Compositional, functional and sensory properties of protein ingredients

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1 Grdute Theses nd Disserttions Grdute College 2009 Compositionl, functionl nd sensory properties of protein ingredients Zr Mtin Nzreth Iow Stte University Follow this nd dditionl works t: Prt of the Nutrition Commons Recommended Cittion Nzreth, Zr Mtin, "Compositionl, functionl nd sensory properties of protein ingredients" (2009). Grdute Theses nd Disserttions This Thesis is rought to you for free nd open ccess y the Grdute College t Iow Stte University Digitl Repository. It hs een ccepted for inclusion in Grdute Theses nd Disserttions y n uthorized dministrtor of Iow Stte University Digitl Repository. For more informtion, plese contct digirep@istte.edu.

2 Compositionl, functionl nd sensory properties of protein ingredients prepred from gs-supported screw-pressed soyen mel y Zr Mtin Nzreth A thesis sumitted to the grdute fculty in prtil fulfillment of the requirements for the degree of MASTER OF SCIENCE Mjor: Food Science nd Technology Progrm of Study Committee: Lwrence Johnson, Mjor Professor Cheryll A. Reitmeier Chrles E. Gltz Iow Stte University Ames, Iow 2009 Copyright Zr Mtin Nzreth, All rights reserved.

3 ii TABLE OF CONTENTS ABSTRACT iii CHAPTER 1. GENERAL INFORMATION 1 Introduction 1 Thesis Orgniztion 2 Literture Review 3 References 25 CHAPTER 2. FUNCTIONAL PROPERTIES OF SOY PROTEIN ISOLATES PREPARED FROM GAS-SUPPORTED SCREW-PRESSED SOYBEAN MEAL 34 Astrct 34 Introduction 35 Experimentl Procedures 36 Results nd Discussion 40 Conclusions 49 CHAPTER 3. FUNCTIONAL PROPERTIES OF JET-COOKED AND HYDROGEN- PEROXIDE-TREATED SOY PROTEIN ISOLATES 52 Astrct 52 Introduction 53 Experimentl Procedures 54 Results nd Discussion 57 Conclusions 67 CHAPTER 4. SENSORY PROPERTIES OF SOY PROTEIN ISOLATE AND GLYCININ-RICH AND β-conglycinin-rich FRACTIONS PREPARED FROM GAS-SUPPORTED SCREW-PRESSED SOYBEAN MEAL 70 Astrct 70 Introduction 71 Experimentl Procedures 73 Results nd Discussion 78 Conclusions 85 CHAPTER 5. GENERAL CONCLUSIONS 89 Generl Discussion 89 Recommendtions for Future Reserch 91 Acknowledgements 92 APPENDIX A. SENSORY PANEL QUESTIONNIARE 93 APPENDIX B. BASIC SENSORY TESTING 94 APPENDIX C. SENSORY SCORE SHEET FOR SOY PROTEIN SAMPLES 96

4 iii ABSTRACT Soy protein products re gining importnce s ingredients in the food industry. A numer of soyen mels hve een investigted s strting mterils for the production of soy protein ingredients. Hexne-extrcted nd flsh-desolventized soyen mels, known s white flkes (WF), re most commonly used, ut hve disdvntges of contining solvent residue nd eing too expensive for processing identity-preserved soyens. Gs-supported screw pressing (GSSP) is new soyen oil-extrction process tht comines screw pressing with injecting cron dioxide (CO 2 ) under pressure. The ojective of the present reserch ws to investigte GSSP mel nd its protein products y determining yields, composition, functionl properties, preservtion methods nd sensory properties. The properties of GSSP mel proteins were compred to trditionl soy protein products produced from WF. For the lortory-scle study, nlyticl, chemicl nd functionlity tests were performed on the strting mterils nd isolted soy proteins. Soy protein isolte (SPI) prepred from GSSP mel hd higher protein yield, ft content, wter-holding cpcity (WHC) nd viscosity, nd etter emulsifiction nd ft-inding properties thn SPIs prepred from WF. The SPIs produced in the pilot plnt were nlyzed for composition nd functionlity. Hydrogen peroxide (H 2 O 2 ) treted SPIs were compred to jet-cooked SPIs. GSSP SPIs did not differ in functionlity from SPIs prepred from WF, except for hving lower soluility nd poorer foming properties. H 2 O 2 used s preservtive improved soluility, emulsifiction nd foming properties nd reduced glycinin nd β-conglycinin (β-con) denturtion. A descriptive sensory pnel study with 12 trined pnelists evluted the rom, flvor nd mouthfeel of SPI nd glycinin-rich (gly-rich) nd β-conglycinin-rich (β-con-rich) soy protein frctions extrcted from oth GSSP mel nd WF. Protein products prepred

5 iv from GSSP mel were similr to protein products prepred from WF except for hving greter mouthcoting. Regrdless of strting mteril, the gly-rich nd β-con-rich frctions hd stronger fishy rom, less floury rom, less rw eny rom nd less floury flvor thn the SPIs. Hunter color LAB dt indicted GSSP mel ws more yellow (higher * vlue) in color compred to WF. SPIs were drker (lower L* vlue) thn thn the gly-rich nd β-conrich frctions. Overll, protein products prepred from GSSP mel were similr in composition, functionl nd sensory properties to protein products prepred from WF. These findings demonstrte tht the GSSP process cn produce deftted mels suitle for mnufcturing soy protein ingredients. Becuse GSSP plnts cn e profitle t low cpcity (50 mt/dy) compred to solvent extrction (3000 mt/dy), GSSP is suitle for processing identitypreserved soyens tht contin vlue-dded trits. Additionl enefits re tht there re no concerns over residul orgnic solvents nd the process complies with orgnic definitions.

