Fractionation of Protein Hydrolysates of Fish and Chicken Using Membrane Ultrafiltration: Investigation of Antioxidant Activity

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1 Appl Biohem Biotehnol (214) 172: DOI 1.17/s Frtiontion of Protein Hydrolystes of Fish nd Chiken Using Memrne Ultrfiltrtion: Investigtion of Antioxidnt Ativity Griel Slete Centenro & Myrim Sls-Melldo & Crl Pires & Irineu Btist & Mri L. Nunes & Crlos Prentie Reeived: 21 Ferury 213 / Aepted: 6 Jnury 214 / Pulished online: 22 Jnury 214 # Springer Siene+Business Medi New York 214 Astrt In this work, hiken nd fish peptides were otined using the proteolyti enzymes α-chymotrypsin nd Flvourzyme. The musle ws hydrolyzed for 4 h, nd the resulting peptides were evluted. Hydrolystes were produed from Argentine roker (Umrin nosi) with degree of hydrolysis (DH) of 25.9 nd 27.6 % nd from hiken (Gllus domestius) with DH of 17.8 nd 2.6 % for Flvourzyme nd α-chymotrypsin, respetively. Memrne ultrfiltrtion ws used to seprte fish nd hiken hydrolystes from Flvourzyme nd α-chymotrypsin sed on moleulr weight utoff of >1,, <1, nd >5, nd <5 D, to produe frtions (F1,, F1, 5, nd F5) with ntioxidnt tivity. Fish hydrolystes produed with Flvourzyme (FHF) nd α-chymotrypsin showed 6.8 nd 5.9 % of peptides with moleulr weight of <3 kd in its omposition, respetively. To hiken hydrolystes produed with Flvourzyme nd α-chymotrypsin (CHC) ws oserved 83 nd 92.4 % of peptides with moleulr weight of <3 kd. The frtion tht showed, in generl, higher ntioxidnt potentil ws F1, from FHF. When dded 4 mg/ml of FHF nd CHC, 93 nd 8 % of lipid oxidtion in ground eef homogentes ws inhiited, respetively. The omposition of mino ids indited higher mino ids hydrophoi ontent nd mino ids ontining sulfuri residues for FHF, whih showed ntioxidnt potentil. Keywords Antioxidnt tivity. Chiken. Fish. Hydrolyste. Peptides. Ultrfiltrtion memrnes G. S. Centenro: M. Sls-Melldo : C. Prentie (*) Lortory of Food Tehnology, Shool of Chemistry nd Food, Federl University of Rio Grnde (FURG), Rio Grnde, Rio Grnde do Sul, Brzil e-mil: dqmprent@furg.r C. Pires: I. Btist : M. L. Nunes Portuguese Institute of Se nd Atmosphere (IPMA, I. P./DMRM), Av. Brsíli, Lison, Portugl

2 2878 Appl Biohem Biotehnol (214) 172: Introdution In reent yers, there hs een n inresing interest in identifying nd hrterizing iotive peptides derived from plnts nd nimls. The iotive peptides re protein frgments tht re intive in the speifi moleulr struture of proteins. After eing relesed y enzymti hydrolysis, they my exert different physiologil funtions [1]. Met nd fish provide vlule soures of protein for mny popultions round the world; furthermore, met nd fish proteins offer huge potentil s novel soures of iotive peptides [2] displying ntioxidnt effets. The hiken met ontined onsiderle mount of histidineontining dipeptides, rnosine (β-lnylhistidine) nd nserine (β-lnyl-1-methylhistidine) [3]. Severl endogenous ntioxidnts, e.g., glutthione, uri id, spermine, rnosine, nd nserine, whih re hrteristi for met hve een studied [4]. Both rnosine (β-lnyl-lhistidine) nd nserine (N-β-lnyl-1-methyl-L-histidine) re ntioxidtive histidyl dipeptides nd the most undnt ntioxidnts in met [5]. The ntioxidnt tivity of these dipeptides my result from their ility to helte trnsition metls [6] nd form omplexes with, e.g., opper, zin, nd olt. These ntioxidtive peptides hve lso een reported to ply role in wound heling, reovery from ftigue nd prevention of diseses relted to stress [7]. Different peptides derived from hydrolyzed food proteins hve demonstrted iotive properties. These peptides hve size of 2 to 2 mino ids [8] nd moleulr weight elow 6, D [9]. Bsed on its struturl properties, its omposition, nd mino id sequene, these peptides my ply multiple roles, suh s immunomodultory [1], ntimiroil [11], ntithromoti [12], hypoholesterolemi [13] ntihypertensive [14], nd ntioxidnt [15 2]. In ddition, severl peptides were lso hrterized for hving multifuntionl properties [7]. Reserh foused on the peptides ontined in protein hydrolystes of niml proteins showed tht they ontin moleules tht n ring helth enefits, whih re promising for nutritionl nd phrmeutil pplitions [21]. Free rdils re speies ontining one or more unpired eletrons, re produed in norml or pthologil ell metolism [22], nd re involved in mny hroni diseses suh s dietes, rdiovsulr nd neurodegenertive diseses nd ner. The superoxide (O 2 ) nd hydroxyl rdil (HO ), known s retive oxygen speies, re some of the free rdils tht re formed in vivo [23] nd n use destrutive nd lethl ellulr effets y oxidizing lipids, proteins, DNA, nd enzymes. In foods, lipid peroxidtion generted y free rdils, is mjor onern during the industril proessing nd storge of produts, s, despite deresing the nutritionl qulity ndfoodsfety[24], they lso led to the development of seondry retion produts whih re potentilly toxi, nd of undesirle flvors nd odors nd lso hnge in texture [17, 25]. The syntheti ntioxidnts suh s utylted hydroxynisole, utylted hydroxytoluene (BHT), propyl gllte, nd tert-utyl hydroquinone re used to inhiit or retrd the formtion of free rdils, preventing lipid oxidtion nd the resulting deteriortion of foods. However, the sfety nd negtive pereption of syntheti ntioxidnts y onsumers restrit their pplitions in food produts [26]. Although the syntheti ompounds re effiient nd of reltively low ost, speil ttention hs een given to nturl ntioxidnts euse of worldwide trend to void or minimize the use of syntheti dditives [24, 27]. The ultrfiltrtion (UF) memrne tehnology hs een of gret importne for the purifition, onentrtion, nd frtiontion of severl produts in vrious res suh s food, phrmeutil, nd iotehnology industries nd hs een one of the est ville tehniques for the enrihment of peptides [28]. However, there is little informtion out the ntioxidnt tivity of musle protein hydrolystes of hiken nd no pper ws found on the ntioxidnt tivity of hydrolystes from Argentine roker (U. nosi). In this reserh, fish nd hiken hydrolystes were produed

