 column chromatography was carried outaccording to the method of Kittiphattanabawon et al [2] andNalinanon et al [8] with a slight modification SC (20mg)were dissolved in 5mL of starting buffer (50mM sodiumacetate buffer pH 48 containing 2M urea) and incubated at60∘C for 30min The mixtures were centrifuged at 20000timesgat room temperature (25-26∘C) for 30min The supernatantwas applied onto a TOYOPEARL CM-650M column (10times 50 cm) previously equilibrated with 10 volumes of thestarting buffer at a flow rate of 30mLh After loading theunbound proteins were washed by the same buffer until A

230

was less than 005 Elutionwas achievedwith a linear gradient

of 0ndash02M NaCl in the same buffer at a flow rate of 30mLhwith a total volume of 100mL The eluant was monitoredat 230 nm and fractions (2mL) were collected The selectedfractions were subjected to SDS-PAGE using 5 separatinggel and 4 stacking gel as previously described

248 Fourier Transform Infrared (FTIR) Spectroscopy Spec-tra of collagens were obtained by using a Bruker ModelEQUINOX 55 FTIR spectrometer (Bruker Ettlingen Ger-many) equipped with a deuterated l-alanine triglycine sul-fate (DLATGS) detector The Horizontal Attenuated TotalReflectance Accessory (HATR) was mounted into the samplecompartmentThe internal reflection crystal (Pike Technolo-gies Madison WI USA) which was made of zinc selenidehad a 45∘ angle of incidence to the IR beam Spectra wereacquired at a resolution of 4 cmminus1 and themeasurement rangewas 4000ndash600 cmminus1 (mid-IR region) at room temperatureAutomatic signals were collected in 32 scans at a resolutionof 4 cmminus1 and were ratioed against a background spectrumrecorded from the clean empty cell at 25∘C Analysis ofspectral data was carried out using the OPUS 30 datacollection software program (Bruker Ettlingen Germany)

249 PeptideMapping Peptidemappings of SC andCCwereperformed according to the method of Nalinanon et al [8]with a slight modification

(1) Lysyl Endopeptidase Hydrolysis The freeze-dried samples(3mg) were dissolved in 05mL of 01M sodium phosphatepH 72 containing 5 (wv) SDS After the addition of 20 120583Lof the same buffer containing 01 120583g of lysyl endopeptidasefromA lyticus to collagen solutions the reactionmixture wasincubated at 37∘C for 60minThe reaction was terminated bysubjecting the reaction mixture to boiling water for 10min

(2) Chymotryptic HydrolysisThe freeze-dried samples (3mg)were suspended in 05mL of 50mM Tris-HCl pH 80 con-taining 10mMCaCl

2The collagenmixture was preincubated

at 37∘C for 15min Then 20120583L of the same buffer containing5 120583g of chymotrypsin was added to collagen solution Themixture was then incubated at 37∘C for 5min The reactionwas terminated by adding 05mL of 5 (wv) SDS (85∘C) tothe reaction mixture and boiling for 10min

Peptides generated by the protease digestion were sub-jected to SDS-PAGE using 75 running gel and 4 stackinggel in the same manner as previously described

25 Statistical Analysis Experiments were performed in trip-licateDatawere presented asmeansplusmn standard deviation anda probability value of lt005 was considered significant Forpair comparison 119905-test was used SPSS statistic programme(SPSS 110 for Windows SPSS Inc Chicago IL USA) wasused for data analysis

3 Results and Discussion

31 Yield and Characteristics of SC Collagen from squidskin was extracted with the aid of pepsin with the yield


Table 1 Yield and characteristics of collagen from the skin ofsplendid squid (SC) and type I collagen from calf skin (CC)dagger

Characteristics SC CCYield ( dry weight) 753 plusmn 120 mdashHydroxyproline (mgg dry sample) 985 plusmn 143aDagger 104 plusmn 245b

Total sugar content ( dry weight) 470 plusmn 039b 145 plusmn 020a

UV absorption peak (nm) 232 plusmn 030a 232 plusmn 013a

119879max (∘C) 341 plusmn 010a 408 plusmn 020b

Δ119867 (Jg) 066 plusmn 004a 120 plusmn 007bdaggerMean plusmn SD from triplicate determinationsDaggerDifferent letters (a and b) in the same row indicate the significant difference(119875 lt 005)

