Food Chemistry 141 (2013) 2343–2354

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Food Chemistry

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Effect of pretreatment on enzymatic hydrolysis of bovine collagenand formation of ACE-inhibitory peptides

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.05.058

⇑ Corresponding author. Tel.: +45 3533 3189; fax: +45 3533 3344.E-mail address: jo@life.ku.dk (J. Otte).

Yuhao Zhang a,b, Karsten Olsen b, Alberto Grossi b, Jeanette Otte b,⇑a College of Food Science, Southwest University, No. 2 Tiansheng Road, Beibei District, Chongqing 400716, PR Chinab Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark

a r t i c l e i n f o a b s t r a c t

Article history:Received 8 January 2013Received in revised form 21 March 2013Accepted 2 May 2013Available online 24 May 2013

Keywords:CollagenBoilingHigh pressureEnzymatic hydrolysisACE-inhibitionDH

Bovine collagen was pre-treated (boiled or high pressure (HP)-treated) and then hydrolysed by 6 prote-ases. The degree of hydrolysis (DH) and the angiotensin-converting enzyme (ACE)-inhibitory activity ofhydrolysates were measured. All enzymes used were able to partly degrade collagen and release ACE-inhibitory peptides. The highest ACE-inhibitory activity was obtained with Alcalase. Pretreatment signif-icantly influenced the DH and ACE-inhibition. For most enzymes, boiling for 5 min resulted in a signifi-cantly higher DH and ACE-inhibitory activity. With Alcalase and collagenase, hydrolysis and release ofACE-inhibitory peptides occurred without any pretreatment, but HP-treatment significantly improvedthe DH and ACE-inhibitory activity. HP did not markedly affect the hydrolysis with the other enzymes.The major peptides obtained with Alcalase were identified; all were released from the triple helix struc-ture of collagen. Many of these peptides had C-terminal sequences similar to known ACE-inhibitory pep-tides. The present results suggest that collagen-rich food materials are good substrates for the release ofpotent ACE-inhibitory peptides, when proper pre-treatment and enzymatic treatment is applied.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Large quantities of by-products from livestock, such as horn,hoof, bone and skin, or fish, such as bone and scales, are producedduring food and meat processing. Presently, a major part of theseby-products are discarded or used as feed. However, these wasteproducts contain a large amount of collagen and thus can be uti-lised as protein resources for novel food materials.

Many types of collagen have been identified, but about 90% ofthe collagen in the body is of type I (Reed, Vernon, Abrass, & Sage,1994; Risteli, Elomaa, Niemi, Novamo, & Risteli, 1993). Collagenmolecules are composed of three a-chains intertwined in the so-called collagen triple-helix. This particular structure, mainly stabi-lized by intra- and inter-chain hydrogen bonds, is the product of analmost continuous repetition of the Gly-X-Y- sequence, where X ismostly proline (Pro) and Y is mostly hydroxyproline (Hyp) (Ichi-kawa et al., 2010). Only the very short N- and C-terminal regions(15–26 amino acid residues), do not form triple helical structures,as they are largely made up of lysine and hydroxylysine (Hyl) resi-dues, as well as their aldehyde derivatives, engaging in intra- andinter-molecular covalent cross-links (Belitz, Grosch, & Schieberle,2009; Bruckner & Vanderrest, 1994). Four to eight collagen mole-cules in cross-section are stabilized and reinforced by covalent

bonds to constitute the basic unit of collagen fibrils (Gomez-Guil-len, Gimenez, Lopez-Caballero, & Montero, 2011).

Major applications of collagens include leather products, cos-metics (due to their good moisturising property) and biomedicalproducts such as wound dressings, implants, and drug carriers, aswell as gelatin production in food-related industries (Nam, You, &Kim, 2008). Gelatin is a soluble protein compound, obtained by par-tial hydrolysis of collagen. Extraction of gelatin is normally done bya chemical pre-treatment with an acid or alkali, which breaks non-covalent bonds so as to disorganise the collagen structure, thus pro-ducing adequate swelling and collagen solubilisation (Djabourov,Lechaire, & Gaill, 1993). Subsequent heating at temperatures above45 �C cleaves the hydrogen and covalent bonds to destabilise thetriple-helix and causes a helix-to-coil transition and thus conver-sion into soluble gelatin (Gomez-Guillen et al., 2002).

Collagen-rich animal by-products can be added value by enzy-matic modification (Kida et al., 1995; Morimura et al., 2002), andover the past decade, collagen-rich discarded materials from themeat and food processing industries, have been found to be valu-able sources of hydrolysates and peptides with bioactive propertiessuch as antioxidant, antihypertensive/ACE-inhibitory activity, anti-microbial and immunomodulatory activities (Aleman, Gimenez,Montero, & Gomez-Guillen, 2011; Aleman, Gimenez, Perez-Santin,Gomez-Guillen, & Montero, 2011; Byun & Kim, 2001; Ding et al.,2011; Ichimura, Yamanaka, Otsuka, Yamashita, & Maruyama,2009; Kim, Byun, Park, & Shahidi, 2001; Molinero, Julia, Erra, Rob-ert, & Infante, 1988; Saiga et al., 2003, 2008; Yang et al., 2009).

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Most studies concerning collagen-derived peptides, have fo-cused on antihypertensive/ACE-inhibitory activity, since ACE-inhibitory properties seem to be associated with the unique aminoacid composition of collagen. It is known that peptides containing aPro or Hyp residue at the C-terminal position, such as Gly-Pro-Hyp-Gly-Thr-Asp-Gly-Ala-Hyp, Gly-Pro-Pro-Gly-Ala-Hyp, Gly-Pro-Pro-Gly-Ala-Hyp, Gly-Ala-Hyp and Gly-Phe-Hyp-Gly-Pro, have strongACE-inhibitory activity (IC50 from 8.6 to 200 lM) (Ichimura et al.,2009; Oshima, Shimabukuro, & Nagasawa, 1979).

However, due to the triple-helical structure, collagen is notaccessible for enzymatic cleavage, except for enzymes belongingto the matrix metallo protease (MMP) family, including variouscollagenases, which contain both a catalytic site and a hemopoxindomain (Chung et al., 2004). Actually, the substrate used in all pre-vious reports about release of ACE-inhibitory peptides from colla-gen was not native collagen but gelatin or an extract of collagenobtained by boiling for >3 h (Saiga et al., 2008) which is probablyalso gelatin. From these gelatineous substrates ACE-inhibitory pep-tides have been released by the use of Alcalase, pronase E, Aspergil-lus protease, trypsin, pepsin and other proteases (Byun & Kim,2001; Ichimura et al., 2009; Kim et al., 2001; Nam et al., 2008; Sai-ga et al., 2008; Zhao et al., 2007). It would, however, be advanta-geous to find a less energy consuming way to pretreat nativeinsoluble collagenous materials to destabilise the triple-helix andexpose inner sites of collagen for enzymatic attack, or to find someenzymes able to attack the triple-helix structure of collageneffectively.