3 Appl Biohem Biotehnol (214) 172: enzymtilly nd frtionted y UF tehnique using memrnes with moleulr weight utoff of 1, nd 5 D. Were otined frtions with different moleulr weight tht were investigted in their ntioxidnt tivity. The ojetive of this study ws to otin peptide frtions from the hydrolystes using memrnes UF, hrterize the hydrolystes nd frtions in reltion to moleulr weight distriution, nd evlute the ntioxidnt tivity of frtions. Mterils nd Methods Rw Mterils The fish speies used ws roker (U. nosi), donted y Pesl S/A industry, loted in Rio Grnde, RS, Brzil. After pture, the fish ws fillet nd the musle (ontining 18.2 % protein) ws mined uniformly nd stored frozen t 2 C in polyethylene gs until use. The frozen hiken musle (ontining 21.4 % protein) ws donted y Minuno Food Co. loted in Ljedo, RS, Brzil, nd stored frozen t 2 C in polyethylene gs until use. Chemils nd Enzymes The enzymes used were Flvourzyme 1 L (mixture of endoprotese nd exopeptidse from Aspergillus oryze), donted y Novozymes Ltin Ameri (Aruri, Brzil) nd α-chymotrypsin (endopeptidse otined from ovine pnres) provided y Sigm-Aldrih Co. (St. Louis, MO, USA). The other regents used in this study were of nlytil grde. Preprtions of Protein Hydrolystes The musle ws homogenized (IKA, RW28, Stufen, Germny) with phosphte uffer.2 mol/l t rtio of 1:3 (w/v), onsidering the protein ontent. Before the retion, suspensions were pre-inuted in jketed glss retor t optimum onditions for eh enzyme (5 C nd ph 7. for Flvourzyme nd 37 C nd ph 8. for α-chymotrypsin) for 2 min. The hydrolysis retion initited y dding enzyme in the mount of 1 % (w/w) with stirring t 6 rpm (IKA, RW28, Stufen, Germny) for 4 h. Smples were tken t preset times (zero, 15, 3, 6, 12, nd 24 min) to mesure the DH ording to the method trinitroenzenesulfoni id (TNBS) desried erlier [29] for determining perentge of hydrolysed peptide onds y mesuring the numer of free mino groups in the superntnt, y the retion with TNBS. A stndrd leuine urve ws prepred from onentrtion of to 1.6 mmol/l. At the end of retion time, the enzyme ws thermlly intivted t 85 C for 15 min with osionl shking. The hydrolystes were entrifuged t 3,5 g (entrifugl Biosystems, MPW-35/35R, PR, Brzil) for 2 min to remove unhydrolysed mteril nd the otined superntnt ws lyophilized (freeze dryer MiroModulyo, Edwrds, Sussex, UK). Fish nd hiken hydrolystes were produed with Flvourzyme (FHF; CHF) nd α-chymotrypsin (FHC; CHC) enzymes. Eletrophoresis For the seprtion of peptides y moleulr weights the tehnique of eletrophoresis on polyrylmide gel ontining sodium dodeyl sulfte (SDS-PAGE) in onentrtion of

