Effects of Chemical Modification and Molecular Weight Distribution on Iron Binding Ability of Phytate-Removal Soybean Protein Isolate Hydrolysate

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1 Advne Journl of Food Siene nd Tehnology 4(2): 78-83, 12 ISSN: Mxwell Sientifi Orgniztion, 12 Sumitted: Jnury 14, 12 Aepted: Ferury 9, 12 Pulished: April, 12 Effets of hemil Modifition nd Moleulr Weight Distriution on Iron Binding Aility of Phytte-Removl Soyen Protein Isolte Hydrolyste Mo-nn Zhng, Gung-rong Hung nd Ji-xin Jing ollege of Life Sienes, hin Jiling University, P.R. hin Astrt: The im of this study ws foused on investigting the effets of hemil modifitions nd moleulr weight distriution of phytte-removl Soyen Protein Isolte (SPI) hydrolyste on Degree of Hydrolysis (DH) nd the iron inding ility. The hemil modifitions inluded id tretments, suinyltion, etyltion nd phosphoryltion. Also the effets of moleulr weight distriution of phosphorylted SPI trypsin hydrolyste on iron inding ility were studied. The results showed tht eti id tretment n inrese signifintly the DH of SPI hydrolyste, ut did not inrese the iron-inding ility of SPI hydrolyste. Phosphoryltion y Sodium Trimetphosphte (STMP) n inrese signifintly the ironinding ility, ut did not hnge the DH. The STMP phosphoryltion retion time of min ws suffiient for improving the iron inding pity nd lkline onditions were enefiil of phosphoryltion retion. The frtion of moleulr size mong 3 to 5 kd exhiited highest iron inding ility, ut there were no signifint different in mino id ompositions etween the four ultrfiltertion frtions. Key words: Amino id omposition, hydrolysis, minerl inding ility, phosphopeptides, phosphoryltion, trypsin INTRODUTION Soyen is one of the rihest soures of nturl protein mong the plnt. Good qulity nd funtionlity of its proteins, hypollergeniity, low ost nd surplus vilility re other dded fetures tht motivted the reserhers to explore its utility in wide rnge of foods inluding the omplementry foods. However, the presene of phytte t different levels in soyen sed produts hs een shown to inhiit tre minerl sorption (Dvidson et l., 4). Phytte is known to hve strong ffinity for zin, iron nd other tre minerls in foods nd feeds. Its ssoition with storge proteins results in reltively high onentrtions of phytte in ommeril soy protein produts, suh s soy mel, tht re minly used for swine nd poultry feeds (Erdmn, 1979). Phytte uses poor sorption of essentil eletrolytes nd minerls nd inds to proteins nd o-preipittes with isoeletri soyen protein isolte (SPI) (Niols nd Lwrene, 7). Iron sorption inresed four to five-fold when phytte ws redued from its ntive mount of to less thn.1 mg/g of isolte (Rihrd et l., 1992). Soyen protein hydrolystes re physiologilly etter thn intt proteins euse their intestinl sorption ppers to e more effetive due to the inrese in soluility nd peptide ontent (Ziegler et l., 1998), thus mking them ttrtive s soure of mino ids or peptides in humn nutrition. Soyen protein hydrolystes hve een shown to hve vrious physiologil tivities, inluding hypolipidemi nd hypoholes-terolemi properties (Ysuyuki nd Yoshikw, ), improvement in rteril ompline nd endothelil funtion (Ringseis et l., 5), insulin resistne nd weight loss in oesity (Hermnsen et l., 1). hemil modiwtions resulted in inrese signiwntly in soluility of the proteins (Zhng et l., 7), the smll peptide ontent with desired ntioxidnt tivity, the nitrogen solule index of whet gluten (Lio et l., ) nd the mount of lium ound to the soyen gloulins (Kumgi et l., 2). A limited trypsin hydrolyste of