of 753 (dry wt) (Table 1) Yield was markedly increasedin comparison with acid solubilised collagen (627) (datanot shown) The result indicated that pepsin was effective inincreasing the extraction efficacy of collagenHydroxyprolinecontent in SC (985mgg dry wt) was lower than that of CC(104mgg dry wt) Rigby [18] reported that hydroxyprolinecontents vary with species environment and body temper-ature of fish Both SC and CC contained the carbohydrateGlucose and galactose are attached to hydroxylysine residuesof the peptide chain by O-glycosidic bonds to form 2-O-120572-D-glucosyl-O-120573-D-galactosyl-hydroxylysine and O-120573-D-galactosyl-hydroxylysine [19] Glycosylgalactosyl hydroxyly-sine in collagen from sea cucumber was reported [20] FromUV-Vis spectra both collagens had the same absorption peakat 232 nm Very low absorbance at 280 nm of both SC andCC (data not shown) indicated the absence of noncollagenousproteins Collagen commonly has a low amount of tyrosinewhich could absorb UV-light at 280 nm [9] Thus collagensfrom the skin of splendid squid were obtained with negligiblecontamination of other proteins

Based on thermal properties analysis themaximum tran-sition temperature (119879max) and total denaturation enthalpy(Δ119867) of SC and CC are presented in Table 1 119879max and Δ119867of SC (119879max = 341

∘C Δ119867 = 0656 Jg) were lower thanthose found in CC (119879max = 408

∘C Δ119867 = 1204 Jg)(119875 lt 005) This might be owing to the lower content of itsimino acids (hydroxyproline and proline) (Table 2) SCshowed higher thermal stability than collagens from coldwater fish including cod (15∘C) and deep-sea redfish (161∘C)[6 10] Thermal stability of SC was similar to that of collagenfrom tropical fish including brownbanded bamboo sharkskin (344∘C) blacktip shark skin (342∘C) black drum skin(342∘C) and sheephead seabream (34∘C) [2 5 21] Thedifference in thermal properties amongst collagens frommammal tropical fish and coldwater fishwas correlatedwiththeir imino acid content (proline and hydroxyproline) bodytemperature and environmental temperature [11 22 23]Thermal stability of collagen is associated with the restrictionof the secondary structure of the polypeptide chain governedby the pyrrolidine rings of proline and hydroxyproline andpartially by the hydrogen bonding through the hydroxylgroup of hydroxyproline [24ndash26]

Table 2 Amino acid composition of collagen from the skin ofsplendid squid (SC) and type I collagen from calf skin (CC)

Amino acid SC CCAlanine 88 119Arginine 56 51Aspartic acidasparagine 57 45Cysteine 0 0Glutamic acidglutamine 83 75Glycine 331 330Histidine 6 5Isoleucine 19 11Leucine 28 23Lysine 12 26Hydroxylysine 17 7Methionine 14 6Phenylalanine 11 3Hydroxyproline 91 94Proline 98 121Serine 35 39Threonine 25 18Tyrosine 5 3Tryptophan 1 3Valine 23 21Total 1000 1000Imino acidsa 189 215aImino acids include proline and hydroxyproline

32 Protein Patterns and Subunit Composition of SC Proteinpatterns of SC determined under reducing condition areshown in Figure 1 SC comprised 120572- and 120573-chains as majorcomponents with low content of 120574-chain Protein pattern ofSC was quite different from that of collagen types I II IIIand V Two different 120572-chains were presented in SC and 120572-chain with MW of 127 kDa was the dominant component Itindicated that SCmight contain at least two types of collagenFrom the elution profiles of SC on the TOYOPEARL CM-650M column (Figure 2) single peak containing differentcollagen components was obtained There were four distinct120572-chains (bands a b c and d) and cross-linked components(120573- and 120574-chains) (fraction numbers 26 and 28)The collagencomponents especially 120572-chain of SC were in accordancewith those of collagen from the skin of squid (Todarodespacificus) which contained two types of collagen type SQ-I and type SQ-II [27] Therefore SC most likely consisted oftype SQ-I and type SQ-II However the components of SCin the present study were quite different from those foundin collagen from the skin of octopus (Octopus vulgaris) andcuttlefish (Sepia lycidas) which comprised two distinct 120572chains 1205721 and 1205722 with a molar ratio of 2 1 [14 28]

33 Amino Acid Composition of SC and CC The amino acidcomposition of SC and CC expressed as residues per 1000total amino acid residues is shown in Table 2 Both collagenshad glycine as their major amino acid (330-331 residues1000residues) and are rich in alanine (88ndash119 residues1000


220170

116

7670

53

M I SC IIIII V

HMC

1205722(V)1205721(V)

120574

120573

1205722(I)1205721(IIIIII)1205723(I)