6 1 CHAPTER 1. GENERAL INFORMATION Introduction The United Sttes produced n estimted 2,973 million ushels of soyens in the 2008 crop yer (U.S. Deprtment of Agriculture, 2009). Approximtely 10% of the soyen crop is used directly for humn consumption. An estimted 4 to 5% of the totl soyen mel is processed into soy protein ingredients (soy flour, soy protein isolte nd soy protein concentrte). Although it seems like smll mount, soy protein food ingredients re gining wide cceptnce in the United Sttes. Soy protein is good qulity protein with the highest protein digestiility corrected mino cid score (PDCAAS) mong the vegetle proteins. Tody, soy protein ingredients re lso gining populrity ecuse recent reserch indictes soy protein hs helth enefits. Trditionl hexne-extrcted nd flsh-desolventized soyen mel, lso known s white flkes (WF), is used in the production of soy protein isolte (SPI). The preferred solvent used for oilseeds extrction is hexne, which is not only flmmle, ut cn e toxic nd expensive (Johnson 1998, Friedrich nd List 1982, Li et l. 2006). Trditionl solvent extrction does not enle the production of orgnic soy protein ingredients ecuse of the lrge scle required to e cost effective. Hence, reserchers hve een developing lterntive oil-extrction processes for identity-preserved processing nd determining the compositionl nd functionl properties of the high-protein soyen mels otined y using these processes. Not only is the sfety of protein ingredients importnt, they lso need to hve good compositionl nd functionl properties so they cn e incorported into different food systems. Flvor is criticlly importnt ttriute when using soy protein ingredients in foods. Consumers ssocite undesirle eny off-flvor with soy protein products

7 2 (Rckis et l. 1979). Hence, studying the sensory ttriutes of ny new soy ingredient product is not only resourceful ut necessry. The present reserch investigtes the potentil for new gs-supported screwpressing (GSSP) process for extrcting oil to produce mel from which highly functionl soy protein products cn e produced. In this process, oil is extrcted from dehulled nd flked soyens y injecting liquefied CO 2 under pressure into screw press, producing soyen mel free of solvent residue tht cn e used to produce functionl soy proteins. When the CO 2 flshes (chnges from liquid to gs soring energy) s it exits the screw press, the temperture is immeditely reduced. Becuse of short exposure time to high tempertures t low moisture, little protein denturtion occurs nd the protein remins highly solule. High soluility protein is needed to extrct soy protein in high yield when mking SPI or frctionted soy protein ingredients. Our hypothesis ws tht GSSP soyen mel cn e used to produce high-qulity SPI nd frctionted soy protein ingredients with similr or etter properties thn protein products prepred from WF. The ojectives the present studies were: 1) to determine the yields nd compositionl nd functionl properties of SPIs produced from GSSP mel nd WF in lortory SPI simultion (proof of concept); 2) to evlute the effects of oilextrction nd preservtion methods on the yields nd compositionl nd functionl properties of SPIs prepred from GSSP mel nd WF in the pilot plnt (scle-up); nd 3) to evlute the sensory properties of SPI nd glycinin-rich nd β-conglycinin-rich frctions produced from GSSP mel (sensory study). Thesis Orgniztion This thesis consists of five chpters nd three Appendixes. Chpter 1 includes generl introduction nd literture review. The ensuing chpters 2, 3 nd 4 re journl mnuscripts to e pulished in the Journl of Americn Oil Chemists Society. Chpter 2 entitled Functionl properties of soy protein isoltes prepred from gs-supported screw-

8 3 pressed soyen mel hs een puliction. Chpter 3 entitled Functionl properties of jet-cooked nd hydrogen-peroxide-treted soy protein isoltes nd chpter 4 entitled titled Descriptive sensory nlysis of soy protein isolte nd glycinin-rich nd β- conglycinin-rich frctions prepred from gs-supported screw-pressed soyen mel will lso e sumitted for puliction. Chpter 5 includes generl discussion summrizing pertinent findings nd recommendtions for future reserch sed on the findings of the present reserch. The Appendixes include relevnt ut not pulishle informtion nd not included in the preceding chpters. Literture Review Soyens The soyen plnt is ntive to southestern Asi, where it ws used for its medicinl properties nd high protein content (Johnson et l. 1992). Since the 20 th century, demnd for soyen oil nd protein from deftted mel hve sustntilly incresed. Soyen mel is widely used for supplementing protein in niml feeds. Soyens rnk highest mong ll food crops for its protein content nd second mong ll legumes for its oil content (Liu 1999). Since the 1950 s, production of soy protein products for humn consumption hs incresed; the United Sttes lone produces more thn 454 million kg per yer of soy products for humn consumption (Endres 2001). Protein products produced from soyens include soy flkes, flour, protein concentrtes, SPI, texturized soy proteins nd spun proteins. These ingredients re used in the production of foods such s ked foods, diry, met, rekfst cerel, infnt formul, s well s diry nd met nlogs (Luss nd Rhee 1995).