the isolted phosphopeptides exhiited n enhned lium inding ility (Yng et l., 4). As hydrolysis proeeded, signifint inreses in surfe hydrophoiity, protein soluility, emulsifying tivity index nd emulsion stility index were oserved in the hydrolystes (Wu et l., 1998). Jing nd Mine (1) found tht the smller frgment of less thn 1 kd nd O-phospho-L-serine did not ind lium to ny signifint extend, while phosphopeptide of 1-3 kd showed higher ility thn sein phosphopeptides to render solule lium. The reserh of the ironhelting peptides from soyen protein hydrolystes demonstrted tht the highest inding mount on the olumn ourred with -3 kd hydrolyste, while the pility of the inding iron ws different in the three moleulr size frtions (Lv et l., 9). orresponding Author: Gung-rong Hung, ollege of Life Sienes, hin Jiling University, P.R. hin 78

2 Adv. J. Food Si. Tehnol., 4(2): 78-83, 12 However, few works hve een pulished fousing on the effet of physil nd hemil modifitions efore hydrolysis of soyen protein with low-phytte ontent on the iron inding ility of the hydrolyste. In this study, SPI hydrolystes with low-phytte ontent were enzymtilly prepred y trypsin. The effets of hemil modifitions of SPI on the degree of hydrolysis nd iron inding ility of hydrolyste, s well s the effet of moleulr weight distriution on iron inding pity of hydrolyste, were investigted. MATERIALS AND METHODS Mterils: SPI ws purhsed in Ferury 11 from lol Food Mrket, Hngzhou ity, Zhejing Provine, hin. It ws immeditely trnsported to the L of Food Siene nd Tehnology, hin Jiling University nd stored t -º until used. Trypsin (2, 5 U/mg), Amerlite 717, suini id, suini nhydride nd Sodium Trimetphosphte (SMTP) were purhsed from Shnghi hemil Regent o. Ltd., hin. Bovine serum lumin ws purhsed from Sigm hemil o. (St. Louis, USA). All other hemils used in the experiments were from ommeril resoure nd of nlytil grde. Phytte removl of SPI: Phytte ws removed from the SPI using nion exhnge resin (Amerlite 717) y the method of Kumgi et l. (4) with some modifitions. In simply, the nion exhnge resin ws wshed suessively with 1. N Hl, distilled wter, 1. N NOH, distilled wter nd 5 mm ph 7.4 Tris-Hl uffer, respetively. The soy protein isolte (.5%, w/v) ws suspended in 5 mm Tris-Hl uffer-resin solution nd stirred t 4º for 2 h. Then the mixture ws filtered through duplex otton loth nd the filtrte ws dilyzed overnight ginst distilled wter t 4/. The ontents were olleted nd lyophilized. hemil modifition of SPI: The phytte-removl SPI solution (5%, w/v) ws prepred nd then ws mixed with different ontent of itri Aid (A), Aeti Aid (AA), Hydrohlori Aid (HA), STMP, Sodium Sulfte (SS), Suini Aid (SA) or Suini Anhydride (SAH) to form suspensions. The suspensions were inuted for 2 h in wter th shker t room temperture. Then the suspension ws entrifuged (, g, min, 4º) nd the superntnt ws dilyzed ginst distilled wter t 4º for overnight to remove the mmonium, phosphors or other ions nd then freeze-dried. SPI without hemil modifition under the sme onditions ws used s the ontrol. Preprtion of SPI hydrolyste: Trypsin ws hosen to hydrolysis SPI to produe iotive peptides. The hydrolysis onditions were: temperture of 37º, time durtion of 1 min, ph 8. nd E/S of 1:5. A 5. g SPI powder ws homogenized with 1. L of uffer solution (5 mm Tris-Hl, ph 8.) nd the homogente ws preheted min t 37º then dding 1. g trypsin to the mixture to egin hydrolysis. At the end of hydrolysis, the solution ws oiled for min to intivte the enzyme nd then entrifuged t 8, g for min t 4º. The superntnt ws olleted for further