4 288 Appl Biohem Biotehnol (214) 172: % [3] ws used. All smples were treted with β-merptoethnol (5 %). Gels were run t onstnt voltge of.2 A for out 1 h nd lter it ws inresed to.4 A for more 1 h. The stining of the gels ourred in solution of methnol (5 %, v/v), eti id glil (6.8 %, v/v), nd Coomssie Brillint Blue-R (1 mg/ml) for 3 h. The gels were distining in solution of eti id (7.5 %, v/v) nd methnol (5 %, v/v), renovting the solution until ler gel ws otined. The moleulr weight of peptides ws determined y omprison with stndrds of moleulr weight of 1 22 kd (Benh MrkTM Protein Ldder, Invitrogen, SP, Brzil). Frtiontion y UF The hydrolystes were frtionted using n UF ell (Advnte, UHP-76, Jpn) under mgneti stirring with pity of 45 ml. UF memrnes regenerted ellulose, 76 mm dimeter (Amion In., Beverly, MA, USA) with moleulr weight utoff of 5 nd 1, D were used. During filtrtion proess pressure ws pplied with nitrogen, s indited y the mnufturer of the memrnes. A smple volume (1 mg/ml) ws pssed through eh memrne where three frtions were olleted: F1, retentte (frtion, >1, D), permete nd retentte F1, 5 (frtion, <1, nd >5 D), nd F5 permete (frtion, <5 D). All frtions were then lyophilized nd nlyzed. Moleulr Weight Distriution y Gel Filtrtion The moleulr weight distriution of the protein hydrolyste ws estimted y gel filtrtion hromtogrphy on fst performne liquid hromtogrphy (FPLC AKTA-Amershm Biosienes, Sweden) equipped with Superdex peptide 1/3 GL, ontining 3 % etonitrile nd trifluoroeti id.1 % s eluent. The void volume of the olumn ws determined with lue dextrn 2. The flow rte ws.5 ml/minute nd redings were mde t 28 nm. For quntittive determintion of the moleulr weights, res of peks of the hromtogrms were integrted. A lirtion urve with rionulese A (13,7 D), protinin (6,5 D), ngiotensin I (1,296 D), nd tryptophn (24 D) ws prepred s desried erlier [31]. Antioxidnt Ativity The ntioxidnt tivity of the frtions of the hydrolystes of fish nd hiken were evluted using different tests tht inluded the sequestrtion ility of the hydroxyl rdil (OH ), the ility to sequester free rdil 2,2-diphenyl-1-piryl-hydrzyl (DPPH), the sequestrtion ility of the ABTS free rdil nd reduing power. Hydroxyl Rdil Svenging Ativity The ility of hydrolystes frtions to inhiit the hydroxyl rdils, formed y Fenton retion y degrdtion of 2-deoxy-D-riose, ws determined [32]. A mixture ontining.2 ml of 1 mmol/l FeSO 4 7H 2 O,.2 ml of 1 mmol/l EDTA,.2 ml of 1 mmol/l 2-deoxy-D-riose,.2 ml of smple (1 mg/ml), nd 1 ml of phosphte uffer solution (.2 mol/l, ph 7.4) ws homogenized with.2 ml of 1 mmol/l H 2 O 2 nd inuted t 37 C for 4 h. Then 1 ml of 2.8 % TCA nd 1 ml of 1 % TBA ws dded, nd the smples were heted in wter th t 1 C for 1 min. Then ooled y immersion in wter th nd ie for 5 min nd the sorne (As) ws red t 532 nm on

5 Appl Biohem Biotehnol (214) 172: spetrophotometer UV/VIS (ATI UNICAM Helios, Alph, UK). The perentge inhiition ws lulted s follow: Inhiition ð% Þ ¼ As ontrol As smple =Asontrol 1 DPPH Free Rdil Svenging Ativity The free rdil svenging effet of DPPH ws mesured s desried erlier [33]withsome modifitions. Smples of 1. ml with different hydrolysed frtions (zero,.5, 1., 2.5, nd 5. mg/ml) were dded in 1. ml of DPPH (.1 mmol/l) in 95 % ethnol. The mixture ws homogenized with vortex (Phoenix, AP-56, Brzil) nd kept 3 min t room temperture. The As of the resulting solution ws mesured t 517 nm in spetrophotometer UV/VIS (ATI UNICAM Helios, Alph, UK). Lower As represents higher sequestering tivity of DPPH, whih ws lulted ording to eqution 1. The vlues of the effetive onentrtion (onentrtion le to inhiit 5 % of the oxidtion (EC 5 )) were lulted from the inhiition perentge. ABTS Rdil Antioxidnt Ativity The ntioxidnt tivity ws mesured ording to Re et l. [34] with some modifitions. 2,2-zinois(3-ethylenzthizoline-6-sulfoni id) (ABTS) ws dissolved in wter t onentrtion of 7 mmol/l. The rdil tion (ABTS + ) ws produed y retion of ABTS stok solution with potssium persulphte (2.45 mmol/l, finl onentrtion) nd keeping the mixture in the drk t room temperture for 16 h efore use. The ABTS + solution ws diluted in sodium phosphte uffer ph 7.4 (5 mmol/l) to n As of.7±.2 to 734 nm. After ddition of 2 ml of smple (5 mg/ml) to 2 ml of diluted ABTS +, the mixture ws homogenized with vortex (Phoenix, AP-56, Brzil) nd inuted in wter th t 3 C for 6 min. The As reding ws rried out t 734 nm in spetrophotometer UV/VIS (Hithi U-21, Tokyo, Jpn). The syntheti ntioxidnt Trolox (solule derivtive of α-toopherol) in onentrtions 1,5 μm (in ethnol) ws used s stndrd nd the results were expressed s Trolox equivlent ntioxidnt pity in mmol/g smple. Reduing Power The ility of hydrolysed frtions to redue Fe 3+ to Fe 2+ ws mesured spetrophotometrilly [35]. A volume of 2 ml of smple (5 mg/ml) ws dded in 2 ml phosphte uffer.2 mol/l (ph 6.6) nd 2 ml of 1 % potssium ferriynide. The mixture ws inuted t 5 C for 2 min, nd therefter ws dded 2 ml of 1 % TCA. A 2-mL liquot ws mixed with 2 ml of distilled wter nd.4 ml of.1 % ferri hloride in test tues. After 1 min of retion, the As of the resulting solution ws red t 7 nm on spetrophotometer UV/VIS (ATI UNICAM Helios, Alph, UK). Asori id (AA) ws used s stndrd for the onstrution of lirtion urve nd reduing power is expressed s mol AA/g of smple. Antioxidnt Ativity in Ground Beef The ntioxidnt tivity in ground eef ws determined s desried y Sknk [36]. Mined met (5 g) ws homogenized with 25 ml of 5 mmol/l HEPES uffer (ph 7.). The mixture ontining.8 ml of suspension of mined met nd.2 ml HEPES uffer or