9 4 Helth enefits Soy protein products re excellent sources of high qulity protein, re low in sturted ft, nd contin dietry fier nd nutrceuticl-vlued isoflvones. Soy protein ingredeints hve een ttriuted numer of eneficil effects on humn helth such s lowering lood cholesterol levels, preventing oesity, providing nutrition nd possily even ply eneficil role in preventing diseses (cncer, osteoporosis, menopusl disorders nd crdiovsculr diseses) (Xio 2007, Mteos-Apricio et l. 2008, Tkmtsu et l. 2003). A rt-feeding study showed tht consuming soy protein diet resulted in 40 to 47% of its iron eing converted to hemogloin iron (Pellett et l. 1990). Consumption of soy protein hs eneficil ffect on renl function (Anderson 2007) nd on reducing weight, diposity (Cope et l. 2008) nd incidence of rest cncer (Wrri et l. 2008). SPIs contin from 88 to 164 mg/100 g of isoflvone (Genovese et l. 2007), which hs lso een reported to provide helth enefits in humns (Xio 2007, Isng nd Zhng 2008, Adlercreutz nd Mzur 1997). Soyens, however, contin ioctive compounds tht my hve dverse helth effects (Isng nd Zhng 2008). In ddition, soyens lso contin digestive enzyme inhiitors, which led to poor digestiility; ut, this cn e eliminted y proper heting (Friedmn nd Brndon 2001). Some other limittions to the widespred consumption of soyens nd its products re its llergenicity (Bllmer-Weer nd Vieths 2008), eny tste nd odors. A study on fctors ssocited with consumers eting helthy rekfst cerel determined consumers void soy-sed products due to unfvorle tste despite widespred promotion of soy s helthy ingredient (Lee et l. 2007).

10 5 Composition Soyens contin pproximtely 40% protein, 20% oil nd 35% crohydrtes on dry sis (Perkins 1995). The mjority of soyen proteins re storge proteins (65-80%) s opposed to functionl or structurl proteins. Soy storge proteins re composed of two primry proteins glycinin (primrily 11S) nd β-conglycinin (primrily 7S), nd their contents vry with soyen vriety nd environmentl conditions under which they re grown. Bsed on soluility, soy proteins they my e further clssified s lumins (wter solule) nd gloulins (slt solule). Most of the soy storge proteins re gloulins nd re deposited in protein odies, which re sphericl in shpe nd rnge in size from 2 to 20 μm (Snyder nd Kwon 1987). Crop yer nd genotype differences in the soyens ffect the reltive proportions of glycinin nd β-conglycinin, nd thus the functionl nd chemicl properties of soy protein products (Khti et l. 2002). Electron microscopy hs shown tht soyens lso hve lipid-contining spherosomes rnging in size from 0.2 to 0.5 μm etween protein odies (Sio nd Wtne 1968). The oil contents of 10 norml soyen genotypes grown in Arknss were reported to rnge from 16.3 to 21.6% nd genotype ffected ftty cid composition (Liu et l. 1995). Soyen flours contin pproximtely 17% solule nd 21% insolule crohydrtes (Perkins 1995). Soyens contin pproximtely 4.1% sucrose, 1.1% rffinose nd 3.7% stchyose, which vry with genetics nd environmentl conditions (Videhi nd Kdm 1989). Soyen flour otined from soyens high in sucrose nd low in stchyose ws similr in protein composition to flour from norml deftted soyens (Dek et l. 2006).

11 6 Mjor soy proteins Glycinin Glycinin mkes up 25-35% of the totl seed protein (Murphy nd Resurreccion 1984) nd is clssified s legumin. Glycinin is hexmer (Fig. 1) of out 360 kd nd composed of 12 polypeptides 6 cidic (34-44 kd) nd 6 sic (20 kd). The polypeptides exist s cidic-sic pirs, linked y single disulfide ond, often clled jelly rolls ecuse of its structure. These pired polypeptides then form two trimers of 6 polypeptides ech, ssocited y hydrophoic nd hydrogen onds. The structures of the two trimers re visile through electron microscopy nd re descried s donuts, ecuse they ssocite with ech other to form the glycnin hexmer (Bdley et l. 1975). These glycinin suunits dissocite under extreme environmentl conditions such s t extreme ph, ionic strength nd het. Two species of glycinin hve een reported to exist, one dissocile t low ionic strength nd the other non-dissocile (Utsumi et l. 1987). Glycinin dentures rpidly, strting t round 90 C. A numer of studies hve een done to identify the cidic-sic peptides, their genetics nd composition (Nielsen 1985, Nielsen et l. 1989, Ststwick et l. 1981, Utsumi et l. 1997) to etter understnd the ehvior of soy proteins in reltion to its structure.

12 7 Figure 1. Structure of glycinin (from Adchi 2003). Stereoviews of the rion digrm. (A) The threefold xis runs perpendiculr to the pper nd is shown y filled tringle. (B) Rion drwing of the hexmer A rotted out the verticl xis. β-conglycinin β-conglycinin is the other mjor storge protein comprising soy protein nd is clssified s vicilin. It hs moleculr mss of kd nd is composed of three suunits α, α' nd β. Erly reports erroneously suggested the presence of 4 th suunit γ (Thnh nd Shiski 1977). These suunits come together to form trimer (Fig. 2). There re no disulfide onds etween the suunits; they ssocite y strong hydrophoic nd hydrogen onds. The trimer contins two cysteine residues, one in the α suunit nd the other in the α' suunit. The trimer lso contins five methionine residues, one in the α suunit nd four in the α' suunit (Utsumi et l. 1997). All three suunits re N-

13 8 glycosylted, α nd α' contin dditionl extension regions (Thnh nd Shiski 1976, Mruym et l. 1998). Figure 2. Structure of β-conglycinin (from Mruym 2001). The rion digrms of the recominnt (A nd B) nd ntive (C nd D) β homotrimers. Mruym et l. (1998) studied the roles of the glycns nd extensions in the folding, ssemly nd structure of β-conglycinin. These regions ply role in estlishing the dimensionl structure of β-conglycinin ut not density or therml stility. They lso suggest tht the extension regions ply role in preventing ggregtion. β-conglycinin dentures slowly with incresing temperture strting t round 70 C.