study. Determintion of protein ontent: The protein ontents were determined y Folin-phenol method (Lowry et l., 1951) using Bovine Serum Alumin (BSA) s stndrd. Degree of hydrolysis: The Degree of Hydrolysis (DH) ws dewned s the perentge of peptide onds leved during retion nd lulted from the rtio of free mino groups to the totl numer of peptide onds in solule ftions, ording to the method desried y Jens (1979): = [solule protein ontent/totl protein protein] Determintion of iron inding ility: Iron inding ility of SPI hydrolyste ws determined y modifition of the ferrozine method (rter, 1971). A 2 ml peptide (8 :g/ml) nd 2 ml of distilled wter were mixed with :L 1 mm FeSO 4. After min, :L 5 mm ferrozine ws dded nd mixed well. The solule iron ws determined. Ultrfiltrtion of SPI hydrolyste: SPI hydrolystes were olleted nd sujeted to ultrfiltrtion memrnes system with moleulr weight utoff of, 5 nd 3 kd, respetively. Four peptide frtions with different moleulr weight rnges were prepred: Frtion I (> kd) Frtion II (5- kd) Frtion III (3-5 kd) Frtion IV (<3 kd) The peptide frtions were lyophilized nd stored t -5º until use. Amino id omposition nlysis: The totl mino id omposition ws determined ording to the method of Heu et l. (3). The protein smples were pled in mpoules nd mixed with 6 N Hl. After seling the mpoule under vuum, the smples were hydrolyzed t 1º for 24 h. The smple were diluted, filtered nd loded on Modle S433D utomti mino id nlyzer (Sykm orp., Eresing, Germny) for mino id nlysis. Sttistil nlysis: The smple tretments in the present study were run in triplite nd ll nlyses were 79

3 Adv. J. Food Si. Tehnol., 4(2): 78-83, 12 Tle 1: Effets of hemil modifition on the degree of hydrolysis (DH) nd iron inding ility Methods of hemil Protein ontent DH Iron inding ility modifition (mg/ml) (%) (:g/mg-protein) ontrol 6.53± ±.65 d 18.47±.13 A 7.± ±.25 d 14.9±.18 e AA 7.26±..37±.14.9±.48 d HA 7.15± ±.36 d 14.15±.5 f STMP 6.6±. d 17.86±.7 d.±.36 SS 6.54± ± ±.27 SA 5.92±.22 d 19.42±. 1.15±.59 h SAH 6.82± ±.39 e 6.79±.58 g The vlues re Men D of triplite mesurement; Vlues with different letters in the sme olumn re signifintly different (p<.5); A: itri id; AA: Aeti id; HA: Hydrohlori id; STMP: Sodium trimetphosphte; SS: Sodium sulfte; SA: Suini id; SAH: Suini nhydride performed in duplite. Dt were expressed s mens±stndrd devitions (SD). The differenes etween the mens of the tretments were ompred y one-wy ANOVA t signifine level of p<.5. Sttistil nlyses were performed with the SPSS pkge (SPSS 13. for windows, SPSS In., higo, IL). RESULTS AND DISUSSION hemil modifition: hemil modifitions of soyen protein produts to hnge their properties hve eome inresingly populr s reserh re in the lst twenty yers. The purpose of these modifitions inlude the redution of phytte ontent, the redution of protese tivity, the inrese or derese in protein soluility to otin etter funtionl properties of soluility-dependent, the inrese in nutritive vlue y ovlent inding of mino ids with sulfur, nd the elimintion undesirle odor or flvor. The methods of hemil modifitions inlude id-lkli denturtion, hemil intivtion of protese inhiitors, redution of phytte onentrtion, suinyltion, etyltion, mleyltion nd phosphoryltion, et. The effets of id tretments, suinyltion, etyltion nd phosphoryltion on the iron inding ility of phytte-removl SPI trypsin hydrolyste were shown in Tle 1. Aeti id tretment n inrese signifintly the DH of SPI hydrolyste, ut did not inrese the iron-inding ility. Phosphoryltion y STMP n inrese signifintly the iron-inding ility nd did not hnge the degree