6 2882 Appl Biohem Biotehnol (214) 172: smple of solution of hydrolyste (hydrolyste in HEPES uffer) t finl onentrtions of 2.5, 5, 1, 2, nd 4 mg/ml were inuted t 37 C for 6 min. After inution, the mixture ws evluted for the formtion of sustnes tht ret with thiorituri id retive sustnes (TBARS). A stok solution of TBA in TCA ws prepred ontining.9 mol/l TCA nd.3 mol/l TBA in.25 mol/l HCl. After slight wrming nd stirring to dissolve the omponents, 3 ml of 2 g/l BHT in solute ethnol were dded to 1 ml TCA/TBA stok solution. At pproprite intervls, liquots of 1. ml of the mixture were dded to tues ontining 2 ml TCA/TBA stok solution nd immeditely homogenized with vortex (Phoenix, AP-56, Brzil). The smples were then heted to oiling for 1 min in wter th, ooled to room temperture nd entrifuged t 1,71 g (Biosystems, MPW-35/35R, PR, Brzil) for 1 min. The As of the superntnt ws mesured t 532 nm. TBARS were quntified from stndrd urve of mlondildehyde using tetrmethoxypropne. Amino Aid Composition of Hydrolyste The determintion of the mino id omposition of smples of roker musle hydrolyste with Flvourzyme nd hiken with α-chymotrypsin were onduted y the Fulty of Mediine of Rieirão Preto (UNESP, Rieirão Preto, Brzil). The mino ids were otined y hydrolysis of the peptides with HCl 6 mol/l for 22 h t 11 C±1 C [37]. Sttistil Anlysis All determintions were performed in triplite. The dt for the different prmeters were ompred using nlysis of vrine using the progrm STATISTICA version 7. (Sttsoft In., Tuls, OK, USA). Signifint differenes (p<.5) etween the results were identified using the Tukey test. Results nd Disussion Enzymti Hydrolysis In this study, roker musle proteins nd hiken were seprtely hydrolyzed with α- Chymotrypsin nd Flvourzyme for the prodution of protein hydrolystes. The hydrolystes were frtionted y UF memrnes. The three frtions otined for eh hydrolyste produed hd their ntioxidnt tivity evluted. The extent of hydrolysis of proteins y proteolyti enzymes ws estimted y the DH yielding in the hydrolyste fish vlues of 25.9 nd 27.6 % nd the hiken hydrolyste, 17.8 nd 2.6 %, with Flvourzyme nd α- Chymotrypsin, respetively. There ws rpid initil inrese in DH, s the time inresed, inditing very high protein levge in the erly hours of retion. Then there ws stiliztion of the hydrolysis retions. The stiliztion or redution in the rte of retion n e explined y inhiition of the enzyme y produts tht re formed in high levels of DH [38]. These produts ompete with the sustrte using none digestion or prtil digestion of proteins. The shpe of the urves of hydrolysis ws similr to tht reported in previous studies with slmon protein [39], tun [4], hiken [41]ndtilpi[28]. With the sme sustrte nd the sme mount of enzyme, α-chymotrypsin showed higher DH vlues for hydrolystes of fish nd hiken thn Flvourzyme. The highest levels of DH otined with α-chymotrypsin suggest tht this enzyme hs more ffinity for the sustrte nd thus more effiient thn Flvourzyme for the prodution of protein hydrolystes of fish musle nd poultry. Generlly,

7 Appl Biohem Biotehnol (214) 172: the lkline proteses suh s α-chymotrypsin exhiit higher tivity thn id or neutrl proteses [42, 43]. Eletrophoresis The hydrolystes were nlyzed through eletrophoresis (SDS-PAGE) in order to determine the moleulr weight profile nd lso to onfirm tht hydrolysis hd een rehed. The eletrophoreti profiles showed tht the enzymti tretments were effetive in the degrdtion of oth fish nd hiken musle, resulting in smll peptides s shown in Fig. 1. After hydrolysis, the presene of two peptide nds ws oserved, with n estimted moleulr weight of 1 kd nd one elow 1 kd, for the fish musle hydrolyste with the enzyme Flvourzyme. Rossini et l. [44] lso oserved the presene of two peptide nds with moleulr weights estimted t 9 nd 14 kd for peptides with ntioxidnt tivity of sein produed with Flvourzyme. Five peptide nds were oserved for the hiken musle hydrolyste with the enzyme α-chymotrypsin. Four of these of higher moleulr weight, eing estimted in the rnge of 35, 27.5, nd 2 kd nd the remining nds with moleulr weight estimted t 15 kd nd less thn 1 kd [44]. In the study of Souissi et l. [38], the eletrophoreti profiles of hydrolystes of Srdinell urit showed moleulr weight rnge less thn 14.2 kd nd two nds with moleulr weights in the rnge of 55 nd 3 kd. Aording to these uthors, this my e due to lrger proteins present in the rw mteril or proteins tht were not totlly hydrolyzed y the enzyme. Fig. 1 Eletrophoreti profile of fish hydrolystes with Flvourzyme (), hiken hydrolystes with α-chymotrypsin (), nd moleulr weight stndrds () C A B