14 9 Soy protein isoltion SPI is one of the protein-rich food ingredients derived from soyen mels. Scientists hve een investigting the isoltion of soy protein s erly s 1903 (Johnson et l. 1992). Soy proteins re isolted on the sis of soluility t different phs (Fig. 3). The trditionl SPI process ws descried y Wolf in The sis steps include soluilizing the protein in WF produced y dehulling the ens, flking, extrcting the oil with hexne nd desolventizing the protein-rich deftted mel y flsh desolventizing to reduce protein denturtion. The proteins re extrcted y soluilizing in wter t 60 C, 10:1 solvent:solids rtio nd ph 8-11, nd removing the insolule fier y centrifuging. The protein is then precipitted y djusting the ph to nd the protein curd is removed from the solule sugrs (whey) y centrifuging. The protein curd is wterwshed nd centrifuged gin. This wshed protein curd is neutrlized to ph 6.8, nd then spry-dried. SPIs re trditionlly prepred from WF, ut recently it hs een shown tht extruded-expelled soyen mel (Wng et l. 2004) nd gs-supported screwpressed (GSSP) soyen mel (Dek et l. 2008) cn e used. Prticle size distriution of the soy flour ffects the yields of SPI otined; the smller the prticle size, the higher the recovery of protein, wheres the purity (protein content) of the SPI is not ffected y prticle size (Russin et l. 2007). The temperture t which soy protein is extrcted does not ffect protein yields nd solids. Extrction temperture nd drying method, however, ffect functionl properties (Dek nd Johnson 2007). Deogr et l. (1992) reported on the ffects on the functionl properties of SPIs tht were heted t different tempertures during isoelectric precipittion.

15 10 Figure 3. Soy protein isoltion procedure (dpted from Dek nd Johnson 2007). Soyen Flour Extrction (60 C, ph 8.5, 45min) H 2 O (10:1) 2 N NOH Spent Flour Centrifuge (30 min, 14000xg, 20 C) Superntnt Precipitte (ph 4.5) 2 N HCl Refrigerte (4 C, overnight) Whey Centrifuge (30 min, 14000xg, 20 C) Protein Curd H 2 O (10:1) 2 N NOH Neutrlize (ph 6.8) Freeze-dry Soy Protein Isolte Frctionting soy storge proteins Glycinin nd β-conglycinin exhiit different functionl properties nd, hence, my hve different uses. Wolf et l. (1962) ws le to frctionte reltively pure glycinin y using cryoprecipittion nd frctiontion, ut the yield ws only 25%. He lso investigted fctors ffecting the purity nd yield of the glycinin frction, including ut not limited to ph, temperture nd extrction rtio (Wolf et l. 1967).

16 11 Another study y Koshiym (1965) reported on procedure to frctionte glycinin nd β-conglycinin. This procedure required numer of steps efore reltively pure frctions could e otined. Thnh nd Shiski (1976, 1977) reported on simpler procedure to frctionte glycinin nd β-conglycinin; this procedure is considered the gold stndrd of lortory soy protein frctiontion. The procedure ws sed on soluility differences of ech protein t different phs. O Keefe et l. (1991) modified Thnh nd Shiski s procedure improving the purity of the β-conglycinin frction, ut with low yields. Ngno et l. (1992) modified Thnh nd Shiski s procedure nd produced >90% pure frctions of glycinin nd β- conglycinin. All the forementioned procedures were developed for lortory use nd not for commercil production. Wu et l. (1999) were le to produce glycinin-rich frction, β-conglycininrich frction nd n intermedite frction ( protein mixture) in the pilot plnt y modifying Ngno s lortory procedure. Their process produced frctions with similr purities to those otined in the lortory. In n effort to eliminte the intermedite frction nd improve the yields of the glycinin-rich nd β-conglycinin-rich frctions, Wu et l. (2000) developed simplified process tht used ph djustment nd ultrfiltrtion to produce glycinin-rich nd β-conglycinin-rich frctions with twice s much yield of the β-conglycinin frction ut with lower purity. Sito et l. (2001) used phytse to id in the frctiontion of β-conglycinin. Rickert et l. (2004) improved the Wu-Ngno s modified procedure nd otined higher β-conglycinin yields, ut with low purity. Khorshid et l. (2007) were le to frctionte glycinin nd β-conglycinin from soymel using cron dioxide t pressure of 30 r, temperture of C nd ph rnge of The Dek nd Johnson procedure (Dek nd Johnson 2005, Dek et l. 2006) developed simplified frctiontion procedure using CCl 2 nd NHSO 3 s the reducing gent. This procedure produced glycinin-rich nd β-conglycinin-rich frctions with >80% purities (Dek et l.

17 ) nd is regrded to e the first commercilly vile soy protein frctiontion process. Electrophoresis Electrophoretic seprtion of soy protein using ion-exchnge chromtogrphy (Thnh nd Shiski 1976) or SDS-polycrylmide (SDS-PAGE) gel electrophoresis (PAGE) (Fontes et l. 1984) is commonly done to identify, seprte nd quntify soy proteins. Electrophoresis identifies the different protein components of SPI including lipoxygense (Lx) (Iwuchi nd Ymuchi 1987), α, α', β nd γ suunits of β- conglycinin (Thnh nd Shiski 1977, Dvies et l. 1985), AB (cidic-sic) suunits nd A (cidic) nd B (sic) suunits of glycinin polypeptides (Nielsen et l. 1985). SDS- PAGE gels cn e successfully run on oth reduced nd ntive SPIs to identify the components listed ove (Petruccelli nd Anon 1995).

