Antioxidant Activity of Hydrolysates Prepared from Flaxseed Cake Proteins Using Pancreatin. Magdalena Karamać*, Anna Kulczyk, Katarzyna Sulewska

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1 Originl rtile Setion: Food Qulity nd Funtionlity Pol. J. Food Nutr. Si., 2014, Vol. 64, No. 4, pp DOI: /pjfns Antioxidnt Ativity of Hydrolystes Prepred from Flxseed Cke Proteins Using Pnretin Mgdlen Krmć*, Ann Kulzyk, Ktrzyn Sulewsk Deprtment of Chemil nd Physil Properties of Food, Institute of Animl Reprodution nd Food Reserh, Polish Ademy of Sienes, Tuwim 10, Olsztyn, Polnd Key words: flxseed protein hydrolystes, pnretin, degree of hydrolysis, ntioxidnt tivity Proteins were isolted from deftted flxseed ke nd hydrolysed with pnretin. The hydrolysis proess ws onduted t stle temperture of C nd ph 7.5, nd monitored with the ph stt method. The otined hydrolystes with degree of hydrolysis (DH) of 5, 10, 15, 20, 25% were investigted in terms of ntioxidnt properties. The rdil svenging tivity ws ssyed ginst DPPH nd ABTS +, the reduing ility with FRAP ssy, nd the pility to ind Fe(II) y retion with ferrozine. SE HPLC nlysis ws used to determine moleulr weight distriution of hydrolysis produts. The ntirdil tivity of pnretin hydrolystes of flxseed proteins ws inresing long with n inresing DH nd for the hydrolyste with DH 25% the EC vlue determined with the DPPH ssy ounted for mg/ssy, nd the ABTS + svenging tivity for mmol Trolox/g. This hydrolyste ws onstituted minly y peptides with low moleulr weights (MW) of kd. In turn, the Fe(II) inding pility inresed from 44.5% to 64.9% in the se of hydrolystes with DH 5 20% nd deresed in the se of the hydrolyste with DH 25%. A similr dependeny ws oserved in the ility of pnretin hydrolystes of flxseed proteins to redue Fe(III). The mximum vlue of reduing ility rehed 0.25 mmol Fe(II)/g for the hydrolyste with DH 20% tht ws predominted y polypeptides nd peptides with MW of D. INTRODUCTION Reently, growing interest hs een oserved in the enzymti hydrolysis of proteins s proess whih ensures the finl produt with enhned iologil tivity. Ample studies hve demonstrted tht enzymti hydrolysis of proteins results in the relese of ntihypertensive (ACE inhiitory), ntioxidtive, immunomodultory, ntimiroil, osteoprotetive, ntilipemi nd opioid peptides [Hrtmnn & Meisel, 2007; Möller et l., 2008; Zmrowiz et l., 2013]. The iotive peptides my e relesed upon immedite effets of proteses on protein sustrtes s well s during gstrointestinl digestion of proteins nd protein hydrolysis y miroorgnisms pplied in fermenttive proessing of foods [Korhonen & Pihlnto, 2006]. The enhned iologil potentil of hydrolyste ompred to the ntive proteins is ffeted y the quntity nd hemil struture of relesed iopeptides. The mino id omposition, mino ids sequene nd the length of the peptide hin determine the enefiil helth effets [Hrtmnn & Meisel, 2007; Smrnyk & Li Chn, 2011]. The size of tive sequenes my vry from two to twenty mino id residues [Korhonen & Pihlnto, 2006]. Certinly, this sequene of mino ids should our in sustrte protein, nd proteses seleted for the hydrolysis proess should e le to * Corresponding Author: E mil: m.krm@pn.olsztyn.pl (M. Krmć) disrupt the pproprite peptide onds nd relese the pproprite frgment of the peptide hin. Highly signifint is lso the djustment of hydrolysis onditions, i.e. temperture (T), ph, initil onentrtion of sustrte (S 0 ), enzyme to sustrte rtio (E/S), nd time (t) [Adler Nissen, 1986; Krmć et l., 2002]. Adler Nissen [1986] demonstrted tht four of these prmeters, i.e. T, S 0, E/S nd t, might e ontrolled with simultneous determintion of hydrolysis degree (DH). For given protein enzyme system, DH monitoring is therefore suffiient for omplete ontrol of the proess onduted t stle ph of the retion medium. Sustrtes pplied in the prodution of enzymti hydrolystes with iologil tivity inlude proteins of oth niml nd plnt origin. It