High hydrostatic pressure (HP) in the range of 100–1000 MPa isused for processing, in order to inactivate microorganisms and im-prove functional properties of proteins (Grossi, Gkarane, Otte, Ertb-jerg, & Orlien, 2012; Knudsen, Otte, Olsen, & Skibsted, 2002). UponHP processing, the balance of intramolecular and solvent–proteininteractions is changed, probably by disturbing the balance of thenon-covalent interactions that stabilize the native conformationof proteins (Torrezan, Tham, Bell, Frazier, & Cristianini, 2007). HPtreatment has been shown to reduce the thermal stability of bo-vine collagen, by dissociating the collagen fibres into fibrils and fi-brils into molecules (Ichinoseki, Nishiumi, & Suzuki, 2006), andalso to accelerate the heat-induced hydrolysis of collagen transfer-ring it into gelatin (Gomez-Guillen, Gimenez, & Montero, 2005).Furthermore, HP could induce hydration of hydrophobic groupsand unfolding of the a-chains of collagen (Potekhin, Senin,Abdurakhmanov, & Tiktopulo, 2009), leading to destabilisation ofthe triple-helix and exposure of inner sites of collagen.

Heat treatment is often used before enzymatic hydrolysis ofproteins to unfold the peptide chain and expose internal cleavagesites (Considine, Patel, Anema, Singh, & Creamer, 2007; Otte,Schumacher, Ipsen, Ju, & Qvist, 1999). However, there is little workaddressing the influence of short-term boiling as a pre-treatmentto enzymatic hydrolysis of collagen.

The aim of the present study is to determine the effect of HP andboiling as pre-treatments on the hydrolysis of collagen by six com-mercial proteases and release of ACE inhibitory peptides. Further-more, peptide profiles were revealed and peptides in the mostactive hydrolysate were identified, and the contribution of theseto the bioactivity discussed.

Table 1Enzymes, buffers and reaction conditions used for the hydrolysis of collagen samples.

Enzyme Optimal condition Buffer

Alcalase pH 8.0, 55 �C 0.1 M phosphate bufferCollagenase pH 7.5, 37 �C 0.1 M phosphate bufferThermolysin pH 8.0, 37 �C 0.1 M phosphate bufferProteinase K pH 7.5, 37 �C 0.1 M phosphate bufferPepsin pH 2.0, 37 �C 0.05 M phosphate bufferTrypsin pH 8.0, 37 �C 0.1 M phosphate buffer

2. Materials and methods

2.1. Materials and chemicals

Bovine collagen from achilles tendon (Type I, Insoluble, C9879),and Alcalase� from Bacillus licheniformis (P4860), proteinase K fromTritirachium album (P6556), thermolysin from Bacillus thermoprote-olyticus rokko (P1512), pepsin from porcine stomach mucosa

(P7000), trypsin from bovine pancreas (T9201), ACE (EC 3.4.15.1,rabbit lung, 0.25 units), o-aminobenzoylglycyl-p-nitro-L-phenylal-anyl-proline (ABZ-Gly-Phe(NO2)-Pro) and o-pthaldialdehyde(OPA) were purchased from Sigma Chemical Co., St. Louis, MO,USA. Collagenase from Clostridium histolytium (P1510), NuPAGE10% Bis–Tris gels, LDS sample buffer and MOPS SDS running bufferwere obtained from Invitrogen A/S, Carlsbad, CA. Precision plusProtein Standard All Blue marker was obtained from Bio-Rad Lab-oratories, Inc., Hercules, CA. Dithiothreitol (DTT) was obtainedfrom Applichem GmbH, Darmstadt, Germany. All chemicals wereof analytical grade or of the highest available purity. Highly puri-fied water (Milli-Q PLUS, Millipore Corporation) was used for prep-aration of all buffers and solutions.

2.2. Samples preparation and pretreatment

Collagen (0.07 g) was suspended in 7 ml of phosphate bufferwith a different pH according to the optimal pH of the enzyme (Ta-ble 1), and kept at 4 �C overnight for hydration. Three sampleswere made for each enzyme, one was left untreated and two weresubjected to boiling and HP, respectively, as pretreatments. Thesamples to be boiled were immersed in a boiling water bath for5 min, then immediately cooled for 10 min by tap water. The sam-ples for HP treatment were quantitatively transferred into polyeth-ylene bags and sealed under vacuum. HP treatment was performedin the pressurising chamber of a QUINTUS Food Processing ColdIsostatic Press QFP-6 (Avure Technologies AB, Västerås, Sweden)at 600 MPa for 15 min. Then, the samples were completely trans-ferred back into tubes and subjected to hydrolysis. The time inter-val between the end of the HP treatment and the start of thehydrolysis was 2 h.

2.3. Hydrolysis of samples

The samples were pre-incubated at the optimal temperature forthe enzyme (Table 1) for 10 min before addition of the enzymesolution. The enzyme-to-substrate ratio was 1:100 (w/w) in allexperiments. Sample aliquots (1 ml) were withdrawn after 1, 30,60, 120, and 240 min of hydrolysis and immediately heated at90 �C for 15 min to inactivate the enzyme. After cooling (20 minon ice) and centrifugation (11,000g for 5 min; Microcentrifuge154.RF, Ole Dich Instrumentmakers ApS, Hvidovre, Denmark), thesupernatant was filtrated through a cellulose acetate filter(0.45 lm pore size, Sartorius, Göttingen) and frozen at �18 �C.The pH of pepsin hydrolysates was modified to 7.0–7.5 using 2 MNaOH before freezing. Blank samples without hydrolysis were pre-pared in phosphate buffer pH 7.5 and pH 2.0, respectively, and keptat 4 �C for 12 h. These were then incubated at 37 �C for 4 h andtreated as the hydrolysate samples.

2.4. Determination of DH

The OPA method used to measure the degree of hydrolysis (DH)was a modification of that described by Spellman, McEvoy,O’Cuinn, and FitzGerald (2003). The OPA reagent (5 mM) was

Y. Zhang et al. / Food Chemistry 141 (2013) 2343–2354 2345

prepared according to the description of Spellman et al. on the dayof use. The assay was carried out by transferring an adequate vol-ume of the hydrolysate sample into a 10 ml tube and adding 2.4 mlof OPA reagent. Sample volumes between 20 and 140 ll were usedto give an absorbance below 1.0. The reaction was allowed to pro-ceed for 10 min to reach a plateau, and then the reaction mixturewas transferred to 3 ml disposable cuvettes (Cat. No. 7590 05,BRAND GMBH + CO KG) and the absorbance was measured at340 nm (Varian Cary 3 UV–visible spectrophotometer, Mulgrave,Australia). A blank containing only 2.4 ml of OPA reagent was alsomade. All determinations were made in duplicate. The DH was cal-culated according to the formula:

DH ð%Þ ¼ 100� n=N;

where n is the average number of peptide bonds hydrolysed and Nis the total number of peptide bonds per protein molecule. n wasfound from the absorbance measurements according to theformula:

n ¼ ðA340sample � A340unhydr :sampleÞ �Md=ec;

where A340 is the absorbance of the samples at 340 nm. M is the mo-lar mass of the protein (Da), and d is the dilution factor and calcu-lated according to the amount of sample used. e is the molarextinction coefficient of OPA at 340 nm (6000 M�1 cm�1) and c isthe protein concentration (10 g/l). A molar mass of bovine type Icollagen of 406.940 Da and the number of peptide bonds per colla-gen molecule (N) of 4287 were used for the calculation. These werefound as a weighted average of two a1(I) chains and one a2(I) chain(P02453 and P02465, respectively, from the UniProt database).