18 13 Figure 4. SPI in grdient ure-sds PAGE gel. Lipoxygense Alph-prime β-conglycinin Alph Bet Glycinin Acidic Bsic Therml ehvior The therml ehvior of protein reltes to its functionl properties nd thus use in foods. Heting soy proteins ove 70ºC cuses protein structures to unfold nd suunits to denture nd dissocite (Morr 1987). Therml ehvior is ffected y numer of fctors including ph, protein concentrtion nd het tretment. Het cogultion time of SPI proteins increses with incresd ph (Ryn et l. 2008). Ntive soy proteins t lkline ph (ph 9) re more stle thn t cidic ph (ph 3.8) (Mohmed nd Xu 2003). Similr results were otined with glycinin, which dentured fster t lower phs (Renkem et l. 1999). Glycinin in het-treted SPI dentured fster t ph 11 thn t ph 7; this chnge in denturtion rte ws not oserved with the β-conglycinin component when the ph ws incresed. It hs een suggested tht glycinin undergoes conformtionl chnges nd is 50% dentured t ph 11 (Petruccelli nd Anon 1996).

19 14 In nother study, therml denturtion of β-conglycinin ws ffected y chnges in ph nd ionic strength y influencing the environment surrounding the protein, which hs greter effects on its ggregtion thn heting (Iwuchi et l. 1991). When heted t 100ºC for 30 min, oth glycinin nd β-conglycinin completely denture. While heting t 80ºC for 30 min cuses β-conglycinin to completely denture, glycinin is more het stle nd is only prtilly dentured (Sorgentini et l. 1995). Functionlity Soluility profile Soluility is the most importnt functionl property ecuse it ffects most other protein functionlities (Bin et l. 2003, Kinsell 1979). The soluility of protein is ffected y mny fctors including its processing history, especilly exposure to het. The soluility of protein decreses with incresing denturtion (Kinsell 1979). Soluility of SPI is ffected y numer of fctors, including ph, ionic strength nd temperture. Soy proteins typiclly exhiit U-shped trend in soluility with respect to ph. Soluility is typiclly high t the extreme ends of the ph scle with little or no soluility round its isoelectric point (pi) of ph 4.5 (Wolf 1983, Kinsell 1979). Temperture increse did not significntly ffect protein soluility, except for few SPIs tht were reported to increse in soluility y 20% when temperture ws incresed to >50ºC (Lee et l. 2003). Dis et l. (2003) investigted the soluility of the reduced cidic nd sic suunits of glycinin in comprison to the suunits of glycinin. They reported tht the cidic suunits re more solule thn those of the glycinin frction, nd the sic components re not solule over ph 3 to 10.

20 15 Wter-holding cpcity Wter-holding cpcity (WHC) is the ility of protein mtrix to hold wter ginst grvity (Kinsell 1979). Dentured proteins in SPI hve higher wter-imiing cpcity thn ntive proteins. This is ttriuted to the unfolding of the dentured protein, which exposes more wter inding sites. In the presence of slts, the WHC of protein increses (y %), slts cuse more protein-protein interction, which results in the proteins ggregting nd precipitting therey inding more wter (Jovnovich et l. 2003). Similr results were reported y Gonzlez et l. (2001), who oserved protein isoltes contining more dentured proteins hd lower soluilities nd higher wter sorption cpcities. WHC is not only ffected y the stte of protein denturtion, ut lso y the extent of denturtion nd the type of protein ggregtion. The two protein frctions (glycinin nd -conglycinin) exhiit different ggregtion properties (Sorgentini et l. 1991). The proportion of the two proteins (glycinin nd -conglycinin) present in soy protein ingredients ffects WHC. Protein-protein interction etween these frctions is gretest when they re present in molr rtio of ~1, which results in low WHC. WHC decresed s the β-conglycinin-to-glycinin rtio incresed (Yo et l. 1988). The presence of sodium chloride decreses WHC of proteins y preventing the protein s polr mino cids from intercting with wter (Yo et l. 1988). Dynmic viscosity Viscosity is the resistnce of protein in solution to flow nd is mesured when exposing the proteins in solution to continuous shering t constnt rte (Dek 2004). This is n importnt property for proteins when incorported in foods like soups, everges, tters nd mets. Kinsell (1979) suggested tht the shpe of the protein is one of mny fctors determining its effect on viscosity, which my e influenced y

21 16 processing tretment. Conformtionl chnges in proteins, such s unfolding cused y lkli nd het, cn ffect soy protein viscosity. Wgner et l. (1992) evluted the rheologicl properties of commercil SPIs nd reported tht moisture nd protein concentrtions re interdependent fctors ffecting rheologicl ehvior. The ddition of sodium chloride nd sodium sulfite slts to SPI shields the protein from intercting with wter, therey, reducing WHC nd incresing viscosity. Therml tretments of SPI dispersions increses viscosity, with oth prtilly nd totlly dentured proteins even with the ddition of slts. This suggests incresed protein-protein interction s protein dentures. Soy protein frctions (glycinin nd -conglycinin) exhiit similr ehvior, viscosity increses with incresed het tretment (Bin et l. 2003). Yo et l. (1988) studied the effects of chnges in the rtio of glycinin nd β-conglycinin in soyens during mturtion on its rheologicl properties. SPI produced from mture seeds ws more viscous thn SPI produced from immture seeds. The lowest viscosity occurred when 35% β-conglycinin nd 65% glycinin ws present. Dis et l. (2003) reported tht the sic suunit reduced y using sodium isulfite, hd the gretest viscosity. All the suunits hd viscosities higher thn tht of intct ntive glycinin, except for the β- mercptoethnol-reduced low-moleculr weight cidic suunit (Dis et l. 2003). Emulsifiction properties Introducing protein to lipid-wter mixture cuses ntive protein structures to unfold. The unfolding exposes hydrophoic regions of the protein to the lipid nd hydrophyllic regions to the wter, thus reducing the surfce tension etween the wter nd oil. This ility is dependent on its structure nd flexiility (Kinsell 1979). Emulsifiction cpcity (EC) nd stility (ES) of soy protein re lowest t the isoelectric points nd increse t phs elow or ove this point. Emulsifiction cpcity