is frequently pplied solution to mnge high protein y produts of the food industry [Krmć et l., 2005; Popovi et l., 2013]. These y produts inlude, mong others, flxseed ke eing residue fter oil solvent extrtion from seeds or seeds pressing. Flxseed ke ontins from 31.3 to 40.9% of protein [Oomh & Mzz, 1993; Mueller et l., 2010]. Its proteins re hrterised y enefiil mino id omposition with reltively high ontents of sprti id, glutmi id, leuine nd rginine [Oomh & Mzz, 1993]. Some reports show tht their nutritive vlue nd mino id profile re omprle to these of soyen proteins [Retfik et l., 2011]. Despite tht, flxseed ke is pplied only s feedstuff nd the protein omponent of flxseed in rrely investigted in the spet of nutritionl pplitions. Copyright y Institute of Animl Reprodution nd Food Reserh of the Polish Ademy of Sienes 2014 Author(s). This is n open ess rtile liensed under the Cretive Commons Attriution-NonCommeril-NoDerivs Liense (

2 228 Antioxidnt Ativity of Pnretin Hydrolystes from Flxseed Cke Proteins So fr, the gretest ttention of reserhers hs een foused on enzymti hydrolystes of flxseed proteins with the ntihypertensive tivity [Udenigwe et l., 2009; Udenigwe & Aluko, 2010; Mrme et l., 2008, 2011]. By using Allse or Flvourzyme it is lso possile to produe flxseed protein hydrolystes with the ntioxidnt tivity [Mrme et l., 2008; Silv et l., 2013]. In turn, Udenigwe et l. [2009] ompred the ntirdil nd nti inflmmtory tivity of low moleulr weight nd tioni peptide frtions isolted from hydrolystes produed using Allse, pepsin, fiin, trypsin, ppin, thermolysin nd pnretin. In view of the ove, the gol of this study ws to isolte proteins from flxseed ke, to produe pnretin hydrolystes with vrious degrees of hydrolysis (DH) nd to determine their ntioxidnt tivity depending on their DH. MATERIAL AND METHODS Mteril The initil mteril in the study ws ke of flxseeds of Reitl ultivr otined from the Oil Prodution Plnt in Grodzisk Wielkopolski (Polnd). Flxseed kes ontined 38.2±0.46% proteins (N 6.25), 10.3±0.78% lipids, 6.4±0.06% sh, 8.4±0.06% moisture (n=3) [AOAC, 1990] nd 36.7% rohydrtes (lulted y differene). Isoltion of proteins from flxseed ke Flxseed ke (. 200 g) ws disintegrted in lortory mill nd oil residues were removed y 3 2 h extrtion with n hexne (1:4, w/v) t room temperture. Protein ws isolted from deftted nd dried ke ording to the proedure desried y Dev & Quensel [1988] with modifition proposed y Udenigwe et l. [2009] nd smll modifitions mde for the purpose of this study. An queous suspension of ke (1:20, w/v) ws djusted to ph 5.0 with 2 mol/l HCl nd pled in wter th with shking t 37 C. Cellulse from Aspergillus niger (Sigm Aldrih, tivity ~0.8 units/mg) ws dded to the suspension in dose of 10 mg/g ke in order to hydrolyse muilge of the ke. The retion ws rrested fter 4 h y hnging ph into 9.5 using 2 mol/l NOH. Proteins were extrted for 2 h in lkline onditions, t room temperture, under ontinuous stirring. After entrifugtion (10 C, 30 min, 4000 g, MPW 3 entrifuge, MPW Med. Instruments, Polnd), the superntnt ws dented nd the preipitte ws suspended in the NOH solution with ph 9.5, nd extrtion ws repeted. Afterwrds, 0.2 mol/l HCl ws dded to the omined superntnts till ph 4.0 hs een rehed. The preipitted proteins were entrifuged t 10 C for 30 min t 8700 g. The preipitte ws then suspended in smll volume of wter, neutrlised (0.2 mol/l NOH), nd dilysed for 48 h t 4 C ginst deionised wter tht ws exhnged 8 times. Dilysis ws onduted using moleulr porous memrne tuing of MW ut off t 6 8 kd (Spetr/Por, Spetrum Lortories, USA). The resultnt isolte ws dried y lyophilistion for ~48 h t C nd mr (FreeZone 6 Liter Freeze Dry System, Lono, USA), nd stored t 4 C. Hydrolysis of flxseed proteins The flxseed protein isolte ws hydrolysed with pnretin (Sigm Aldrih). A protein suspension in wter (1:20 w/v) ws djusted to ph 7.5, nd then the enzyme ws dded in dose of 15 mau/g. The