The theoretical maximum DH obtainable with some enzymeswas calculated from the specificity of the enzyme and the numberof such residues present in the collagen a-chains mentioned aboveas an average from two a1 and one a2 chains. This was performedusing the Peptide Cutter software (http://web.expasy.org/pep-tide_cutter/).

2.5. Determination of ACE-inhibitory activity

The ACE-inhibitory assay was performed in microtitre platesusing ABZ-Gly-Phe(NO2)-Pro as the substrate, as described by Sent-andreu and Toldra (2006) with some modifications. Aliquots(50 ll) of hydrolysates and of ACE solution (50 ll; 6 mU/ml in150 mM Tris-buffer, pH 8.3) were added into the well. The micro-titre plate was shaken for 10 s and incubated at 37 �C for 10 min.The reaction was started by addition of 200 ll of preheated(37 �C) substrate solution (0.45 mM ABZ-Gly-Phe(NO2)-Pro in150 mM Tris-Base buffer, pH 8.3, containing 1.125 mM NaCl). Fluo-rescence measurements were taken every min for 30 min (TecanGenios Plus, Tecan US, Durham, North Carolina, USA) using excita-tion and emission wavelengths of 360 and 415 nm, respectively.The control sample (100% ACE activity) contained Tris–Base bufferinstead of hydrolysate. Blank samples containing only substrate, orsubstrate and sample (and buffer instead of ACE) were also made.All samples were assayed in triplicate.

The slope of the fluorescence vs time curve was taken as a mea-sure of the ACE activity, and the ACE inhibition (%) was calculatedas:

ACE� inhibition ¼ ½100� 100� ðasample � ablankþsampleÞ=ðacontrol

� ablank� � 100%;

where the acontrol and asample are the slopes for the control and sam-ples, respectively, and ablank and ablank+sample are the slopes fromthe blanks with ACE replaced by 150 mM Tris–Base buffer.

2.6. SDS–PAGE analysis of solubilised proteins and fragments

Four control samples, unhydrolysed but subjected to the vari-ous pre-treatments, and the proteinase K hydrolysates made withHP pre-treatment were analysed by SDS–PAGE. The control samplewith pH 2 was prepared in 0.05 M phosphate buffer, pH 2.0, andleft at 4 �C for 12 h to evaluate the degradation of collagen by acid.Samples (25 lL) were prepared with 65 lL of LDS sample buffer,and 10 lL of 1.0 M DTT, and boiled for 5 min. The samples wereimmediately cooled in an ice bath and centrifuged (15,000g for5 min). Forty microlitres of unhydrolysed samples and 15 lL ofhydrolysed samples were loaded on the gel, together with 5 lL ofstandard markers (Precision plus Protein Standard All Blue mar-ker). NuPAGE 10% Bis–Tris gels were used according to the manu-facturer’s instructions in a XCell II Mini-Cell (EI 9001, Novex, SanDiego, CA). Electrophoresis was run at 200 V for 55 min in cassettescontaining the MOPS SDS running buffer. The gel was stained with0.1% Coomassie brilliant blue R-250 (2% concentrated phosphoricacid, 15% ammonium sulphate, 17% ethanol, 0.1% Coomassie bril-liant blue) for at least 3 h on a rocking table and destained inMilli-Q water for at least 1 h. The gels were photographed by acharge-coupled device (CCD) camera (Raytest, Camilla II, Strauben-hardt, Germany). The intensity of the bands was quantified usingPhoretix 1D software, version 2003.02. A minimum profile back-ground was subtracted. The concentration of protein in the bandswas determined as the peak area of the selected bands in the laneprofile.

2.7. Characterisation and identification of peptides by LC–MS

Selected samples from all the enzymatic hydrolysates and un-treated samples were analysed by LC–MS/MS as described by Otte,Shalaby, Zakora, Pripp, and EI-Shabrawy (2007) using an Agilent1100 MSD Trap with Chemstation for LC 3D systems Rev. B.01.03(Agilent Technologies 2001–2005) and Trap Control software ver-sion 5.3 (Bruker Daltonics GmbH 1998–2005). Sample volumes of25 lL were injected and eluted in 75 min using a gradient with100% A (0.1% TFA in water) for 5 min followed by a linear increaseto 60% B (0.1% TFA in 90% acetonitrile) over the next 70 min. MSspectra were recorded using the range 50–2000 m/z and the targetmass 1521, and Auto MS (2) spectra were recorded from two pre-cursor ions with the Smart Parameter Setting on.

Data was processed by Bruker Daltonics Data Analysis version3.3. All MS(n) compounds from each hydrolysate were searchedfor matching proteins using Mascot Version 2.1 (Matrix ScienceLtd., London, UK) with selection of trypsin or no enzyme. Further-more, Biotools software (Version 3.0, Bruker Daltonics, Bremen,Germany) was used to assign peptide masses to particular frag-ments of collagen. Bovine type I collagen a1 and a2 sequenceswere obtained from the UniProt database. Hydroxylated Pro resi-dues as identified by various researchers (Fietzek, Furthmay, &Kuhn, 1974; Fietzek & Kuhn, 1974; Fietzek & Rexrodt, 1975; Fie-tzek et al., 1979; Hulmes, Miller, Parry, Piez, & Woodhead-Gallo-way, 1973; Shirai et al., 1998) were included. The masses of themajor peptides in the hydrolysates made with Alcalase weresearched against these sequences, and the match with MS(2) frag-ments resulting from the possible peptides was assessed with theBiotools software.

2.8. Statistical treatment of data

Statistical analysis was performed using SPSS 13.0 for Windows(SPSS Inc, Chicago, IL). The overall effect of pretreatment, enzymeand hydrolysis time on DH and ACE-inhibitory activity was testedby the general linear models procedure, and the mean DH and ACEinhibition values for each variable were compared by the Tukey’s

2346 Y. Zhang et al. / Food Chemistry 141 (2013) 2343–2354

studentised range test. Difference in the DH and ACE inhibitoryactivity at the 3 pretreatments and 5 times of hydrolysis was alsotested for each enzyme by analysis of variance and compared byTukey’s studentised range test. Correlations between DH andACE-inhibitory activity were tested using Microsoft excel.