Figure 1 SDS-PAGE pattern of collagen from the skin of splendidsquid (SC) under reducing condition HMC M I II III andV denote high-MW cross-linked components high-MW proteinmarkers and collagen type I type II type III and type V respec-tively

24 28

ab cd

22

2426

28

3031

23

SC 22 23 24 26 28 30 31

HMC

120572212057211205723

0ndash02 M NaCl

000020040060080100120140160180200

Abso

rban

ce at

230

nm

0

5

10

15

20

Con

duct

ivity

(mS c

mminus

1 )

120574

120573

5 10 15 20 25 30 35 40 45 500Fraction number (2mL)

Figure 2 Elution profile of collagen from the skin of splendid squid(SC) on the TOYOPEARL CM-650M ion-exchange column Thefractions indicated by numbers were examined by SDS-PAGE using5 separating gel and 4 stacking gel HMC denotes high-MWcross-linked component

residues) proline (98ndash121 residues1000 residues) andhydroxyproline (91ndash94 residues1000 residues) Generallyglycine is about one-third of the total amino acid residuesProline and hydroxyproline constitute about one-fifthand alanine is for about one-ninth of the total aminoacids in collagen [29] Furthermore they had relatively lowcontents ofmethionine lysine phenylalanine hydroxylysinehistidine tyrosine and tryptophan and no cysteine Whencomparing amino acid composition between both collagens

Abso

rban

ce

Am

ide A

Am

ide B

Am

ide I

I

Am

ide I

IIAm

ide I

CC

SC

1077

cmminus

1

1453

cmminus

1

3400 3000 2600 2200 1800 1400 1000 6003800Wavenumber (cmminus1)

Figure 3 FTIR spectra of collagen from the skin of splendid squid(SC) and type I collagen from calf skin (CC)

SC had much lower content of alanine and proline Theresults were in agreement with collagen from octopus arm(Callistoctopus arakawai) and outer skin of cuttlefish (Sepialycidas) [13 14] Additionally the same results were alsoobserved in collagen from the skin of other fish speciessuch as arabesque greenling [8] blacktip and brownbandedbamboo shark [2 21] bigeye snapper [11 24] and unicornleatherjacket [30] The imino acid content (proline +hydroxyproline) of SC was 189 residues1000 residueswhich was much lower than that of calf skin collagen (215residues1000 residues) However it was much higher thanthat of skin collagen from cold water fish species (154ndash160residues1000 residues) such as cod [9] arabesque greenling[8] and deep-sea redfish [10]The imino acid of fish collagenshas been known to correlate with the water temperatureof their normal habitat [1] The sensitivity of collagen toheat is associated with its superhelical structure that ismaintained by the conformational restrictions imposed bythe pyrrolidine rings of the imino acids (hydroxyprolineand proline) [26] Hydroxyproline plays an important rolein stabilization of the helix structure by preventing rotationof the N-C bond [1] With reference to hydroxyprolinecontent the conversion factor of hydroxyproline to collagenfrom the skin of splendid squid was calculated to be 920The conversion factors of collagen from other sources suchas ornate threadfin bream (1272) [31] and Baltic cod skin(147) [6] have been reported This factor might be useful forestimation of the collagen content in raw material and theyield of extraction as well as purity assessment of resultantcollagen

34 Fourier Transform Infrared (FTIR) Spectra of SC and CCFTIR spectra in the range 4000ndash650 cmminus1 of SC and CC arepresented in Figure 3 SC exhibited FTIR spectrum in whichthe absorption bands were situated in the amide band regionincluding the peak of amide I amide II amide III amideA and amide B The wavenumber of each amide band wasquite similar to that found in collagens from the skin ofother fish [2 8 21 24 32] FTIR spectra of both collagens


were slightly different It might be caused by the differencein extraction process used their amino acid compositionsand amino acid sequence between both collagens Amide Abands of SC and CC were observed at 3290 and 3296 cmminus1respectively This band is generally associated with the N-H stretching vibration and shows the existence of hydrogenbonds Doyle et al [33] reported that a free N-H stretchingvibration commonly occurs in the range of 3400ndash3440 cmminus1When the NH group of a peptide is involved in a hydrogenbond the position is shifted to lower frequencies Amide Bband of SC (2921 cmminus1) and CC (2933 cmminus1) was related toasymmetrical stretch of CH

2stretching vibration [34] The

differences in wavenumber and amplitude of amides A andB found in SC and CC indicated that secondary structure ofboth collagens might be different in some extents