8 2884 Appl Biohem Biotehnol (214) 172: During the hydrolysis, levge of the protein involves greter struturl hnge, in whih the protein is hydrolyzed into smller peptide units [45]. Compring the eletrophoreti profiles, it ws found tht FHF (DH=25.9 %) showed nds with moleulr weights lower thn the CHC (DH=2.6 %), inditing tht the enzyme Flvourzyme fvored the hydrolysis of the musle proteins. Frtiontion y UF The use of the UF memrne is suitle to otin protein hydrolystes with rnges of desired moleulr weight [46]. During the UF proess in two stges, three different frtions (from F1,, F1, 5, nd F5 D) were otined using memrnes with utoff of 1, nd 5 D. The ultrfiltrted (F1, 5 nd F5 D) nd retentte (F1, D) were rih in peptides with moleulr weights in ordne with the utoff of its respetive UF memrne used. However, smll perentge of peptides, ove or elow the utoff of eh memrne were found in the nlyzed frtions. Aording to some uthors, the memrnes don t perform seprtions with totl effiieny, nd some lsses of moleulr weights n e found in severl frtions. A omplited phenomenon euse of the physil nd hemil ftors ours during the filtrtion of solutions ontining proteins nd proteineous solids, solule nd insolule, whih uses onentrtion polriztion nd fouling/logging of the memrne, nd derese in flow [47]. Given their hydrophoiity, intertions etween proteins nd memrnes my our nd re inherent in the proess. Depending on prtile size, the proteins n enter the pores of the memrne or remin s lyer in gel form forming deposit on the surfe, signifintly inresing the resistne to filtrtion [28]. Moleulr Weight Distriution The gel filtrtion hromtogrphy, using FPLC system ws used to study the distriution profiles of the moleulr weight of frtions of fish (FHF nd FHC) nd hiken (CHF nd CHC) protein hydrolystes otined through UF memrnes. From the hromtogrphi dt (Figs. 2 nd 3), there ws redution of high moleulr weight peptides. Considering the whole hydrolystes efore the UF proess (dt not shown), it n e seen tht 6.8 % of the FHF nd 5.9 % of the FHC onsisted of smll peptides, smller thn 3 kd. Similr vlues were reported in hydrolystes otined from silver rp with pnretin nd Neutrse, showing tht 69.3 nd 64.7 % of the peptides hd moleulr weight less thn 3 kd, respetively [48]. However, for CHF nd CHC, 83 nd 92.4 % of ll peptides hd moleulr weight less thn 3 kd, respetively. These results lso gree with dt pulished erlier [49], who report tht hydrolystes of Pollhius virens, speies of the od fmily produed with the enzyme Allse, onsisted of 9 % of peptides less thn 3 kd. In generl, the peptide frtions otined from hiken musle (Fig. 3) showed higher perentge of low moleulr weight peptides in reltion to peptide frtions of the hydrolyste otined from fish musle (Fig. 2). Although the reltive proportions of the peks vried ording to the porosity of the memrne used, the moleulr weight distriutions were similr when ompring the frtions with the sme enzymes, oth s fish nd hiken peptides. From these results, it is verified tht the musle fish nd hiken hydrolystes were proly formed from mixture of peptides of smll size. All frtions, oth fish nd hiken hydrolystes showed high onentrtions of peptides (ove 8 %) ording to the utoff of the memrne used, exept for F1,, whih retined

9 Appl Biohem Biotehnol (214) 172: A F F F B 2 F F F Fig. 2 Moleulr weigth distriution of frtion of fish hydrolystes (F1,, F1, 5, nd F5) with Flvourzyme () nd α-chymotrypsin (). 1, Rionulese A (13,7 D); 2, protinin (6,5 D); 3, ngiotensin I (1,296 D); nd 4, tryptophn (24 D) A F F F mu B F F F Fig. 3 Moleulr weigth distriution of frtion of hiken hydrolystes (F1,, F1 5, nd F5) with Flvourzyme () nd α-chymotrypsin (). 1, Rionulese A (13,7 D); 2, protinin (6,5 D); 3, ngiotensin I (1,296 D); nd 4, tryptophn (24 D)

10 2886 Appl Biohem Biotehnol (214) 172: high onentrtion of peptides with smller moleulr weight thn the porosity of the memrne. This is proly due to the memrne fouling. Antioxidnt Ativity of Frtions Although there re mny methods to evlute the ntioxidnt pity in food systems, none of these n e used s n offiil stndrd method. Therefore, it is suggested tht eh evlution e done using vrious mesurement tehniques, in different medi nd oxidtion onditions [5]. In generl, onsidering the evlution methods employed, it ws oserved tht ll frtions exhiited ntioxidnt tivity. Hydroxyl Rdil Svenging Ativity The sequestrtion ility of the hydroxyl rdil ws investigted y the method of oxidtion of 2-deoxyriose. Figure 4 shows the effet of sequestrtion of the hydroxyl rdil presented y the frtions of the hydrolystes of fish nd hiken musle. The frtions otined from the hydrolystes of fish were more effetive thn the frtions otined from hiken hyrdrolystes in the sequestrtion ility of the hydroxyl rdil. F1, 5, otined from the FHF hs rehed 55.3 % inhiition with 1 mg/ml (Fig. 4). Of the three frtions otined for eh hydrolyste using UF memrnes, F1, 5 showed the highest inhiition for oth nd fish hiken hydrolystes, exept for the F1, 5 of CHF (Fig. 4). Furthermore, fish hydrolystes otined with Flvourzyme were more effiient in sequestering ility of the rdil OH thn those otined with α-chymotrypsin (Fig. 4). As for the hiken hydrolystes, the ehvior ws different nd opposite where the α-chymotrypsin produed frtions with more effiient ntioxidnt power. DPPH Free Rdil Svenging Ativity Figure 5 shows the sequestrtion ility of DPPH of frtionted hydrolystes. The frtion F1 for oth fish nd hiken hydrolystes produed with Flvourzyme nd α- Chymotrypsin showed greter ility to sequester most of the DPPH rdil onentrtions. The EC 5 vlues ple of inhiiting 5 % of the oxidtion were 2.1 nd 1.4 mg/ml for the A Inhiition (%) , d F1 F1-5 F5 B Inhiition (%) , F1 F1-5 F5 Flvourzyme Chymotrypsin Flvourzyme Chymotrypsin Fig. 4 Hydroxyl rdil svenging tivity of frtions of fish () nd hiken () hydrolystes. Vlues represent the men±sd of three determintions. Different letters indite signifint differenes (p<.5)