3. Results and discussion

3.1. Overall effects of pre-treatment and enzymatic hydrolysis on DHand ACE-inhibitory activity

As expected, the control samples without enzyme showed a DHbelow 0.1% and displayed no ACE-inhibitory activity, except for thesample incubated at low pH which showed a low DH and ACE-inhibitory activity. This might be due to breakage of some bonds,including covalent bonds and hydrogen bonds, under the acid con-ditions (Kolodziejska, Skierka, Sadowska, Kolodziejski, & Niec-ikowska, 2008). After hydrolysis, with all the enzymes used, theDH increased and a high ACE-inhibitory activity was obtained(Figs. 1–3). The reason that all the enzymes, and not only matrixmetalloproteinases (MMP) (collagenase), were able to degrade col-lagen might be that part of the collagen had been broken down intogelatin, during manufacture or subsequent incubation, making itmore accessible to other enzymes.

Taking all results together, there was a significant effect of allparameters tested, i.e. the pre-treatment used before hydrolysis,the enzyme used, and the time of hydrolysis, on the DH andACE-inhibitory activity of hydrolysates (Table 2).

3.1.1. Overall effect of pre-treatmentWhen considering all enzymes and hydrolysis times, boiling

was the pre-treatment giving rise to the highest DH and ACE-inhib-itory activity of the collagen hydrolysates (Table 2). This could bedue to some inner sites in the triple-helix structure being exposedduring boiling resulting in cleavage of part of the hydrogen bondsand covalent bonds, resulting in helix-coil transition of collagen(Zhang, Li, & Shi, 2006). Overall, the HP pretreatment significantlyimproved the DH of collagen hydrolysates in comparison to the

0

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Fig. 1. DH and ACE-inhibitory activities of collagen hydrolysates made with Alcalase (a asamples. Pre-treatments were boiling for 5 min (dark grey) and HP processing at 600 Mletters inside columns, and difference within the three samples at one hydrolysis time i

untreated samples, however the release of ACE-inhibitory peptideswas significantly reduced (Table 2). The reason that a HP treatment(600 MPa, 15 min), performed less than 2 h prior to hydrolysis, didlead to a general increase in the release of ACE-inhibitory peptidesmight be that the triple-helix structure of collagen was not weak-ened by this treatment. The helical collagen structure is saturatedwith intramolecular hydrogen bonds, and there is one (or two)hydrogen bond between the –CO and –NH groups of the mainchains per every three amino acid residues of collagen (Ramachan& Chandras, 1968). During HP, some intramolecular hydrogenbonds may be disrupted and some new hydrogen bonds withwater molecules formed. However, the new hydration structureby hydrogen bonding still could stabilize the triple-helix structureof collagen (Potekhin et al., 2009). Consequently, the inner Pro-richpart of collagen, which gives rise to ACE-inhibitory peptides (Min-kiewicz, Dziuba, & Michalska, 2011), were not exposed after the HPtreatment. Alternatively, the structural changes caused by the HPtreatment were partly reversed before hydrolysis. On the otherhand, the significantly higher DH in the HP pretreated samplesthan in the untreated samples might be due to HP causing somestable changes in the non-triple helix structure of collagen, makingthem more readily accessible to the enzymes.

3.1.2. Overall effect of hydrolysis timeWhen all the enzymes were considered together, there was a

significant effect of hydrolysis time from 1 to 240 min on the DH,which increased significantly with time (Table 2). The ACE-inhibi-tory activity of hydrolysates also increased with hydrolysis time,but the maximum was reached after 60 min in most cases. In gen-eral, peptides were quickly released during hydrolysis to reach halfof the maximum DH and ACE-inhibitory activity after 30 min ofhydrolysis (Figs. 1–3), however this varied with the enzyme used.

3.1.3. Overall effect of the enzyme usedWhen all pre-treatments are taken into account, Alcalase was

the most effective enzyme in hydrolysing collagen and giving riseto ACE-inhibitory peptides, followed by collagenase and then pro-teinase K (Table 2). The lowest DH was found in the hydrolysates

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nd b) and collagenase (c and d) from untreated (light grey) and pre-treated collagenPa (medium grey). Statistical difference (p < 0.05) within all samples is denoted bys denoted with letters above columns.

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Fig. 2. DH and ACE-inhibitory activities of collagen hydrolysates made with thermolysin (a and b) and trypsin (c and d) from untreated (light grey) and pre-treated collagensamples. Pre-treatments were boiling for 5 min (dark grey) and HP processing at 600 MPa (medium grey). Statistical difference (p < 0.05) within all samples is denoted byletters inside columns, and difference within the three samples at one hydrolysis time is denoted with letters above columns.

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Fig. 3. DH and ACE-inhibitory activities of collagen hydrolysates made with proteinase K (a and b) and pepsin (c and d) from untreated (light grey) and pre-treated collagensamples. Pre-treatments were boiling for 5 min (dark grey) and HP processing at 600 MPa (medium grey). A blank sample (without enzyme) was included to evaluate thedegradation of collagen in phosphate buffer with pH 2.0 (darkest grey). Statistical difference (p < 0.05) within all samples is denoted by letters inside columns, and differencewithin the three samples at one hydrolysis time is denoted with letters above columns.

Y. Zhang et al. / Food Chemistry 141 (2013) 2343–2354 2347

made with pepsin, but the ACE-inhibitory activity of thesehydrolysates was higher than of those made with trypsin (Ta-ble 2). These results show that even though a high DH was ob-tained with some proteases, the specificity of the enzyme wascontributing to determining the ACE-inhibitory activity of the re-leased peptides.

3.2. Effects of pre-treatment and hydrolysis time on DH and ACE-inhibitory activity for each enzyme

3.2.1. Alcalase and collagenaseThe hydrolysates made with Alcalase and collagenase showed

the highest DH of all hydrolysates (Fig. 1), and the DH and ACE-

Table 2Effects of pre-treatment, enzyme, and hydrolysis time on DH and ACE-inhibitory activity of collagen hydrolysates. Overall results.

Factor Significancelevel (p)a

Effect on DHb Effect on ACE-inhibitory activityb

Pretreatment <0.0001 Boiled > HP-treated > Untreated Boiled > c Untreated > HP-treatedEnzyme <0.0001 Alcalase > collagenase > Proteinase

K > trypsin > thermolysin > pepsinAlcalase > Proteinase K > collagenase P d

thermolysin P pepsin > trypsinHydrolysed time <0.0001 240 > 120 > 60 > 30 > 1 min 240 P 120 P 60 > 30 > 1 minPretreatment⁄hydrolysis time <0.0001Hydrolysis time⁄enzyme <0.0001Enzyme⁄pretreatment <0.0001Pretreatment⁄hydrolysed

time⁄enzyme<0.0001

a Analysis based on type III sum of squares.b Compared by Tukey’s studentized range test.c >Significantly higher than (p < 0.05).d PHigher than, but not significantly different (p > 0.05).

2348 Y. Zhang et al. / Food Chemistry 141 (2013) 2343–2354

inhibitory activity were significantly affected by the pretreatment(Table 3).