22 17 nd stility re lso higher for the -conglycinin-rich protein frction thn for the glycinin-rich frction over the ph rnge 2-10 (Aoki et l. 1980). Emulsifiction stility nd ctivity hve good liner correltion with the surfce hydrophoicity of the - conglycinin-rich frction. The more hydrophoic the surfce of the protein, the etter the emulsifiction properties. The surfce hydrophoicity of the -conglycinin-rich frction increses with het denturtion (Kto et l. 1983). Bin et l. (2003) reported tht EC of the β-conglycinin-rich frction is higher thn the glycinin-frction when compring two SPI extrction processes. Dis et l. (2003) studied the emulsifiction ehviors of the individul suunits of glycinin nd reported tht the cidic suunit nd sic suunit oth hve higher EC s thn the intct ntive glycinin, with the low-moleculr-weight cidic suunit eing the highest. The glycinin: -conglycinin rtio ffects the ility of the soy protein to emulsify. Considerle differences in this rtio hve een oserved mong vrious soyen genotypes. Genotypes with the high glycinin: -conglycinin rtios nd low β-conglycinin concentrtions hve high emulsifiction ctivity index (EAI). It hs lso een reported tht glycinin when in the monomeric form enhnces emulsion stility nd tht the rtio of monomeric nd dimeric glycinin is importnt for emulsion stility (Pesic et l. 2005). Emulsion stility index (ESI) is higher for soy protein frctions with isoelectric points etween 5.6 nd 5.1 thn etween 5.1 nd 4.5 (Chove et l. 2001). At higher protein concentrtions (~ mg/ml), SPI hs etter emulsion forming ility t ph 6 thn t ph 7 (Sntigo et l. 1998). Improved SPI emulsifiction properties re oserved when ph is incresed from 7 to 9. This ws ttriuted to incresed slt content when ph is chnged or due to chnges in degree of protein ssocition-dissocition. Exposing SPI to short therml tretments improves emulsifiction properties. (Petruccelli nd Anon 1996). Another study found heting improved emulsifiction properties of SPI compred to its ntive form; heting incresed hydrophoicity.

23 18 Reducing disulfide onds of SPI y chemicl tretments with ure or gunidine hydrochloride lso improved the emulsifiction properties of the SPI (Nir et l. 1994). Foming properties In order to e le to crete fom, the protein needs to unfold, dsor to the irwter interfce nd reduce surfce tension of wter. Surfce hydrophoicity is highly correlted with foming power. There is no correltion etween surfce hydrphoicity with foming stility, which might e ffected y denturtion of the protein rther thn surfce hydrophoicity (Kto et l. 1983). Foming properties decline s result of decresed dsorption t ph 5, which is close to the isoelectric point of soy protein. Interfcil chrcteristics improve with incresing ionic strength, even t cidic ph. Close reltionships exist etween foming cpcity nd diffusion of soy gloulin to the ir-wter interfce nd etween foming stility nd surfce pressure. At ph 7, β- conglycinin hs etter foming cpcity nd stility thn glycinin (Ruiz-Henestros et l. 2007). Yu nd Dmodrn (1991) found foms prepred with -conglycinin hve lower foming stility thn foms prepred with SPI nd glycinin. Chnging the proportions of glycinin nd -conglycinin in SPI did not improve foming properties (Petruccelli nd Anon 1995). Soyen mel extrction Oil is extrcted from soyens leving ehind protein-rich mel tht is gining importnce for producing food ingredients for humn consumption. The importnt properties for n extrction solvent re; high solvent power, nontoxicity, nonflmmility, low specific het, low het of vporiztion nd low cost (Johnson 1998). Soyen mels otined y using different oil extrction processes contin different mounts of neutrl oil nd my contin different mounts of polr lipids (Wu

24 19 nd Wng 2003). The oil extrction process lso ffects its protein composition nd structure, which results in differences in SPI functionlity. Hexne extrction Hexne is the most commonly used solvent for soyen oil extrction. The resulting mels re known s white flkes (WF) when the solvent is evported y flsh desolventiztion, vpor desolventiztion, or downdrft desolventiztion. Using hexne s solvent is expensive nd its vilility cn e uncertin. It is highly flmmle nd explosive when in contct with ir nd source of ignition. Hexne is lso not selective when extrcting oil from soyens; hence the extrcted oil requires further refining dding cost (Friedrich nd List 1982). Commercil hexne-extrcted, tosted, deftted soy flour nd SPI contined 90 to 410 μg/g nd 6 μg/g residul hexne, respectively (Honig et l. 1979). Extruding-expelling Extruding-expelling (EE) hs the dvntge over solvent extrction ecuse it requires low cpitl costs, simple mchinery, no solvent, nd cn e used for smll-scle identity-preserved processing. EE, however, cuses extensive het-denturtion of the proteins resulting in protein products with very poor functionl properties. Hydrotherml cooking (jet cooking) of the extruded soyen mel hs improved the functionl properties of het-dentured proteins y disrupting protein ggregtes (Wng et l. 2004). SPI yields from EE soyen mels re lower thn tht otined from WF. The yield of SPI from WF is proportionl to decrese in protein dispersiility index (PDI) or nitrogen soluility index (NSI) of the EE soyen mel. Heywood et l. (2002) investigted nd reported on the functionl properties of EE mels. The protein content of