retion ws run under stle ph nd temperture ( C) using the ETS 822 end point titrtion system working in the ph stt mode (Rdiometer Anlytil, Denmrk). Bsed on the volume of 0.5 mol/l NOH used in the retion, the degree of hydrolysis (DH) ws determined. to the eqution provided y Adler Nissen [1986]: B M DH = P h tot 100% where: B se onsumption (ml), M molrity of the se (mol/l), α verge degree of dissoition of the α NH 2 groups, P mss of protein (g), nd h tot totl numer of peptide onds in the protein (meqv Leu NH 2 /g protein). A series of hydrolyti retions were rried out tht were rrested when DH rehed 5, 10, 15, 20 nd 25%, y heting t 100 C for 5 min. The ontent of protein in the isolte (82.2±0.39%, n=6) ws determined with the Kjeldhl s method (N 6.25) [AOAC, 1990], wheres the totl numer of peptide onds in proteins (h tot ) ws ssyed sed on the numer of the NH 2 groups relesed during idi hydrolysis of protein in 6 mol/l HCl t 105ºC nd determined using the spetrophotometri method with o phthlldehyde (OPA) [Pnsiuk et l., 1998]. SE HPLC seprtion The SE HPLC seprtion of hydrolystes ws onduted on TSKgel G2000SW XL olumn (5 μm, mm, Tosoh Biosiene, Tokyo, Jpn) oupled with Shimdzu HPLC system: LC 10AD Vp pump, SPD M10A Vp photo diode rry detetor, nd SCL 10A Vp system ontroller (Kyoto, Jpn). Aetonitrile : wter : trifluoroeti id (45:55:0.1 v/v/v) ws pplied s moile phse with flow rte of 0.5 ml/min. Then, 20 μl portions of hydrolyste solutions with the onentrtion of 5 mg/ml or the solution of mixture of moleulr weight (MW) stndrds (Sigm Aldrih): lumin from hiken egg white (45 kd), ytohrome C (12.4 kd), protinin (6.5 kd), ngiotensin II ette (1.046 kd), leuine enkephlin (0.556 kd), VAL TYR VAL (0.379 kd) nd GLY TYR (0.238 kd) were injeted onto the olumn. Detetion ws monitored t 216 nm. ABTS ssy The ility of hydrolystes to intivte ABTS + ws determined with the method of Re et l. [1999] with modifition enling rrying out the retion on multi well pltes. ABTS (96 mg) ws tivted in 2.45 mmol/l solution of potssium persulfte for 16 h. The stok solution ws diluted in wter t the rte of 1: (v/v). Hydrolystes were dissolved in 0.1 mol/l phosphte uffer (ph 7.0) in the onentrtion of 2 mg/ml. Then, 10 µl portions of the smple were pplied onto 96 well plte nd 200 µl of ABTS solution heted to 30ºC were dded. The plte ws fixed in n Infinite M1000 miroplte reder (Ten, Switzerlnd) nd shken. The -

3 M. Krmć et l. 229 sorne ws red out fter 6 min t λ=734 nm. The results were lulted using the stndrd urve for Trolox (r=0.999) or redued L glutthione (r=0.991) nd expressed in mmol of stndrd equivlents per g of hydrolyste. DPPH ssy The DPPH svenging tivity of the hydrolystes ws ssyed with the method of Brnd Willims et l. [1995] djusted to sorne detetion in multi well pltes. The hydrolystes were dissolved in 0.1 mol/l phosphte uffer (ph 7.0) in onentrtions rnging from 0.4 mg/ml to 2 mg/ml. To 100 µl of hydrolyste solutions, there were dded 100 µl of 0.3 mol/l solution of DPPH in methnol. The retion ws ontinued for 30 min t room temperture. Asorne ws red out t λ=517 nm using n Infinite M1000 miroplte reder. Solutions of Trolox nd redued L glutthione were pplied onto the plte in prllel. Curves of the dependeny of % svenged DPPH on smple onentrtion were plotted for eh hydrolyste nd stndrd. The resultnt urves enled determining the EC vlue tht indites the quntity of hydrolyste (mg/ssy) neessry to intivte % of DPPH rdils. FRAP ssy The reduing ility of hydrolystes ws estimted vi the ferri reduing ntioxidnt power (FRAP) ssy [Benzie & Strin, 1996]. To this end, 40 μl portions of hydrolystes dissolved in deionized wter (1 mg/ml) or solutions of stndrd ntioxidnts Trolox ( mg/ml) nd redued L glutthione (0.125 mg/ml), were pplied onto 96 well plte. Afterwrds, 200 μl portions of heted to 37 C regent onsisting of: 0.3 mol/l ette uffer with ph 3.6, 10 mmol/l 2,4,6 Tris(2 pyridyl) s trizine in 40 