of hydrolysis. Phosphoryltion is one of the most importnt modifitions of food proteins to inrese in soluility nd derese in pi of the proteins, therey hnging the funtionl properties, espeilly ners the pi of the originl proteins. Beuse phophte groups n e tthed to the oxygen of seryl, threonyl, sprtyl ($roxyl) nd tyrosyl residues nd vi nitrogen to lysyl (,-mino) nd histidyl (1 nd 3) residues. STMP hd een reported to e very effetive phosphoryltion regent of SPI (Lee et l., 5). The phosphoryltion onditions with STMP, inluding temperture, ph, ddition quntity nd time, would ffet the DH nd iron inding ility of 26 () 3 28 () STMP (%) Time (min) () o Temperture ( ) (d) ph Fig. 1:,,, d: Effets of phosporyltion onditions with Sodium Trimetphosphte (STMP), STMP onentrtion (), time (), temperture () nd ph (d), on the degree of hydrolysis (DH) of Soyen Protein Isolte (SPI) hydrolyste. Vlues with different letters re signifintly different (p<.5) 8

4 Adv. J. Food Si. Tehnol., 4(2): 78-83, 12 Iron inding ility ( µ g/mg-protien) () d STMP (%) Iron inding ility ( µ g/mg-protien) () 12 8 d Time (min) Iron inding ility ( µ g/mg-protien) () o Temperture ( ) Iron inding ility ( µ g/mg-protien) (d) ph Fig. 2:,,, d: Effets of phosporyltion onditions with Sodium Trimetphosphte (STMP), STMP onentrtion (), time (), temperture () nd ph (d), on iron inding ility of Soyen Protein Isolte (SPI) hydrolyste. Vlues with different letters re signifintly different (p<.5) the phytte-removl SPI hydrolyste. The retion onditions were optimized, in order to stisfy iron inding ility of SPI hydrolyste, s shown in Fig. 1 nd Fig. 2. The retion onditions did not improve signifintly the DH of hydrolyste (Fig. 1). The iron inding ility of phosphoryltion SPI hydrolyste ws performed with inresing STMP onentrtions, t phs of 6 to 11. As shown in Fig. 2, the ddition of STMP up to 2.5% (w/v) indued proportionl ugmenttion in the iron inding ility level, whih might e used y the inrese in phosphoryltion levels. Our result ws in greement with the dt otined in reent report (Lee et l., 5), whih the lium inding ility of SPI peptides ws improved with STMP ddition. The phosphoryltion retion time of min ws suffiient for improving the iron inding ility, s shown in Fig. 2. Alkline onditions (ph 9. or higher) were enefiil of phosphoryltion retion, whih improved the iron inding pity of phytte-removl SPI hydrolyste (Fig. 2d). Moleulr weight distriution nd mino id ompositions: The reltionship etween the moleulr weight distriution of protein hydrolyste nd their minerl inding property or enhne of minerl sorption is not fully understood. It hs een reported tht smller frgment of less thn 1kD phosphopeptides derived from hen yolk phosvitin is unle to ind lium to signifint extent (Jing nd Iron inding ility ( µ g/mg-protien) d < > Moleulr weight distriution (kd) Fig. 3: Effets of moleulr weight distriution on iron inding ility of Soyen Protein Isolte (SPI) hydrolyste. Vlues with different letters re signifintly different (p<.5) Mine, 1). Lee nd Song (9) purified n iron inding non-peptide (55 D) from hydrolystes of porine lood plsm protein. However, tripeptide nd hexpeptide from et-sein enhned iron sorption signifintly y o-2 ells (hud et l., 7). In the present study, the effets of the moleulr size of the phosphopeptides derived from phosphorylted SPI nd its susequent trypsin hydrolyste were studied. The hydrolystes were ultrfilterted into four frtions with moleulr weight utoff of, 5 nd 3 kd, respetively. The iron inding ilities of the four frtions were determined, s shown in Fig. 3. The frtion III with 81