Amides I II and III peak of SC was found at wavenum-ber of 1643 1541 and 1233 cmminus1 respectively Payne andVeis reported that [35] amide I peak associated with C=Ostretching vibration or hydrogen bond coupled with COOminusMoreover it is a sensitive marker for the secondary structureof proteins analysis in FTIR analysis [36] while amide IIpeak resulted from N-H bending vibration coupled with CNstretching vibration [37] Generally amides I II and III ofpeptide linkage have been found at 1600ndash1690 1480ndash1575and 1229ndash1301 cmminus1 respectively [37] Muyonga et al [32]reported that shifts of amides I II and III peaks to lowerwavenumbers are associated with increased intermolecularinteractions (by hydrogen bonding) in collagen The ratiobetween amide III and 1454 cmminus1 peak of SC was 102Generally the ratio of approximately 10 reveals the triple-helical structure of collagens [38] The result indicated thatthe triple helical structuremight be slightly affected by pepsindigestion during collagen extraction When pepsin cleavedtelopeptide regions of tropocollagen the secondary structureof the resulting PSC might be altered to some degree [2439] Collagen from the skin of splendid squid exhibitedabsorptions at 1031 1060 and 1081 cmminus1 which arise fromthe C-OH stretching vibrations of the carbohydrate moietiesattached to the protein [40]The similar result was also foundin CC The result suggested that the collagens might containcarbohydrates which are attached to hydroxylysine residuesof the polypeptide chain by O-glycosidic bonds The resultwas also in accordance with total sugar content (Table 1)

35 Peptide Mapping of SC and CC The peptide maps ofSC digested by chymotrypsin or lysyl endopeptidase incomparison with that of CC are shown in Figure 4 Majorcomponents including 120572 120573- 120574-chains and high-MW cross-linked components of SC were hydrolysed into degradationpeptides with MW of 683 532 478 387 and 347 kDa andlow MW peptides (Lane 4) whilst CC was hydrolysed intoMW 1086 1004 855 719 674 and 387 kDa and low MWcomponents after being treated with chymotrypsin (Lane 5)For peptide maps of collagens digested by lysyl endopepti-dase major components of CC were much degraded intolowerMWpeptides especially peptides withMWof 338 kDaand lower MW components (Lane 6) In contrast lowernumber of degradation bands was found in SC (Lane 7)

220170

116

7670

53

HMC

M CC SC CCCC SCSC

Original ChymotrypsinLysyl

1205722

12057211205723

120574

120573

endopeptidase

Figure 4 Peptide maps of collagen from the skin of splendidsquid (SC) and type I collagen from calf skin (CC) digested bylysyl endopeptidase or chymotrypsin M and HMC denote high-MW protein markers and high-MW cross-linked componentsrespectively

The result suggested that SC was more tolerant to hydrolysisby lysyl endopeptidase than CC This might be due to thehigher content of lysine in CC (Table 2) Chymotrypsincatalyses the hydrolysis of peptide bonds on the carboxyl sideof hydrophobic amino acid residues such as phenylalaninetyrosine tryptophan and leucine [41] whilst lysyl endopep-tidase prefers to hydrolyse peptide bonds at the carboxyl sideof lysyl residues [42] Due to the specificity of cleavage sitesof both proteases the marked difference in peptide maps ofSC and CC was observed The result revealed that primarystructure of SC andCCwas completely different especially interms of their sequence and the composition of amino acids

4 Conclusion

Collagen could be extracted from the skin of splendid squidwith the aid of pepsin The resultant collagen was glycopro-tein and comprised SQ-I and SQ-II components Howeveridentification of collagen type using additional separatingtechnique or immunochemical detection or enzymatic treat-ment should be further conducted The information gainedfrom this study is beneficial for utilisation of squid skinbyproduct in food nutraceutical and cosmetic applications

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the Thailand Research Fund(TRF) and Office of the Higher Education Commission forProject no MRG5680116 to Dr Sitthipong Nalinanon andtheTRFDistinguishedResearchProfessorGrantThe authors


would like to express their appreciation to King MongkutrsquosInstitute of Technology Ladkrabang for support

References

[1] E A Foegeding T C Lanier andH O Hultin ldquoCharacteristicsof ediblemuscle tissuesrdquo in FoodChemistry O R Fennema Edpp 879ndash942 Marcel Dekker New York NY USA 1996

[2] P Kittiphattanabawon S Benjakul W Visessanguan HKishimura and F Shahidi ldquoIsolation and Characterisationof collagen from the skin of brownbanded bamboo shark(Chiloscyllium punctatum)rdquo Food Chemistry vol 119 no 4 pp1519ndash1526 2010