The DH increased quickly from 1 to 30 min of hydrolysis, withAlcalase and reached a DH of around 12% after 60 min. Visually,the insoluble collagen in the hydrolysis mixture disappeared, andan almost clear solution resulted within 10 min. This shows thatthe Alcalase preparation is able to degrade collagen, although itdoes not belong to the MMP family of proteases. Other studies haveshown similar results (Cheng, Liu, Wan, Lin, & Sakata, 2008; Liu,Chen, Su, & Zeng, 2011). Alcalase is a member of the serine S8endoproteinase family, which has a broad specificity, and there-fore, degraded the triple-helix structure of collagen within shorttime. After 1 min of hydrolysis, the boiled sample showed the high-est DH and best ACE-inhibitory activity compared to the HP treatedand the untreated sample (Fig. 1). Boiling causes damage to the tri-ple-helix structure of collagen (Zhang et al., 2006), thus, this sam-ple was hydrolysed faster and the ACE-inhibitory peptides releasedmore rapidly. Interestingly, the HP pretreatment improved the DHsignificantly from 30 to 240 min of hydrolysis (Fig. 1). The ACE-inhibitory activity was also slightly higher after HP treatment inhydrolysates taken after 30 min of hydrolysis with Alcalase, andreached 88.5%. However, from 60 min of hydrolysis, pre-treatmenthad no significant influence on the ACE-inhibitory activity (80.8–90.4%) of the Alcalase hydrolysates, which implies that Alcalasehad completely degraded the triple-helical structure and releasedthe ACE-inhibitory peptides in all the samples. This level of activityis slightly better than those obtained by Liu et al. (2011) and Chenget al. (2008) who used Alcalase for the production of ACE inhibitorypeptides from sea cucumber collagen and chicken bone protein,respectively.

Collagenase belongs to MMPs, which initially cleave collagens I,II and III at specific Gly-Leu or Gly-Ile bonds located approximatelythree quarters along the chain from the N-terminus (Chung et al.,2004). This cleavage is critical for the initiation of further collagenbreakdown since it causes damage to the triple-helix and exposureof more peptide bonds. In this way collagenase unwinds the triple-

Table 3Influence of collagen pre-treatment on DH and ACE-inhibitory activity of collagenhydrolysates obtained by each enzyme.

Enzyme Effect on DHa Effect on ACE-inhibitory activitya

Alcalase HP > Boiled P c Untreated Boiled P HP > UntreatedProteinase K Boiled>b HP > Untreated Boiled P Untreated P HPCollagenase Boiled > HP > Untreated Boiled > HP > UntreatedThermolysin Boiled > HP > Untreated Boiled > Untreated > HPTrypsin Boiled > Untreated > HP Boiled > Untreated > HPPepsin Boiled > Untreated > HP Boiled > Untreated P HP

a Compared by Tukey’s studentized range test.b >Significantly higher than (p < 0.05).c PHigher than, but not significantly different (p > 0.05).

helical structure first, before hydrolysing the peptide bonds. Sincethis is a slow process, the hydrolysis reaction is slow, which is alsoreflected in the DH which increases throughout hydrolysis (Fig. 1c).Reports on the use of collagenase to hydrolyse collagenous mate-rial and produce active peptides are scarce. Oshima et al. (1979)hydrolysed gelatin by collagenase for various times up to 24 h,and the ACE inhibitory activity of hydrolysates was time-depen-dent with the maximum obtained after 24 h of hydrolysis. Their re-sults confirm that the hydrolytic process with collagenase is slow,and we therefore expect, that an even higher DH and ACE-inhibi-tory activity might be obtained if the hydrolysis time is prolonged.Pre-treatment by boiling significantly improved the hydrolysis,and also increased the ACE-inhibitory activity of hydrolysates ta-ken from 1 to 120 min (Fig. 1 d). After 240 min of hydrolysis, aDH of 18.1% was obtained, and the corresponding ACE-inhibitoryactivity was 80.5%. This suggests that the triple-helical structurewas damaged by boiling and collagenase could hydrolyse the pep-tide bonds directly without unwinding first. Interestingly, the HPtreatment before hydrolysis also increased the DH compared tothe untreated samples. However, this was not reflected in a signif-icantly higher ACE-inhibitory activity, which implies that triple-helical structure still persisted after the HP pretreatment. Thepretreatment had no significant influence on the ACE-inhibitoryactivity of collagenase hydrolysates after 240 min of hydrolysis,which suggests that collagenase had completely unwound the tri-ple-helical structure and released the ACE-inhibitory peptides inall the samples at that time.

For both enzymes, Alcalase and collagenase, the DH of the HP-treated sample was significantly lower than that of the untreatedsample when hydrolysis started, which means that, after HP treat-ment, the conformation of collagen did not allow collagenase tounwind or Alcalase to degrade the triple-helix as efficiently. How-ever, the DH increased for the HP treated samples, indicating thatafter HP, the unwinding and degradation, respectively, of the tri-ple-helix structure during incubation was facilitated. This mightbe due to HP causing aggregation of some parts of collagen, andonce these were damaged by initial hydrolysis, they were quicklyhydrolysed.

3.2.2. Thermolysin and trypsinThe ability of thermolysin and trypsin to hydrolyse collagen

was very limited, the DH reaching only about 1% in the untreatedand HP treated samples (Fig. 2). The advantage of boiling as apretreatment was most evident for these enzymes, with significanteffects on DH obtained with both enzymes (Fig. 2). During hydro-lysis with these enzymes, the DH increased with time, especially inthe boiled sample, however, it remained low. The highest DH inboiled samples was around 7%, which is close to the theoreticalDH (8.3%) obtainable with trypsin but much lower than expected

Y. Zhang et al. / Food Chemistry 141 (2013) 2343–2354 2349

for thermolysin (14.8%), which has a much broader specificity. Astudy using a sequence analogue to the collagen a1-chain f772-780 (Gly-Pro-Gln-Gly-Nph-Ala-Gly-Gln-Val, where Nph is 4-nitro-phenylalanine) showed that the rate of thermolysin hydrolysis ofthe Nph-Ala bond was reduced 15-fold in the native peptide com-pared with the heat denatured peptide, demonstrating that the tri-ple-helical conformation inhibits general proteolysis of susceptiblesequences (Fields, 1995). Another study (Zhang et al., 2006) con-firms that native collagen is hardly digested by trypsin due to itsstable triple helix, whereas denatured products such as gelatin iseasily attacked by trypsin, explaining why boiling was a better pre-treatment than the others.

The higher amount of peptides released from the boiled colla-gen sample was also reflected in a significantly higher ACE-inhibi-tory activity of these samples (Fig. 2), reaching quite high values,with a maximum around 70% for the trypsin hydrolysates andaround 75% for the thermolysin hydrolysates. The specificity oftrypsin is mainly towards basic amino acids, and the presence ofa positively charged amino acid at the ultimate C-terminal positionmay contribute significantly to the inhibitory potency of peptidesto ACE (Otte et al., 2007). Thermolysin, which is specific for manyhydrophobic amino acid residues, should generate peptides withthe most favourable amino acids (hydrophobic) at the C-terminalposition, explaining the slightly higher ACE-inhibitory activity ob-tained with this enzyme.