mmol/l HCl nd 20 mmol/l FeCl 3 6H 2 O in the rtio of 5:1:1 (v/v/v), were dded to the smples. After 30 min inution t 37 C, sorne ws deteted t λ=593 nm (Infinite M1000 plte reder). Results were expressed in mmol of Fe(II) per g of hydrolyste/stndrd sed on stndrd urve plotted for FeSO 4 7H 2 O (r=1). Fe(II) inding tivity The ility of hydrolystes to ind Fe(II) ions ws determined using the spetrophotometri method with ferrozine [Krmć & Pegg, 2009]. The retion ws rried out on 96 well plte. After mixing 200 μl of queous solutions of hydrolystes (2 mg/ml) with 20 μl of 0.4 mmol/l FeCl 2 4H 2 O, 40 μl of 5 mmol/l ferrozine were dded to wells. Solutions sorne ws red out fter 10 min t λ=562 nm using n Infinite M1000 plte leder. The perentge of ound Fe(II) ws lulted for eh smple. Sttistil nlysis All nlyses were rried out in t lest three replitions. Results in figures re presented s men vlues with stndrd devition. The results of ntioxidnt tests were nlysed y one wy ANOVA sttistil model with Tukey s multiple omprison test (GrphPd Prism version 6.04 for Windows, GrphPd Softwre, USA). The differenes were onsidered signifint t p<0.05. RESULTS AND DISCUSSION The pplied onditions of hydrolysis (initil onentrtion of sustrte 20%, pnretin dose 15 mau/g, temperture C, ph 7.5) enled produing hydrolystes with DH 5, 10, 15, 20 nd 25%. The mximum DH vlue 25% is omprle to the vlues otined during pnretin hydrolysis of proteins from seeds of other plnts [Vldez Ortiz et l., 2012; Betnur Anon et l., 2014]. Moleulr weight distriution of produts of flxseed proteins hydrolysis with pnretin ws presented in Figure 1. Pnretin is mixture of enzymes tht ontins proteses hrterised y mjor tivities of trypti, hymotrypti nd elstse types, nd displying roxypeptidse tivity [Andrimihj et l., 2013]. Owing to tht, it is ple of relesing simultneously polypeptides with vrious lengths of the peptide hin nd single mino ids from proteins. Hene, the hydrolyste with the lowest DH (5%) ontined produts of hydrolysis with wide rnge of moleulr weights (MW) from to 12.4 kd (Figure 1A). The hromtogrm shows lso pek with retention time of 12.9 min whih orresponds to proteins with MW 45 kd. An inrese of DH to 10% used n inrese in the mount of hydrolysis produts with MW kd. Further inrese in DH vlue ws ompnied y tngile suessive derese of peks of the polypeptides with the highest moleulr weights ( kd) nd n enlrgement of peks orresponding to lower MW. The hydrolyste with DH 20% ws predominted y ompounds with MW kd. In turn, sed on the hromtogrm from the seprtion of the hydrolyste with DH 25% it my e onluded tht it ontined minly peptides with MW kd (Figure 1E). Suessive degrdtion of polypeptides to oligopeptides nd mino ids in the ourse of pnretin hydrolysis of penut mel ws demonstrted y Zheng et l. [2013]. These uthors frtionted hydrolystes sed on MW nd oserved tht the lrge peptide frtion with MW >5 kd deresed drstilly, while the smller peptide frtions of 3 5 kd nd <3 kd inresed. The ntirdil tivity of pnretin hydrolystes of flxseed proteins with vrious DH vlues ws nlysed ginst two syntheti rdils: ABTS + (Figure 2) nd DPPH (Figure 3). In the se of oth rdils, grdul inrese ws noted in rdil svenging tivity long with n inresing DH of hydrolystes. A similr orreltion regrding ABTS + nd DPPH ws demonstrted for hydrolystes of hot nd old pressed penut mels treted with pepsin followed y pnretin [Zheng et l., 2013]. Also the ABTS + svenging tivity of ukwhet proteins digested with the sme system of enzymes ws inresing in the ourse of hydrolysis [M et l., 2010]. A few studies demonstrted tht frtions of peptides with the lowest MW seprted from pnretin hydrolystes were hrterised y high ntirdil tivity ginst ABTS + or DPPH [M et l., 2010; Alshi et l., 2014]. Results of our study re onsistent with this oservtion. The hydrolyste with DH 25%, whih ws the most effetive in rdils svenging, ontined minly smll peptides with MW kd.