[3] J H Muyonga C G B Cole and K G Duodu ldquoCharacterisa-tion of acid soluble collagen from skins of young and adult Nileperch (Lates niloticus)rdquo Food Chemistry vol 85 no 1 pp 81ndash892004

[4] D W S WongMechanism and Theory in Food Chemistry VanNostrand Reinhold Company Inc New York NY USA 1989

[5] M Ogawa M W Moody R J Portier J Bell M A Schexnay-der and J N Losso ldquoBiochemical properties of black drum andsheepshead seabream skin collagenrdquo Journal of Agricultural andFood Chemistry vol 51 no 27 pp 8088ndash8092 2003

[6] M Sadowska I Kołodziejska and C Niecikowska ldquoIsolationof collagen from the skins of Baltic cod (Gadus morhua)rdquo FoodChemistry vol 81 no 2 pp 257ndash262 2003

[7] S Nalinanon S Benjakul and H Kishimura ldquoCharacterizationof collagen from the skin of unicorn leatherjacket (Aluterusmonoceros) solubilized by albacore tuna pepsinrdquoKingMongkutrsquosAgro-Industry Journal vol 3 no 1 pp 53ndash70 2011

[8] S Nalinanon S Benjakul and H Kishimura ldquoCollagens fromthe skin of arabesque greenling (Pleurogrammus azonus) solu-bilized with the aid of acetic acid and pepsin from albacore tuna(Thunnus alalunga) stomachrdquo Journal of the Science of Food andAgriculture vol 90 no 9 pp 1492ndash1500 2010

[9] R Duan J Zhang X Du X Yao and K Konno ldquoProperties ofcollagen from skin scale and bone of carp (Cyprinus carpio)rdquoFood Chemistry vol 112 no 3 pp 702ndash706 2009

[10] L Wang X An Z Xin L Zhao and Q Hu ldquoIsolation andcharacterization of collagen from the skin of deep-sea redfish(Sebastes mentella)rdquo Journal of Food Science vol 72 no 8 ppE450ndashE455 2007

[11] P Kittiphattanabawon S Benjakul W Visessanguan T NagaiandM Tanaka ldquoCharacterisation of acid-soluble collagen fromskin and bone of bigeye snapper (Priacanthus tayenus)rdquo FoodChemistry vol 89 no 3 pp 363ndash372 2005

[12] S Nalinanon S Benjakul W Visessanguan and H KishimuraldquoUse of pepsin for collagen extraction from the skin of bigeyesnapper (Priacanthus tayenus)rdquo Food Chemistry vol 104 no 2pp 593ndash601 2007

[13] T Nagai K Nagamori E Yamashita and N Suzuki ldquoCollagenof octopus Callistoctopus arakawai armrdquo International Journalof Food Science and Technology vol 37 no 3 pp 285ndash289 2002

[14] T Nagai E Yamashita K Taniguchi N Kanamori and NSuzuki ldquoIsolation and characterisation of collagen from theouter skin waste material of cuttlefish (Sepia lycidas)rdquo FoodChemistry vol 72 no 4 pp 425ndash429 2001

[15] M Nagarajan S Benjakul T Prodpran P Songtipya and HKishimura ldquoCharacteristics and functional properties of gelatinfrom splendid squid (Loligo formosana) skin as affected byextraction temperaturesrdquo Food Hydrocolloids vol 29 no 2 pp389ndash397 2012

[16] E Fournier ldquoBasic protocol determination of reducing andnonreducing sugars using the phenol-sulfuric acid assayrdquo inHandbook of Food Analytical Chemistry R E Wrolstad T EAcree E A Decker et al Eds pp 653ndash655 JohnWiley amp SonsHoboken NJ USA 2005

[17] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[18] B J Rigby ldquoAmino-acid composition and thermal stability ofthe skin collagen of the antarctic ice-fishrdquo Nature vol 219 no5150 pp 166ndash167 1968

[19] H D Belitz W Grosch and P Schieberle Food ChemistrySpringer Berlin Germany 2009

[20] M Saito N Kunisaki N Urano and S Kimura ldquoCollagenas the major edible component of sea cucumber (Stichopusjaponicus)rdquo Journal of Food Science vol 67 no 4 pp 1319ndash13222002

[21] P Kittiphattanabawon S Benjakul W Visessanguan and FShahidi ldquoIsolation and properties of acid- and pepsin-solublecollagen from the skin of blacktip shark (Carcharhinus lim-batus)rdquo European Food Research and Technology vol 230 no3 pp 475ndash483 2009