25 20 SPI prepred from EE mel ws out 80%, which ws significntly lower thn the stndrd (>90% d) for SPI prepred from WF. The SPI prepred from EE mel hd similr or etter functionl properties thn SPI prepred from WF (Wng et l. 2004). The residul oils nd PDIs of EE mels rnge from 7.0 to 11.7% nd 32 to 50, respectively. Crowe et l. (2001) studied the oil contents nd PDIs of extruded mels when vrying extrusion conditions nd reported the residul oil contents of extruded mels could rnge from 4.7 to 12.7% nd the PDIs from 12.5 to Mels extruded t lower tempertures chieved higher PDIs thn the mels with higher residul oil contents. In mels extruded t tempertures of <117 C, lipoxygense ws present. Screw-pressing The screw-pressing process is similr to extruding-expelling except the extruding step is replced with cooking in stck cooker or rotry-tue dryer efore the soyen is put into the press, resulting in extensive het denturtion. Wng nd Johnson (2001) evluted extruded-expelled nd screw-pressed mels nd reported tht the screw-pressed mels hd higher oil contents nd lower protein nd moisture contents, nd lower PDIs compred to solvent-extrcted mels. Alcohol extrction L Hocine et l. (2006) showed tht ethnol extrction nd queous extrction cn e used s lterntives to hexne extrction. The SPIs produced from these mels hd protein contents of 90 nd 84%, respectively, nd their functionl properties were similr to those of SPIs prepred from WF. Decresed emulsifiction ctivity ws oserved with the SPIs prepred from soyen mels prepred y lterntive mens to hexne extrction. Improved emulsion stility nd foming properties were oserved with

26 21 queous-extrcted SPIs. Decresed ft-holding cpcity ws oserved with SPIs prepred from methnol-extrcted soyens (L Hocine et l. 2006). Supercriticl CO 2 (SC-CO 2 ) extrction In n ttempt to eliminte orgnic solvent residue in extrcted oil or mels used for humn consumption, liquid or SC-CO 2 extrction hs een investigted. These processes hve the dvntge in tht CO 2 is esily removed from the mel, it is nontoxic, inexpensive, nd non-polluting, nd cn e used for smll-scle ( 50 mt/d) or identitypreserved production unlike lrge (>3000 mt/d) solvent-extrction plnts (Yu et l. 2007, Li et l. 2006, Sthl et l. 1980). SC-CO 2 extrction cn e used to extrct isoflvones from soyen mels ut results in lower totl isoflvone yield when compred to solvent extrction, it is more pplicle to the extrction of cetylglucoside nd glycone (Ko et l. 2008). Yu et l. (2007) were le to produce isoflvone-rich SPI from SC-CO 2 deftted soy mel. A 10% SC-CO 2 /ethnol mixture ws le to completely extrct the phospholipids present in deftted soyen mel (Montnri et l. 1997). One disdvntge of using SC-CO 2 extrction is tht soyen oil is not s solule in SC-CO 2 compred to hexne (Li et l. 2006). The soluility of soy lecithin in SC-CO 2 increses s pressure increses when temperture is held constnt. Oil soluility decreses with incresing temperture t constnt pressure. Cpitl costs re quite high due to the need for high-pressure vessels nd no mens yet exists to get lrge volumes of solids into nd out of the high-pressure vessels continuously. Therefore, only tch systems re ville. SC-CO 2 hs een commercilly used for extrcting high-vlue products such s in decffeinted coffee nd flvor concentrtes.

27 22 Gs-supported screw-pressing (GSSP) GSSP is recently developed process y Crown Iron Works (St. Pul, MN, USA) nd SfeSoy Technologies (Ellsworth, IA, USA). CO 2 is injected into screw press s displcement fluid to increse oil removl, therey chieving low residul oil contents in mel (3-6% d). The CO 2 flshes when exiting the press to tmospheric pressure cooling the mel to chieve low protein denturtion nd high PDIs (>70). GSSP mel ws used to produce SPI in high yields nd hving unique functionl properties (Dek et l. 2008). Het-treted SPIs Inctivtion of lipoxygense nd trypsin inhiitors (TI) using het tretments denture nd insoluilze proteins results in poor functionlity (Kinsell 1979). Het tretment cuses dissocition, denturtion nd ggregtion of soy protein (Sorgentini et l. 1995). Nki nd Lichn (1986) investigted het tretments to improve SPI functionl properties. Wng nd Johnson (2001) reported tht hydrotherml cooking, high-sher stem-infusion tretment, improved the functionl properties of soy protein y disrupting lrge protein ggregtes. Het tretment in the presence of lkli improved some functionl properties SPI, such s soluility nd emulsifiction ctivity (Wu et l. 1999). High-pressure tretment pplied to SPI t n pproprite protein concentrtion cn lso e used to modify functionl properties (Wng et l. 2008). SPIs treted with cid experienced structurl chnges nd did not result in much improvement in functionl properties. Exposure to mild cid for specific time results in some denturtion of glycinin. Denturtion nd dissocition results in reduced soluility, incresed foming cpcity nd stility (Wgner et l. 1996).