4 230 Antioxidnt Ativity of Pnretin Hydrolystes from Flxseed Cke Proteins A B C D E 7 F FIGURE 1. SE HPLC seprtion of flxseed ke protein hydrolystes with different degree of hydrolysis (DH): A 5%, B 10%, C 15%, D 20%, E 25% nd moleulr weight stndrds F: 1 lumin from hiken egg white (45 kd), 2 ytohrome C (12.4 kd), 3 protinin (6.5 kd), 4 ngiotensin II ette (1.046 kd), 5 leuine enkephlin (0.556 kd), 6 VAL TYR VAL (0.379 kd), 7 GLY TYR (0.238 kd).

5 M. Krmć et l. 231 ABTS ntioxidnt tivity mmol Trolox/g mmol gluthtione/g d FIGURE 2. ABTS + ntioxidnt tivity of flxseed ke protein hydrolystes with different degree of hydrolysis. The rs with different letters re signifintly different t p<0.05. EC (mg/ssy) d Trolox Glutthione Aording to the proedure y Re et l. [1999] nd ommon prtie, the ABTS + ntioxidnt tivity of hydrolystes ws expressed in Trolox equivlents. In the se of protein hydrolystes, peptides re the potentil ntioxidnt ompounds, hene results of the ABTS test in our study were dditionlly expressed in equivlents of redued glutthione s n exmple of ntioxidnt with hemil struture of peptide (Figure 2). The otined results expressed per oth stndrds were similr nd rnged from to mmol Trolox/g nd from to mmol glutthione/g. Similr vlues rehing μmol Trolox/g were hieved y Ng et l. [2013] for trypsin hydrolystes from plm kernel ke proteins with DH 30 %. Slightly higher vlues were reorded y Zheng et l. [2013] for old nd hot pressed penut mels digested with pepsin pnretin system, i.e. 9 nd 527 μmol Trolox/g, respetively. In turn, ommeril isolte of pe proteins hydrolysed with ppin demonstrted signifintly lower ABTS + svenging tivity 28 mmol Trolox/kg d.m. [Zili et l., 2012]. The lowest EC vlue mg/ssy determining the mximum DPPH svenging tivity ws noted for the flxseed protein hydrolyste with DH 25%. The EC vlues for Trolox nd redued glutthione were, respetively, 28.6 nd 13.2 times lower thn the vlue ssyed for the most tive hydrolyste. Only negligily lower ntirdil tivity ginst DPPH ompred to redued glutthione ws oserved y Alshi et l. [2014] for nol mel protein hydrolyste otined using pnretin. Glutthione ws hrterised y. 7 fold lower EC thn the hydrolyste. In ontrst, signifintly lesser differenes in DPPH svenging tivity of hydrolystes nd stndrd were reported y other uthors [Pownll et l., 2010; Ajiol et l., 2011]. However, in these investigtions results of DPPH ssy were expressed s % of svenged rdil. FIGURE 3. DPPH svenging tivity of flxseed ke protein hydrolystes with different degree of hydrolysis. The rs for hydrolystes with different letters re signifintly different t p< d e FRAP mmol Fe(II)/g Fe(II) ound (%) Trolox Glutthione 0 FIGURE 4. Ferri reduing ntioxidnt power (FRAP) of flxseed ke protein hydrolystes with different degree of hydrolysis. The rs for hydrolystes with different letters re signifintly different t p<0.05. FIGURE 5. Fe(II) inding ility of flxseed ke protein hydrolystes with different degree of hydrolysis. The rs with different letters re signifintly different t p<0.05.