[22] TNagaiN Suzuki andTNagashima ldquoCollagen fromcommonminke whale (Balaenoptera acutorostrata) unesurdquo Food Chem-istry vol 111 no 2 pp 296ndash301 2008

[23] A M Pearson and R B YoungMuscle and Meat BiochemistryAcademic Press Inc San Diego Calif USA 1989

[24] S Benjakul Y Thiansilakul W Visessanguan et al ldquoExtractionand characterisation of pepsin-solubilised collagens from theskin of bigeye snapper (Priacanthus tayenus and Priacanthusmacracanthus)rdquo Journal of the Science of Food and Agriculturevol 90 no 1 pp 132ndash138 2010

[25] K A Piez and J Gross ldquoThe amino acid composition of somefish collagens the relation between composition and structurerdquoThe Journal of Biological Chemistry vol 235 no 4 pp 995ndash9981960

[26] D Liu GWei T Li et al ldquoEffects of alkaline pretreatments andacid extraction conditions on the acid-soluble collagen fromgrass carp (Ctenopharyngodon idella) skinrdquoFoodChemistry vol172 pp 836ndash843 2015

[27] S Mizuta R Yoshinaka M Sato and M Sakaguchi ldquoIsolationand partial characterization of two distinct types of collagen inthe squid Todarodes pacificusrdquo Fisheries Science vol 60 no 4pp 467ndash471 1994

[28] S Kimura Y Takema and M Kubota ldquoOctopus skin collagenIsolation and characterization of collagen comprising twodistinct alpha chainsrdquo The Journal of Biological Chemistry vol256 no 24 pp 13230ndash13234 1981

[29] G Balian and J H Bowes ldquoThe structure and properties ofcollagenrdquo inThe Science and Technology of Gelatin A G Wardand A Courts Eds pp 1ndash31 Academic Press London UK1977

[30] M Ahmad S Benjakul and S Nalinanon ldquoCompositionaland physicochemical characteristics of acid solubilized collagenextracted from the skin of unicorn leatherjacket (Aluterusmonoceros)rdquo Food Hydrocolloids vol 24 no 6-7 pp 588ndash5942010

[31] S Nalinanon S Benjakul H Kishimura and K Osako ldquoTypeI collagen from the skin of ornate threadfin bream (Nemipterushexodon) characteristics and effect of pepsin hydrolysisrdquo FoodChemistry vol 125 no 2 pp 500ndash507 2011


[32] J H Muyonga C G B Cole and K G Duodu ldquoFouriertransform infrared (FTIR) spectroscopic study of acid solublecollagen and gelatin from skins and bones of young and adultNile perch (Lates niloticus)rdquo Food Chemistry vol 86 no 3 pp325ndash332 2004

[33] B BDoyle E R Blout andEG Bendit ldquoInfrared spectroscopyof collagen and collagen like polypeptidesrdquo Biopolymers vol 14no 5 pp 937ndash957 1975

[34] Y Abe and S Krimm ldquoNormal vibrations of crystalline polyg-lycine Irdquo BiopolymersmdashPeptide Science Section vol 11 no 9 pp1817ndash1839 1972

[35] K J Payne and A Veis ldquoFourier transform IR spectroscopyof collagen and gelatin solutions deconvolution of the amideI band for conformational studiesrdquo Biopolymers vol 27 no 11pp 1749ndash1760 1988

[36] W K Surewicz and H H Mantsch ldquoNew insight into proteinsecondary structure from resolution-enhanced infrared spec-trardquo Biochimica et Biophysica Acta (BBA) vol 952 no 2 pp115ndash130 1988

[37] S Krimm and J Bandekar ldquoVibrational spectroscopy and con-formation of peptides polypeptides and proteinsrdquo Advances inProtein Chemistry vol 38 pp 181ndash364 1986

[38] A M D G Plepis G Goissis and D K Das-Gupta ldquoDielectricand pyroelectric characterization of anionic and native colla-genrdquo Polymer Engineering and Science vol 36 no 24 pp 2932ndash2938 1996

[39] P Kittiphattanabawon S Benjakul W Visessanguan and FShahidi ldquoIsolation and characterization of collagen from thecartilages of brownbanded bamboo shark (Chiloscyllium punc-tatum) and blacktip shark (Carcharhinus limbatus)rdquo LWTmdashFood Science and Technology vol 43 no 5 pp 792ndash800 2010

[40] C Petibois G Gouspillou K Wehbe J-P Delage and GDeleris ldquoAnalysis of type I and IV collagens by FT-IR spec-troscopy and imaging for a molecular investigation of skeletalmuscle connective tissuerdquo Analytical and Bioanalytical Chem-istry vol 386 no 7-8 pp 1961ndash1966 2006