11 Appl Biohem Biotehnol (214) 172: A 1 8 F1 F1-5 F5 F1 F1-5 F5 B 1 8 F1 F1-5 F5 F1 F1-5 F5 Inhiition (%) 6 4 Inhiition (%) Conentrtion (mg/ml) Conentrtion (mg/ml) Fig. 5 DPPH rdil svenging tivity of frtions of fish () nd hiken () hydrolystes. Solid line, Flvourzyme; dotted line, α-chymotrypsin. Vlues represent the men of three determintions F1, of FHF nd FHC, respetively (Fig. 5). For F1,, of the CHF nd CHC, the EC 5 vlues were 2.9 nd 1.9 mg/ml, respetively (Fig. 5). It ws lso oserved tht the ility to sequester the DPPH rdils presented y frtions of CHF, CHC, nd FHF ws dose dependent, in greement with erlier results [51]. This tendeny ws not oserved in the frtions otined from the FHC, i.e., when the smple onentrtions were inresed ove 2.5 mg/ml, the power of inhiition ws not inresed euse t higher onentrtions hydrolystes my hve used pro-oxidnt effet. ABTS Free Rdil Svenging Ativity The method of pture of the ABTS rdil is widely used to determine ntioxidnt tivity nd is reported s n ssy pplile to disolortion of hydrophili ompounds nd lipophili ompounds, inditing redution of olor nd redution of the ABTS rdil [52]. Figure 6 shows the ility nd sequestrtion of ABTS rdil of frtionted hydrolystes. The frtion F1,, oth for FHF nd CHF showed greter ility to sequester ABTS rdil, while for CHC nd FHC the higher ntioxidnt potentil is displyed y F1, 5. A F1 1, F1-5 F5,8 TEAC (mmol/g),6,4 B 1, TEAC (mmol/g),8,6,4 d F1 F1-5 F5,2,2, Flvourzyme Chymotrypsin, Flvourzyme Chymotrypsin Fig. 6 ABTS rdil svenging tivity of frtions of fish () nd hiken () hydrolystes. Vlues represent the men±sd of three determintions. Different letters indite signifint differenes (p<.5)

12 2888 Appl Biohem Biotehnol (214) 172: Peptides produed from the hydrolysis with different enzymes my vry in their ility to pture ABTS free rdil. These uthors lso found tht the ility to sequester free rdil ABTS of Printhus mrnthus hydrolystes ws higher in the frtion tht ontined peptides with moleulr weight of 1,7 D [53]. Reduing Power The reduing power is the most effetive method to ssess the ility of n ntioxidnt in donting eletrons [54]. Figure 7 shows the reduing power of the frtions of the fish nd hiken hydrolystes. All frtions of the hydrolystes showed some ntioxidnt tivity. The frtions of the hydrolystes prepred with fish musle showed the gretest reduer potentil thn the frtions of the hydrolystes prepred with hiken musle. This is proly due to higher DH vlues displyed y the fish hydrolystes (25.9 nd 27.6 % for FHC nd FHF respetively) or due to inresed vilility of hydrogen ions produed during the hydrolysis of protein [54]. Aording to these results, we n sy tht F1, otined from FHF nd CHF ws the prt tht most ontriuted to the tivity of these hydrolystes. Moreover, F1, 5 ws the frtion tht showed the gretest ontriution to the ntioxidnt tivity of hydrolystes produed with α-chymotrypsin, following the sme trend s the ntioxidnt tivity mesured with ABTS rdil ssy. In this study, strong ntioxidnt tivity in F1, nd F1, 5 D ws oserved. In generl, for the frtions otined from the hydrolystes of fish higher vlues of ntioxidnt tivity of the enzyme Flvourzyme were oserved. However, for the frtions otined from hiken hydrolystes, etter results were presented y the enzyme α-chymotrypsin. These oservtions seem to gree with the findings for the whole hydrolystes. Smll peptides re often relted to higher ntioxidnt tivity, however this ws not oserved in this study [55]. However, hydrolystes of mkerel (Somer ustrisius) with moleulr weight of pproximtely 1,4 D, showed greter in vitro ntioxidnt tivity ginst lipid peroxidtion, sequestrtion ility of rdil DPPH nd reduing power, thn peptides with moleulr weights in the rnge of 9 2 D [56]. Hke ones hydrolystes ws produed nd ws oserved tht the frtion with moleulr weight in the rnge of 1, 3, D, otined fter UF, showed the highest ntioxidnt potentil [57]. These results suggest tht the ntioxidnt tivity of the protein or peptide depends, not only on its moleulr weight, ut lso on nture nd the omposition of the A umol AA/g smple ,d d F1 F1-5 F5 B 5 F1 F1-5 F5 umol AA/ g smple ,,, Flvourzyme Chymotrypsin Flvourzyme Chymotrypsin Fig. 7 Reduing power of frtions of fish () nd hiken () hydrolystes. Vlues represent the men±sd of three determintions. Different letters indite signifint differenes (p<.5)