Although HP pre-treatment, before hydrolysis with thermolysinresulted in a significantly higher DH than for the untreated sample(Table 3), the ACE-inhibitory activity of the hydrolysates obtainedwith both enzymes from the HP treated collagen samples was sig-nificantly lower than for the untreated sample (Table 3, Fig. 2b andd). HP in this case obviously was not beneficial, maybe because itlead to aggregation of parts of the collagen, which were accessibleto these enzymes, and thus caused a different composition of pep-tides in the hydrolysates.

3.2.3. Proteinase K and pepsinAs expected, these two enzymes also hydrolysed collagen to a

limited degree (Fig. 3), but the effect of pre-treatment, especiallyboiling, was not as marked as for thermolysin and trypsin. TheACE-inhibitory activity of these two enzymes was also not muchaffected by the pre-treatment (Fig. 3)

The DH of hydrolysates, made with proteinase K, increased withtime for the untreated and HP treated samples, whereas the boiledsample reached a maximum (around 5%) after 60 min. The ACE-inhibitory activity only increased significantly from 1 to 30 min,and reached a value close to 80%. This value is close to the valueobtained by Arihara, Nakashima, Mukai, Ishikawa, and Itoh(2001) for hydrolysates of porcine skeletal muscle proteins (majorcomponents were myosin, actin and collagen) made with protein-ase K (82.1% after 18 h of hydrolysis). So proteinase K seems tocleave quickly what can be cleaved, followed by a very slow in-crease in DH, which does not affect ACE-inhibitory activities. Thismight be due to preferential cleavage of the non-helical parts ofcollagen by this enzyme, since this part also has more susceptiblepeptide bonds than the helical part. For example, the theoreticalDH of proteinase K upon hydrolysis of the a1-chain from bovinecollagen is 23.94%, while the values are 18.85% and 35.27% forhydrolysis of the triple-helix and the non-triple-helix regions,respectively.

Boiling of the collagen sample, before hydrolysis, did increasethe DH significantly, but the ACE-inhibitory activity was onlyslightly affected. Interestingly, the HP treatment improved theDH, but the ACE-inhibitory activity of these samples was slightlylower (not significant) than of the untreated samples (Table 3). Thismight be because the triple helix region of collagen was not hydro-lysed by proteinase K and therefore the strong ACE-inhibitory

peptides were not released after the HP pretreatment. The protein-ase K hydrolysates were further investigated by SDS–PAGE analysis.

During hydrolysis with pepsin, the DH increased with time forall samples, but remained below 3%. From 1 to 30 min, the ACE-inhibitory activity of all samples also increased significantly, thennon-significantly afterwards. The final ACE-inhibitory activitieswere below 70%. The effect of boiling on DH and ACE-inhibitoryactivity was not significant, whereas the effect of HP treatmentwas to lower the DH (significantly) and the ACE-inhibitory activity(non-significantly). This might be due to the acid extracting some ofthe collagen or gelatine, especially in combination with boiling. Inthe HP treated sample, these proteins do not seem to be extractedto the same degree. Maybe HP in the acidic conditions leads to for-mation of aggregates that are not dissolved or not easily degradedby pepsin. The control sample, kept in phosphate buffer with pH2.0 overnight at 4 �C, had a DH around 0.7%, showing that part ofthe collagen was degraded by the acid during this process. TheDH of the peptic hydrolysates was the lowest of all hydrolysates.This is consistent with the theoretical DH obtainable with pepsinon collagen of 6.88%, which is also the lowest within all enzymes.Thus, very few sites in collagen seem to be susceptible to pepsin.

3.3. Relation between DH and ACE-inhibitory activity

In many cases, the values for DH and ACE-inhibition both in-creased with time of hydrolysis or with a certain pre-treatment.However, as discussed above, this was not always the case. To ex-plore the relationship between DH and ACE-inhibitory in more de-tail, results from all hydrolysates and hydrolysates with selectedenzymes are plotted in Fig. 4. The ACE-inhibitory activity of allhydrolysates increased with DH up to around 4%, after which itseemed almost unaffected by further increases in DH (Fig. 4a). Thistrend could partly stem from the fact that 90% ACE-inhibition wasnear the maximum measurable with this method. A similar trendhas been observed in the studies by Walsh et al. (2004) and Niel-sen, Martinussen, Flambard, Sørensen, and Otte (2009) for milkprotein-derived ACE-inhibitory peptides. The correlation betweenDH and ACE-inhibition could best be described by a logarithmicfunction with the equation y = a � (bcx), where y is the ACE-inhibi-tion, x is the DH (%), and a, b, and c are the regression parameters.Using this equation it was also possible to calculate the DH atwhich half of the maximal ACE-inhibition was obtained (DH50),in this case 1.1%. The same trend was seen for most of the individ-ual hydrolysates, i.e. those made with collagenase (with a DH50 of2.5%; Fig. 4b), thermolysin, trypsin, and proteinase K (not shown).In contrast, a more linear relationship was seen between the DHand ACE-inhibitory activity for the hydrolysates made with Alca-lase and pepsin (Fig. 4 c and d), which reached the highest andthe lowest DH, respectively. As discussed before, the ACE-inhibi-tory peptides were quickly released during hydrolysis with Alca-lase, and the values for DH and ACE-inhibition of thesehydrolysates were beyond 10% and 80%, respectively, after30 min and were not significantly increased after 60 min. There-fore, most of the points in Fig. 4c are in the upper part, giving ashallow linear relation (with DH50 around 4.3%). Pepsin hydrolysis,on the other hand, resulted in only low DH and the points were atthe lower end (Fig. 4d) where both the DH and ACE-inhibitoryactivity still increased, giving a steep linear correlation, and in thiscase DH50 was around 1%.

3.4. Characterisation of collagen molecules and degradation productsin pretreated collagen samples and hydrolysates

3.4.1. Polypeptides detected by SDS–PAGE analysisThe effect of the various pre-treatments on the solubilisation of

individual collagen molecules and primary degradation products

0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20

0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20

0

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inhi

bitio

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inhi

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inhi

bitio

n (%

)

DH (%)

0

20

40

60

80

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ACE-

inhi

bitio

n (%

)

DH (%)

A B

C D

Fig. 4. Relationship between DH and ACE-inhibitory activities in all hydrolysates (A) and hydrolysates obtained with selected enzymes (B–D). (B) Collagenase hydrolysates;(C) Alcalase hydrolysates; (D) pepsin hydrolysates. Lines shown were fitted to data using the equation ACE-inhibition = a � (bcDH), where a, b and c are regression parameters.

Fig. 5. SDS–PAGE gel showing the proteins and fragments in unhydrolysed collagensamples with the various pre-treatments (lanes 2–5) and of collagen pretreatedwith HP and then hydrolysed with proteinase K (PK) for various times (lanes 6–10).M, molecular weight marker; UN, untreated collagen; B, boiled for 5 min; HP, highpressure treated at 600 MPa; acid, incubated in phosphate buffer; pH 2, overnight.