[41] R Boyer ldquoEnzyme II cofactors regulation and catalytic RNArdquoin Conceptes in Biochemistry R Boyer Ed p 178 JohnWiley ampSons 2006

[42] P A Jekel W J Weijer and J J Beintema ldquoUse of endopro-teinase Lys-C from Lysobacter enzymogenes in protein sequenceanalysisrdquo Analytical Biochemistry vol 134 no 2 pp 347ndash3541983

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


the skin byproduct is generated in large quantity It has beenmainly used for fish meal or fertilizer with a low marketvalue Nagai et al [14] reported that skin waste materialfrom cuttlefish contained high amount of collagen Hencesquid skin can be served as an alternative source of collagenHowever no information regarding the collagen from theskin of splendid squid (Loligo formosana) has been reportedTherefore the objective of this study was to isolate andcharacterise the collagen from the skin of splendid squid (Lformosana) a byproduct from squid processing

2 Materials and Methods

21 Chemicals Bovine haemoglobin 120573-mercaptoethanol(120573ME) pepsin from porcine stomach mucosa (EC 34231)(750 unitsmg dry matter) and type I collagen from calf skinwere purchased from Sigma Chemical Co (St Louis MOUSA) Coomassie Blue R-250 and NNN1015840N1015840-tetramethylethylene diamine (TEMED) were procured from Bio-RadLaboratories (Hercules CA USA) Bovine serum albu-min was obtained from Fluka (Buchs Switzerland) High-molecular-weight protein marker was purchased from GEHealthcare UK Limited (Buckinghamshire UK) TOY-OPEARL CM-650M was purchased from Tosoh Corpo-ration (Tokyo Japan) 120572-Chymotrypsin from bovine pan-creas (EC 34211) lysyl endopeptidase (EC 342150) fromAchromobacter lyticus and type II III and V collagensfrom porcine cartilage skin and placenta respectively wereobtained from Wako Pure Chemical Industries Ltd (TokyoJapan)

22 Preparation of Splendid Squid Skin Splendid squid(Loligo formosana) were obtained from the dock in Samut-sakhonThailandThe squidwere packed in polyethylene bagkept in ice with a solidice ratio of 1 2 (wv) and transportedto the Faculty of Agro-Industry King Mongkutrsquos Instituteof Technology Ladkrabang Bangkok Thailand within 1 hAll procedures were performed at 0ndash4∘C Upon the arrivalthe squid skin was removed and washed with cold waterdrained and cut into small pieces (05 times 05 cm2) using thescissor To remove noncollagenous proteins the skin wasmixed with 01M NaOH using a samplealkaline solutionratio of 1 10 (wv) The mixture was stirred continuously for6 h The alkaline solution was changed every 2 h Then thealkaline treated skin was washed with cold water until neutralor faintly basic pH of wash water was obtained The treatedskin was then defatted with 10 (vv) butyl alcohol with asolidsolvent ratio of 1 10 (wv) for 18 h and the solvent waschanged every 6 h Defatted skin was then washed with 10volumes of cold water for 3 times prior to lyophilisation

23 Isolation of Collagen from the Skin of Splendid SquidPepsin-solubilised collagen from the skin of splendid squidwas isolated following the method of Nalinanon et al [12]with some modifications All procedures were performedat 4∘C To extract the collagen the lyophilised skin wassoaked in 05M acetic acid with a samplesolution ratio of1 250 (wv) in the presence of porcine pepsin (10 g100 g

lyophilised skin) The mixture was gently stirred for 72 hfollowed by centrifugation at 20000timesg for 1 h using thecentrifuge (Sorvall Legend Mach 16R Thermo Fisher Sci-entific Germany) Then the supernatant was immediatelyprecipitated by the addition of NaCl to a final concentrationof 26M in 005M Tris-HCl (pH 75) The mixture wasallowed to stand for 1 h for pepsin inactivation The resultantprecipitate was collected by centrifugation at 20000timesg for1 h and dissolved in 10 volumes of 05M acetic acid Thesolution obtained was dialysed against 10 volumes of 01Macetic acid in a dialysis bag with a molecular weight cutoff of14 kDa for 48 h with the change of dialysis solution every 6 hSubsequently the solution was dialysed with 60 volumes ofdistilled water The changes of dialysis water were performeduntil neutral pH of dialysate was obtained The dialysate waslyophilised and referred to as ldquopepsin-solubilised collagenfrom the skin of splendid squid SCrdquo SC was subjected toanalyses The yield of collagen was calculated based on theweight of SC in comparison with that of dry defatted skin