6 232 Antioxidnt Ativity of Pnretin Hydrolystes from Flxseed Cke Proteins The ntioxidnt tivity of ompounds is determined not only y their rdil svenging tivity ut lso y their pility to prtiipte in redox retions, nd to e more speifi y their reduing ility. Therefore, nother test ws onduted to determine the pility of pnretin hydrolystes of flxseed proteins to redue Fe(III) ions to Fe(II) (Figure 4). The lowest reduing power ws demonstrted for the hydrolyste with DH 5%. The ility of hydrolystes with DH 5 20% to redue Fe(III) ws inresing suessively. The reduing power of the hydrolyste with DH 25% ws lower thn tht of the hydrolyste with DH 20%. The lower reduing tivity of the hydrolyste ontining minly peptides with low MW is quite unexpeted. In mny works, frtiontion of hydrolystes demonstrted tht smller size peptides exhiited etter reduing ility thn high MW frtions [Li et l., 2008; Ajiol et l., 2011]. It should, however, e notied tht differenes oserved in reduing ility etween prtiulr hydrolystes of flxseed proteins were smll. The FRAP vlues determined for pnretin hydrolystes of flxseed proteins rnged from 0.21 to 0.25 mmol Fe(II)/g. The stndrd ntioxidnts redued glutthione nd Trolox ted signifintly stronger nd were hrterised y, respetively,. 7.5 fold nd 17 fold higher reduing ility thn the nlysed hydrolystes. Even weker reduing tivity ompred to glutthione ws demonstrted y Afrin ym en seed proteins hydrolysed with Allse nd y pe protein hydrolyste produed using Thermolysin [Ajiol et l., 2011; Pownll et l., 2010]. It is known tht redued forms of some trnsition metl ions elerte oxidtive proesses y prtiipting in the Fenton/Her Weiss retions tht result in the formtion of very retive hydroxyl rdils [Borg, 1993]. The tion of ntioxidnts onsists in heltion of ions of pro oxidtive metls. In the se of pnretin hydrolystes of flxseed proteins with vrious DH, their ility to ind with Fe(II) ws nlysed in the study (Figure 5). Likewise in the se of reduing ility, Fe(II) omplextion y hydrolystes ws inresing long with their DH inresing from 5% to 20%, nd for the hydrolyste with the highest DH it ws oserved to derese. The mximum vlue of ound Fe(II) rehed 64.9%, nd the miniml 44.5%. Zhng et l. [2014] investigted the effet of DH on iron inding pity of soy protein hydrolystes nd demonstrted tht, likewise in our study, the ility of Flvourzyme hydrolystes to ind Fe(II) rehed the mximum vlue t some DH nd then deresed. In turn, Ajiol et l. [2011] noted tht the metl ion helting effet of peptide frtions with MW <1 kd nd frtions with higher MW did not differ sttistilly. CONCLUSIONS Flxseed ke proteins treted with pnretin exhiited ntioxidnt tivity. Pnretin hydrolystes were ple of svenging DPPH nd ABTS +, reduing Fe(III) ions nd inding Fe(II). Their ntioxidnt tivity depended on their degree of hydrolysis (DH). The study showed tht the most tive svengers of free rdils were flxseed protein hydrolystes with the highest degree of hydrolysis. However, the reduing ility nd ility to form omplexes with metl ions rehed the mximum vlues in the se of the hydrolyste with slightly lower DH. It my, therefore, e onluded tht depending on the mehnism of tion the most effetive ntioxidnts re pnretin hydrolystes of flxseed proteins with DH 20 25%. Their ntioxidnt tivity is sried to peptides with MW kd relesed from proteins. ACKNOWLEDGEMENTS We grtefully knowledge the finnil support from the Ntionl Siene Centre (Polnd) for (projet No. 2011/01/B/NZ9/00253). REFERENCES 1. Adler Nissen J., Enzymti Hydrolysis of Food Proteins. 1986, 1 st ed., Elsevier Applied Siene, London, pp ; Ajiol C.F., Fshkin J.B., Fgemi T.N., Aluko R.E., Effet of peptide size on ntioxidnt properties of Afrin ym en seed (Sphenostylis stenorp) protein hydrolyste frtions. Int. 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