2350 Y. Zhang et al. / Food Chemistry 141 (2013) 2343–2354

was investigated by SDS–PAGE (Fig. 5). Two a-chains and a b-com-ponent (dimer of a-chains) were found in all unhydrolysed sam-ples, since they were heated at 90 �C for 15 min to inactivate theenzyme (lanes 1–3). The boiled sample had slightly less b-compo-nent and more components with smaller molecular weights (be-low 100 kDa), indicating that boiling promotes degradation ofcollagen chains. Interestingly, the sample that had been subjectedto HP treatment was quite similar to the others. However, thissample contained less of the lower molecular weight protein frag-ments (below 100 kDa), showing that less of the subunits (a�andb�components) of collagen were degraded by heating after HP pre-treatment. In contrast, the control sample that had been incubatedovernight in phosphate buffer, pH 2.0, (lane 4) contained almost noa- and b-components and in turn contained several componentswith a molecular weight below 75 kDa, and even as low as 15–20 kDa. This shows that the acid had cleaved part of the covalentbonds and agrees with the fact that treatment of collagen with acidcauses extraction of gelatin (Gomez-Guillen et al., 2005).

The hydrolysates taken after 30 min and 120 min of hydrolysiswith proteinase K were particularly interesting; the HP-treatedsample had a lower DH but a higher ACE-inhibitory activity thanthe boiled samples. SDS–PAGE analysis of the HP treated sampleshydrolysed with proteinase K is shown in Fig. 5, lanes 5–9. Similarbands were seen at all hydrolysis times (B1 to B8). All the proteinswith a MW > 100 kDa (compare to untreated samples) had beendegraded after 1 min of hydrolysis. Data analysis by Phoretix 1Dshowed that only two bands (B2 and B5) increased with time ofhydrolysis. This might be due to the band intensity measured,resulting from a balance between the formation, by degradationof native collagen, and further degradation into peptides with asmaller molecular weight. The DH of this sample was significantlyincreased from 120 to 240 min (Fig. 3), so some of the small

peptides, that could not be displayed in this gel must have beenfurther degraded during this period. The discrepancy with respectto effect of HP on DH and ACE-inhibitory activity must be sought inthe composition of the low molecular weight peptides.

Y. Zhang et al. / Food Chemistry 141 (2013) 2343–2354 2351

3.4.2. Peptide profiles of hydrolysatesAll peptide profiles, except those from the pepsin-hydrolysates,

showed the presence of a multitude of small peptides (eluting be-tween 10 to 50 min). Peptide profiles of selected hydrolysates areshown in Fig. 6. There was a very good agreement between theamount of peptides in the profiles and the DH in the hydrolysatesas a function of pre-treatment. The hydrolysates obtained afterboiling, except with Alcalase and pepsin, contained a much higherconcentration of peptides than those from the untreated and HP-treated samples, as shown for the thermolysin hydrolysates inFig. 6a, confirming the higher DH in these samples. In fact, forthe Alcalase hydrolysates, a slightly higher peptide concentrationwas seen in the hydrolysates made after HP treatment (Fig. 6b),in accordance with the higher DH in these hydrolysates (Fig. 1a).Conversely, for hydrolysis with pepsin, the HP-treated sample gave

0

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ance

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0 10 20 30 40 50 60 70

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0 10 20Rete

Fig. 6. Peptide profiles of collagen hydrolysates made with (a) thermolysin (60 min), (b) Ano pre-treatment; middle panel: boiled for 5 min; lower panel: HP treated.

Retentio

0 5 10 15 20 25

Abs

orba

nce

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727.

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, 797

.485

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795

.492

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.4602.

372

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556

.252

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570.

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379.

1

839.

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641.

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60.2

Fig. 7. Detailed peptide profile (reversed-phase HPLC) of the collagen hydrolysate with tand hydrolysis with Alcalase for 30 min. Major masses in peaks are given above peaks.

the lowest amount of peptide material (Fig. 6c), in accordance witha lower DH in these samples (Fig. 3c). Apart from the differentamounts, the profiles were quite similar, indicating that in generalthe same peptide bonds were accessible to Alcalase irrespective ofthe pre-treatment. The peptides released after pepsin hydrolysiseluted between 50 and 65 min, suggesting that these were muchbigger than those released by the other enzymes. Furthermore, aslightly different peptide profile for the HP-treated sample(Fig. 6c) suggests that some parts of collagen (giving rise to pep-tides with the longest elution time) were less accessible to pepsinafter the HP treatment.

The peptide profiles of the collagenase and proteinase K hydrol-ysates were practically identical (except for the amount) for thethree pre-treatments, meaning that these enzymes hydrolysedthe same peptide bonds, just at a different rate. Interestingly, the

30 40 50 60 70ntion time (min)

C

0 10 20 30 40 50 60 70

lcalase (30 min), and (c) pepsin (60 min) after various pre-treatments. Upper panel:

n time (min)

30 35 40 45 50 55 60

779.

4

1154

.7

1202

.7

636.

2, 1

055.

5, 1

549.

8

1048

.5

1102

.911

62.6

, 539

.3

898.

5871.

5879.

4, 9

01.4

, 821

.4

837.

511

05.6

841.

4, 8

77.4

he highest ACE-inhibitory activity, which was obtained after pre-treatment with HP

Table 4Masses and tentative identification of major peptides in the Alcalase hydrolysate (30 min) of collagen pre-treated with HP.

Rt (min) Mass obs. [M+H]+ Suggested peptide (score) Origin Mass calc. [M+H]+

13.2 288.1 LVG (11) a2(263–265) 288.19215.4 872.4 GDRGDAGPK (151) a1(581–589) 872.42216.1 570.3 RGPSGP (71) a1(253–258) a2(528–533) 570.29916.2 499.2 GPAGPT (51) a1(626–631) 499.25116.6 554.3 RGPAGP (86) a2(972–977) 554.30516.9 1088.5 TGSPaGSPGPDGK (261) a1(379–390) 1088.48517.4 511.1 GPPGPA (15) a1(212–217,296–301,392–397,458–463,527–532,608–613,677–682,734–739,764–769) a2(688–693) 511.25117.5 528.3 GVAGPK (41) a1(338–343) 528.31418.3 641.3 RGAPGPA (142) a1(349–355) 641.33718.6 460.2 MGPR (23) a2(366–369) 460.23419.3 724.3 RGPPGPQ (93) a1(82–88) 724.37419.6 556.2 PGPAGAA (94) a1(214–220) 556.27320.2 602.3 PGSRGL (33) a2(321–326) 602.32620.6 558.3 GLPGAK (43) a1(371–376), a2(223–228) 558.325