13 Appl Biohem Biotehnol (214) 172: different peptide frtions produed, properties suh s hydrophoiity, the ility to trnsfer eletrons from the mino id residues in sequene nd hydrolysis onditions [27, 49]. The presene of the ntioxidnt tivity using in vitro ssys is sed on different mehnisms nd reflets the ntioxidnt properties of the multifuntionl peptide. However, neither the struture tivity nor ntioxidnt mehnism re yet fully understood [58]. In generl, the frtiontion improved the ntioxidnt properties of hydrolystes. Although ntioxidnt properties were deteted in ll tested peptide frtions, the highest moleulr weight frtions from hydrolystes produed with Flvourzyme nd α-chymotrypsin presented the highest DPPH svenging tivity. Antioxidnt Ativity in Ground Beef The FHF nd CHC hydrolystes, whih showed good response to the ntioxidnt tivity in vitro were tested in model system of mined eef. The hydrolystes were dded to the homogenized mined eef in different onentrtions, nd lipid oxidtion ws mesured. The retion ws mesured y monitoring the TBARS, nd the results re shown in Fig. 8. Aording to some uthors, the homogenized mined eef n e n effetive mens to investigte the protetive effets of wter-solule ntioxidnts ginst lipid peroxidtion [59]. The ddition of mined eef, FHF nd CHC inhiited respetively, 72 nd 73.8 % of lipid oxidtion dded t onentrtion of 2 mg/ml. When 4 mg/ml of CHC nd FHF ws dded, 93 nd 8 % of lipid oxidtion ws inhiited, respetively. It ws oserved tht the hydrolyste produed with Flvourzyme ws more effiient t inhiiting the oxidtion nd thus n e used s good soure nturl ntioxidnt in met systems. The study of Sknk et l. [35] showed 69.7 % inhiition of the oxidtion s mined eef with peptides otined from lium seinte using the sme onentrtion. Wheres Rossini et l. [44] showed 1 % inhiition of oxidtion using 2 mg/ml sein peptides otined with Flvourzyme. Lee nd Hendriks [58] rehed 76.2 % inhiition with 2 mmol/l of rnosine ( dipeptide endogenous found in skeletl musle of most vertertes). Fig. 8 Effet of fish (FHF) nd hiken (CHC) hydrolystes on the formtion of TBARS in ground eef homogentes. Vlues represent the men±sd of three determintions. Different letters indite signifint differenes (p<.5) TBARS (ug MDA/g smple) d d e Flvourzyme Chymotrypsin e f e Control 2, Conentrtion (mg/ml)

14 289 Appl Biohem Biotehnol (214) 172: Amino Aid Composition of Hydrolyste The mino id omposition of the fish musle hydrolyste otined from Flvourzyme (FHF) nd hiken musle prepred with α-chymotrypsin (CHC) is shown in Tle 1. It ws found tht 34. nd 32.9 % of the mino ids in the FHF nd CHC, respetively, were omprised of hydrophoi mino ids. For hydrolystes of proteins nd peptides, the greter the hydrophoiity, the higher the lipid soluility, nd therefore the ntioxidnt tivity inreses [6]. The highest mount of histidine ws oserved in CHC. Histidine hs the ility to sequester rdil lipid y the imidzole ring [61]. However, the mount of mino id ontining sulfur residues (ysteine nd methionine) ws higher for FHF thn CHC, whih my hve ontriuted more intensely to the higher ntioxidnt tivity of this hydrolyste long with the higher ontent of hydrophoi mino ids, espeilly leuine, lnine, vline, nd isoleuine. Similr results were oserved for sein hydrolystes with Flvourzyme, whih the mount of hydrophoi mino ids ws 35.6 % (exluding tryptophn) [44]. It ws found tht 39.1 nd 4.8 % of hydrophoi mino ids in protein hydrolystes of silver rp musle (Hypophthlmihthys molitrix) were prepred with Flvourzyme for 1.5 nd 4 h of hydrolysis, respetively [62]. Conlusions The different frtions otined showed ppreile levels of ntioxidnt tivity nd rdil sequestering ility. The results showed tht the frtion of fish hydrolyste otined with the enzyme Flvourzyme with moleulr weight greter thn 1, D, presented the highest ntioxidnt tivity. The hydrolyste otined from fish musle with Flvourzyme hd Tle 1 Amino id omposition of the fish (FHF) nd hiken (CHC) hydrolystes (milligrms per grm protein) Amino id omposition of whole hydrolystes Not determined Amino ids (%) FHF CHC Asprti id 91.9± ±.6 Threonine 43.5± ±.2 Serine 45.3± ±3.6 Glutmi id 167.8± ±2.5 Glyine 48.8± ±.1 Alnine 63.3± ±.3 Vline 5.6± ±3. 1/2 ystine 4.6±.1 3.3±.3 Methionine 35.7± ±3.3 Isoleuine 45.6± ±.7 Leuine 85.1± ±1.2 Tyrosine 19.8± ±3.7 Phenyllnine 4.4±.3 4.9±.7 Lysine 1.2± ±3.5 Histidine 24.5± ±.7 Tryptophn Arginine 47.3± ±1. Proline 85.7± ±.6