5 Adv. J. Food Si. Tehnol., 4(2): 78-83, 12 Bound iron ontent ( µ g/ml) PP SPI hydrolyste Protein ( µ g/ml) Fig. 4: The iron inding ilities of Soyen Protein Isoltes (SPI) hydrolyste nd sein Phosporyltion Peptide (PP). Vlues with different letters re signifintly different (p<.5) Tle 2: Amino id omposition of soyen protein isolte (SPI) nd peptide frtions y ultrfiltrtion of trypsin hydrolyste Amino SPI Frtion I Frtion II Frtion III Frtion IV id (%) ( %) (%) (%) (%) Asp Thr Ser Glu Gly Al ys Vl Met Ile Leu Tyr Phe Lys His Arg Pro Totl..... molr rtio The moleulr weight distriution of four frtions were Frtion I (> kd), Frtion II (5- kd), Frtion III (3-5 kd) nd Frtion IV (<3 kd), respetively moleulr size of 3-5 kd showed highest iron inding pity. seinophosphopeptides (PP), whih ontin inding sites, phosphoseryl nd roxyl, for different minerls, hve een widely studied mong metl-inding peptides. In present study, the iron inding ility of the frtion III of phytte-removl SPI hydrolyste with moleulr size of 3-5 kd ws ompred with tht of PP t different onentrtions, s shown in Fig. 4. At low peptides ontent, the SPI peptides hd the sme iron inding ility s PP. However, t high peptides ontent, PP hd higher iron inding pity tht SPI peptides. The mino id ompositions of SPI trypsin hydrolyste nd four ultrfilertion frtions were shown in Tle 2. There were no signifint different in mino id ompositions etween the four frtions. But the frtion III with moleulr size of 3-5 kd exhiited higher iron inding pity thn tht of other frtions (Fig. 3), whih might e relted to its higher phosphoryltion level. ONLUSION hemil modifitions ffeted the iron inding pity of phytte-removl SPI trypsin hydrolyste. Phosphoryltion with STMP inresed signifintly the iron inding ility of SPI hydrolyste. Severl frtions with different moleulr size exhiited ovious different iron inding ility. The middle moleulr size (3-5 kd) frtion hd highest iron inding ility. REFERENES rter, P., Spetrophotometri determintion of serum iron t the sumirogrm level with new regent (ferrozine). Anl. Biohem., 4: hud, M.V.,. Izumi, Z. Nhl, T. Shuhm, P.L. Binhi nd O. De Freits, 7. Peptides isolted from in vitro digests of milk enhne iron uptke y o-2 ells. J. Agri. Food hem., 55: Dvidson, L., E.E. Zeigler, P. Kstenmyer, P.V. Del nd D. Brly, 4. Dephytinistion of soy en protein isolte with low ntive phyti id ontent hs limited impt on minerl nd tre element sorption in helthy infnts. Br. J. Nutr., 91: Erdmn, J.W., Oilseed phyttes-nutritionl implitions, J. Am. Oil hem. So., 56: Hermnsen, K., M. Sondergrd, L. Hoie, M. rstensen nd B. Brok, 1. Benefiil effets of soy-sed dietry supplement on lipid levels nd rdiovsulr risk mrkers in type 2 dieti sujets. Dietes re, 24: Heu, M.S., J.S. Kim nd F. Shnhidi, 3. omponents nd nutritionl qulity of shrimp proessing yproduts. Food hem., 82: Jens, A.N., Determintion of degree of hydrolysis of food protein hydrolystes y trinitroenzeno sulfoni id. J. Agri. Food hem., 27: Jing, B. nd Y. Mine, 1. Phosphopeptide derived from hen egg yolk phosvitin: Effet of moleulr size on the lium-inding Properties. Biosi. Biotehnol. Biohem., 65: Kumgi, H., S. Ishid, A. Koizumi, H. Skuri nd H. Kumgi, 2. Preprtion of phytte-removed demidted soyen gloulins y ion exhngers nd hrteriztion of their lium-inding ility. J. Agri. Food hem., 5: Kumgi, H., A. Koizumi, N. Sto, Y. Ishikw, A. Sud, H. Skuri nd H. Kumgi, 4. Effet of phytteremovl nd demidtion of soyen proteins on lium sorption in the in situ rts. Bioftors, 22:

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