854.4 GEGGPQGPR (52) 1(191–199) 854.41221.6 923.5 EPGPAGLPGP (135) a1(312–321) 923.44721.7 809.4 GPSGPPGPK (320) a1(260–268) 809.41522.3 855.4 AGRPGEVGP (131) a1(754–762) 855.43222.4 795.4 GPRGPAGPS (272) a2(970–978) 837.45822.9 379.1 RGF (11) a1(106–108, 331–33,517–519,808–810); a2(84–86,369–371) 379.20923.6 797.4 VGPPGPSGN (23) a1(721–729) 797.37924.8 584.4 GLPGPK (21) a1(575–580) 584.34025.1 839.4 GRVGPPGPS (268) a1(719–727) 839.43726.9 727.3 RGPPGPM (130) a1(832–838) 727.35626.9 811.2 PSGPQGIR (71) a2(917–924) 811.44228.4 841.4 GPPGPSGISG (275) a2(715–724) 841.40528.4 877.4 VGPPGPPGPA (84) a1(760–769) 877.44129.2 1105.6 GPIGPVGARGPAG (16)b a1(914–926) 1105.61129.7 837.5 GRPGPIGPA (158) a2(394–402) 837.45832.3 879.4 TGPIGPPGPA (94) a1(604–613) 879.45732.4 901.4 LTGSPGSPGP (38) a1(378–387) 901.42632.6 821.4 GPPGPAGPR (42) a1(527–535) 821.42633.9 871.5 GERGLPGVA (19) a2(799–807) 871.46334.2 898.5 GVVGLPGQR (348) a1(797–805) 898.51035.8 539.3 GPIGPV (49) a1(914–919) 539.31936.3 1162.6 GAVGPAGAVGPRGP (44) a2(904–917) 1162.63337.3 1102.5 GFPGSPGNIGPA (167) a2(370–381) 1102.51639.3 1048.5 PGPPGPAGAAGPA (638) a1(211–223) 1048.50640.5 636.2 PGSRGF (14) a1(328–333) 636.31040.8 1055.5 GPAGPPGFPGAV (531) a1(176–187) 1055.51642.1 1202.7 GERGFPGLPGPS (77) a1(806–817) 1202.58045.4 1154.7 GKEGPVGLPGID (276) a2(382–393) 1154.60546.5 779.4 GPAGPPGPI (65) a1(638–691) 778.409

a P with boldface means Hyp (hydroxylated proline).b This sequence could not be confirmed.

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Y. Zhang et al. / Food Chemistry 141 (2013) 2343–2354 2353

peptide amount in the hydrolysate made with thermolysin fromthe HP treated collagen was higher than that from the untreatedsample (Fig. 6a), in accordance with the higher DH in these sam-ples (Fig. 2a). The peptide profiles for the thermolytic and trypticcollagen hydrolysates made after HP-treatment were slightly dif-ferent from those of the untreated sample, indicating that someparts of the molecule may have been more exposed after the HPtreatment and a more complete hydrolysis was achieved. However,the peptides released from these parts, obviously, were not thosegiving rise to the highest ACE-inhibitory activity (Fig. 2b).

3.4.3. Identification of peptides released by AlcalaseThe highest ACE-inhibitory activity was exerted by the hydroly-

sate obtained from the HP treated collagen subsequently hydroly-sed by Alcalase (30–240 min of hydrolysis), so one of thesehydrolysates was characterised in more detail. The peptide profileof the hydrolysate obtained after 30 min of hydrolysis is shown inFig. 7, with the masses of the major peptides indicated.

A tentative identification of these peptides was undertakenwith the results, as shown in Table 4. All identified sequences werefrom the triple helix structure of collagen. This shows that Alcalasecan degrade the triple helix structure of collagen effectively, prob-ably due to its broad specificity. Twenty-nine of the peptides iden-tified from the Alcalase hydrolysate contained Pro or Hyp at one ofthe three C-terminal positions (Table 4). And many of the peptidescontained hydrophobic amino acids at one or more of the three C-terminal positions. These could all have high ACE-inhibitory activ-ity, since the C-terminal tripeptide plays a predominant role incompetitive binding to the active site of ACE especially if this hasa high hydrophobicity (Byun & Kim, 2002; Gomez-Guillen et al.,2011) and especially in the presence of proline. This is further sub-stantiated by the fact that the peptide GPIGPV (retention time35.8 min, Table 4) has the same C-terminal as GPV, which has beenisolated from bovine gelatin and shown to have an IC50 of 4.67 lM(Kim et al., 2001). Furthermore, the peptides EP(Hyp)GPAGLPGP(retention time 21.6 min) and LTGSPGSPGP (retention time32.4 min) share the three C-terminal amino acids with the peptideGFPGP (IC50 = 91 lM) obtained from porcine skin gelatin by a pro-tease from Aspergillus oryzae (Ichimura et al., 2009) and withGAPGLPGP (IC50 = 29 lM) obtained from chicken collagen by pro-tease FP (Saiga et al., 2008). The peptide RGPAGP (Retention time16.6 min) just has an additional N-terminal Arg residue in compar-ison to GPAGP, which possesses ACE-inhibitory activity (Ichimuraet al., 2009). The peptides mentioned may thus contribute to theACE-inhibitory activity of this hydrolysate.

Several of the peptides identified further contained Lys or Arg atthe ultimate C-terminal position, which should contribute to theinhibitory potency (Hernandez-Ledesma, Contreras, & Recio,2011). Therefore, at least 40 of the peptides released by Alcalase,from bovine collagen, are expected to have good ACE-inhibitoryactivity. Among them nine of the peptides identified seem particu-larly interesting, i.e. GDRGDAGPK, GVAGPK, MGPR, GEGGPQGPR,GPSGPPGPK, GLPGPK RGPPGPM GPPGPAGPR and GPAGPPGPI,since they contain both Lys, Arg or a very hydrophobic amino acidat the ultimate C-terminal position and Pro at the penultimate po-sition. None of these 9 peptides have previously been reported inthe literature, and the ACE-inhibitory activity remains to bedetermined.

4. Conclusions

The present results confirm that collagen is a good substrate forrelease of potent ACE-inhibitory peptides. With Alcalase and colla-genase the ACE-inhibitory peptides can be released without anypretreatment. When other enzymes are used, a pretreatment, such

as boiling for 5 min, is necessary to obtain a good yield of activepeptides. Interestingly, HP-treatment, which is receiving increas-ing interest for protein denaturation, also significantly influencedthe outcome of the subsequent hydrolysis, but the effect was notthe same for all enzymes. HP-treatment significantly improvedthe DH and ACE-inhibitory activity of hydrolysates made withAlcalase and collagenase, whereas the opposite was the case forhydrolysates made with trypsin and pepsin. With proteinase Kand thermolysin, a HP pre-treatment increased DH but not theACE-inhibtory activity. The highest ACE-inhibitory activity was ob-tained from HP-treated collagen subsequently hydrolysed withAlcalase for 30 min, and all identified sequences from this hydroly-sate were from the triple helical structure of collagen. These resultsimply that collagen rich by-products from animal processingplants might be turned into valuable bioactive peptides by properpre-treatment and enzymatic treatment.

Acknowledgements

We gratefully acknowledge the financial support from the Chi-nese Government through the scholarship under the State Scholar-ship Fund for Yuhao Zhang, and the support from the DanishStrategic Research Council through the NOVENIA project. We thankCristian De Gobba for skillful help with the ACE assay. We are alsothankful to Kirsten Sjøstrøm, Jie Yin and Sisse Jongberg, Depart-ment of Food Science, for their technical assistance and advice.

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