24 Characterisation of Collagen fromthe Skin of Splendid Squid

241 Hydroxyproline Content Collagenwas dehydratedwithacetone and then hydrolysed in 6M HCl at 110∘C for 24 hprior to the determination of hydroxyproline content usingthe colorimetricmethod as described byNalinanon et al [12]The hydroxyproline content was calculated and expressed asmgg collagen

242 Total Sugar Content Total sugar content of collagenwas determined according to the phenol-sulfuric methodas described by Fournier [16] with a slight modificationCollagen was dissolved in 05M acetic acid to obtain afinal concentration of 6mgmL and further stirred untilcompletely solubilised To determine total sugar content col-lagen sample (250120583L) was mixed with 500120583L of 4 phenoland 25mL of 96 sulfuric acid The mixture was allowedto stand at room temperature for 30min The absorbanceof the reaction mixture was then measured at 490 nm Tocalculate the concentration of sugar the calibration curvewas performedusingD-glucose (0ndash04mgmL) as a standardTotal sugar content was calculated and expressed as (dryweight basis)

243 UV Absorption Measurement Collagen was dissolvedin 05M acetic acid to obtain a concentration of 1mgmLThe solution was placed into a quartz cell with a pathlength of 1 cm UV absorption spectrum of collagen wasmeasured using a spectrophotometer (UV-1800 ShimadzuKyoto Japan) Prior to measurement the base line wasset with 05M acetic acid The spectrum was obtained byscanning the wavelength in the range of 190ndash350 nm with ascan speed of 50 nmmin at room temperature

244 Differential Scanning Calorimetry (DSC) DSC analysisof collagen samples was carried out following the methods ofNalinanon et al [12] with a slight modification The samples






230





























220170

116

7670

53

M I SC IIIII V

HMC

1205722(V)1205721(V)

120574

120573

1205722(I)1205721(IIIIII)1205723(I)


24 28

ab cd

22

2426

28

3031

23

SC 22 23 24 26 28 30 31

HMC

120572212057211205723

0ndash02 M NaCl

000020040060080100120140160180200

Abso

rban

ce at

230

nm

0

5

10

15

20

Con

duct

ivity

(mS c

mminus

1 )

120574

120573




Abso

rban

ce

Am

ide A

Am

ide B

Am

ide I

I

Am

ide I

IIAm

ide I

CC

SC

1077

cmminus

1

1453

cmminus

1











220170

116

7670

53

HMC

M CC SC CCCC SCSC


1205722

12057211205723

120574

120573

endopeptidase



4 Conclusion




Acknowledgments




References





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






230





























220170

116

7670

53

M I SC IIIII V

HMC

1205722(V)1205721(V)

120574

120573

1205722(I)1205721(IIIIII)1205723(I)


24 28

ab cd

22

2426

28

3031

23

SC 22 23 24 26 28 30 31

HMC

120572212057211205723

0ndash02 M NaCl

000020040060080100120140160180200

Abso

rban

ce at

230

nm

0

5

10

15

20

Con

duct

ivity

(mS c

mminus

1 )

120574

120573




Abso

rban

ce

Am

ide A

Am

ide B

Am

ide I

I

Am

ide I

IIAm

ide I

CC

SC

1077

cmminus

1

1453

cmminus

1











220170

116

7670

53

HMC

M CC SC CCCC SCSC


1205722

12057211205723

120574

120573

endopeptidase



4 Conclusion




Acknowledgments




References





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

















220170

116

7670

53

M I SC IIIII V

HMC

1205722(V)1205721(V)

120574

120573

1205722(I)1205721(IIIIII)1205723(I)


24 28

ab cd

22

2426

28

3031

23

SC 22 23 24 26 28 30 31

HMC

120572212057211205723

0ndash02 M NaCl

000020040060080100120140160180200

Abso

rban

ce at

230

nm

0

5

10

15

20

Con

duct

ivity

(mS c

mminus

1 )

120574

120573




Abso

rban

ce

Am

ide A

Am

ide B

Am

ide I

I

Am

ide I

IIAm

ide I

CC

SC

1077

cmminus

1

1453

cmminus

1











220170

116

7670

53

HMC

M CC SC CCCC SCSC


1205722

12057211205723

120574

120573

endopeptidase



4 Conclusion




Acknowledgments




References





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of







220170

116

7670

53

HMC

M CC SC CCCC SCSC


1205722

12057211205723

120574

120573

endopeptidase



4 Conclusion




Acknowledgments




References





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



References





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article characteristics of pepsin-solubilised...

Documents