15 Appl Biohem Biotehnol (214) 172: higher ontent of hydrophoi mino ids nd sulfur mino ids, leding to greter inhiition of lipid oxidtion when used in met system. Aknowledgments This work ws supported y CAPES of Brzil through sholrship grnted to the first uthor y PhD Progrm in Brzil with the Foreign Internship-PDEE (Proess BEX: 76/1-4) nd developed t the Fisheries nd Mrine Reserh Institute (IPMA, I. P./DMRM) in Lison, Portugl. The uthors lso thnk support from the Europen Projet Chill-On (FP ) nd CNPq of Brzil (Grnt 3555/26-2). Referenes 1. Srmdi, B. H., & Ismil, A. (21). Antioxidtive peptides from food proteins: review. Peptides, 31, Kim, E. K., Lee, S. J., Jeon, B. T., Moon, S. H., Kim, B., Prk, T. K., Hn, J. S., & Prk, P. J. (29). Purifition nd hrteristion of ntioxidtive peptides from enzymti hydrolystes of venison protein. Food Chemistry, 114, Chn, K. M., & Deker, E. A. (1994). Endogenous skeletl musle ntioxidnts. Critil Reviews in Food Siene nd Nutrition, 34, Deker, E. A., Livisy, S. A., & Zhou, S. (2). Mehnisms of endogenous skeletl musle ntioxidnts: hemil nd physil spets. In E. A. Deker, C. Fustmn, & C. J. Lopez-Bote (Eds.), Antioxidnts in musle foods (pp. 25 6). New York: Wiley. 5. Guiotto, A., Cldern, A., Ruzz, P., & Borin, G. (25). Crnosine nd rnosine-relted ntioxidnts: review. Current Mediinl Chemistry, 12, Brown, C. E. (1981). Intertions mong rnosine, nserine, ophidine nd opper in iohemil dpttion. Journl of Theoretil Biology, 88, Young, J. F., Therkildsen, M., Ekstrnd, B., Che, B. N., Lrsen, M. K., Oksjerg, N., & Stgsted, J. (213). Novel spets of helth promoting ompounds in met. Met Siene, 95, Meisel, H., & Fitzgerld, R. J. (23). Biofuntionl peptides from milk proteins: minerl inding nd ytomodultory effets. Current Phrmeutil Design, 9, Sun, J., He, H., & Xie, B. J. (24). Novel ntioxidnt peptides from fermented mushroom Gnoderm luidum. Journl of Agriulturl nd Food Chemistry, 52, Guthier, S. F., Pouliot, Y., & Sint-Suveur, D. (26). Immunomodultory peptides otined y the enzymti hydrolysis of whey proteins. Interntionl Diry Journl, 16, MCnn, K. B., Shiell, B. J., Mihlski, W. P., Lee, A., Wn, J., Roginski, H., & Coventry, M. J. (26). Isoltion nd hrteriztion of novel ntiteril peptide from ovine α S1 -sein. Interntionl Diry Journl, 16, Shimizu, M., Swshit, N., Morimtsu, F., Ihikw, J., Tguhi, Y., Ijiri, Y., et l. (28). Antithromoti ppin-hydrolyzed peptides isolted from pork met. Thromosis Reserh, 123, Zhong, F., Liu, J., M, J., & Shoemker, C. F. (27). Preprtion of hypoholesterol peptides from soy protein nd their hypoholesterolemi effet in mie. Food Reserh Interntionl, 4, Ji, J., M, H., Zho, W., Wng, Z., Tin, W., Luo, L., & He, R. (21). The use of ultrsound for enzymti preprtion of ACE-inhiitory peptides from whet germ protein. Food Chemistry, 119, Mendis, E., Rjpkse, N., & Kim, S. K. (25). Antioxidnt properties of rdil-svenging peptide purified from enzymtilly prepred fish skin geltin hydrolyste. Journl of Agriulturl nd Food Chemistry, 53, Wu, H. C., Pn, B. S., Chng, C. L., & Shiu, C. Y. (25). Low-moleulr-weight peptides s relted to ntioxidnt properties of hiken essene. Journl of Food nd Drug Anlysis, 13, Je, J. Y., Lee, K. H., Lee, M. H., & Ahn, C. B. (29). Antioxidnt nd ntihypertensive protein hydrolystes produed from tun liver y enzymti hydrolysis. Food Reserh Interntionl, 42, Aewsiri, T., Benjkul, S., Visessngun, W., Wiereng, P. A., & Gruppen, H. (21). Antioxidtive tivity nd emulsifying properties of uttlefish skin geltin tnni id omplex s influened y types of intertion. Innovtive Food Siene nd Emerging Tehnologies, 11, Bougtef, A., Nedjr-Arroume, N., Mnni, L., Rvlle, R., Brki, A., Guillohon, D., & Nsri, M. (21). Purifition nd identifition of novel ntioxidnt peptides from enzymti hydrolystes of srdinelle (Srdinell urit) y-produts proteins. Food Chemistry, 118, Centenro, G. S., Melldo, M. S., & Prentie-Hernández, C. (211). Antioxidnt tivity of protein hydrolystes of fish nd hiken ones. Advne Journl of Food Siene nd Tehnology, 3,

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