28 23 Preservtion No chnges were oserved in the suunit composition of soyens stored either under mild, cold or mient conditions nd s result no significnt differences in functionl properties of SPI were detected when compred to SPI from freezer-stored soyens (Liu et l. 2008). Botright nd Hettirchchy (1995) reported higher soluility of spry-dried SPIs compred to freeze-dried SPIs. Dek nd Johnson (2007) studied the effects of spry-drying, freeze-drying nd freezing-thwing on the functionlity of SPIs. The preservtion method significntly ffected SPI functionlity. Sensory properties Despite helth enefits ssocited with consuming soy protein, soy protein ingredients re still not widely ccepted due to poor sensory chrcteristics, cused minly y off-flvors (McLeod nd Ames 1988). A numer of studies hve reported on hexnl (Fujimki et l. 1965, Ari et l. 1970) in reltion to the eny, grssy flvor it imprts to soyens nd soy products (Wilkens nd Lin 1970, Solin et l. 2005). O Keefe et l. (1991) reported on the numer of sites in soy glycinin nd β-conglycinin tht ind to hexnl, which chnges ccording to uffer conditions. They suggested tht the inding of hxnl rings out structurl chnges to protein. Hexnl does not contriute to the eny rom individully ut does so in comintion with other chemicl compounds (Bott nd Chmers 2006). Other compounds responsile for the eny odor of soy include 1-hexnol, trns- 2-nonenl, 1-octen-3-ol, trns,trns-2-4-decdienl, trns,trns-2-4-nondienl, cetophenone 2-pentyl pyridine nd dimethyl trisulfide (Botright nd Lei 1999). Zhou nd Cdwllder (2004) investigted the inding of hexne, 1-hexnol nd hexnl to SPI under controlled reltive humidity.

29 24 An erlier study (Mttick nd Hnd 1969) indentified ethyl ketone to e responsile for the green en odor nd flvor of soy. Botright nd Lei (2000) determined the components responsile for odor of SPIs in solutions using sttic or dynmic hedspce nlyses with GC/MS techniques. This is the first study to report methnethiol to e one of the responsile odornts. 2-Pentyl pyridine (2-pp) is responsile for the strong grssy rom detected y using GC nd cuses throt-ctching tste (Botright nd Crum 1997). Anderson nd Wrner (1976) reported tht cidsensitive soy proteins hd greter ffinity for grssy-eny flvor. Klrener et l. (1974) oserved tht lipoxygense hydroperoxides nd their decomposition products contriuted to grssy-eny flvor. Comining hexenl, methnethiol, 2-pentyl furn nd dimethly trisulfide (DMTS) reproduced the odornts detected in the hedspce top n queous SPI slurry (Ang nd Botright 2003). Zhou nd Botright (1999) studied the effect of ph during SPI production on flvor due to 2-pp. They reported incresed 2-pp levels t ph 7, which is lower t ph 4.5 or 9. Other fctors tht contriute to the unfvorle flvor or soy products include itterness nd stringency. Ari et l. (1966) identified phenolic cids from deftted soy flour tste sour, itter nd stringent. Activted cron nd ion exchnge removed the phenolic compounds from soy protein extrcts, which improved flvor ut did not improve itterness or stringency (How nd Morr 1982). Still other studies reported tht soy isoflvones nd soy sponins re responsile for stringent nd/or itter tste in soy products (Tsukmoto et l. 1995). Mlonyl-β-glucoside isoflvones nd DDMPconjugted sponins cuse itterness nd off-flvor (Aldin et l. 2006). Less processed or het-treted soy products re more stringent. Other studies hve suggested the itter, rncid nd eny off-flvors re cused y the oxidiztion of unsturted ftty cids (Sess nd Rckis 1977). Sess et l. (1976) isolted three phosphtidylcholines from residul lipid in hexne-deftted soy flkes, which contriutes to the itter tste of soy.

30 25 A numer of ttempts hve een mde to eliminte the ojectionle eny flvor of soy products. Use of het tretment hs chieved little success. Breeding hs lso een used to eliminte lipoxygense(kitmur 1993), which reduced the eny flvor found in the soy products (Koyshi et l. 1995). Extrcting soy flkes with queous lcohol removed the ojectionle flvors in soy (Bker et l. 1979). SPIs produced from deftted soy flkes nd wshed with queous lcohol hd improved flvor profiles thn SPIs produced from unwshed soy flkes (Hu et l. 2003). SPI produced from ethnol zeotrope-extrcted flkes, tosted or untosted, hd less grssy or eny ttriutes ut were still itter in tste (Honig et l. 1976). A 66% reduction in eny flvor ws reported in SPI produced from hexne/cetic-cid-treted soyen mel (Swmylingpp nd Srinivs 1994). Mheshwri et l. (1995) reported tht liquid cron dioxide is the lest effective nd SC-CO 2 is most effective in removing voltile off-flvors from SPI. The zeotropic mixture of hexne nd solute ethnol produces flkes with little ojectionle flvors y removing most residul lipids, which oxidize nd led to ojectionle flvors (Sess et l. 1969). A descriptive sensory pnel reported 19 different chemicls with eny roms nd flvors similr to tht of soy. Beny flvor ws descried s musty/erthy, musty/dusty, sour romtics, green/pod pe, nutty or even rown. Three lcohols, two ketones, one ldehyde nd one pyrzine hd eny chrcteristics t low concentrtions (Vr-Uol et l. 2004). Another descriptive sensory pnel used the following descriptors to descried commercil SPI smples in wter (10% w/v) s cerel, mlty, flour pste, rosted, sweet romtic, crdord nd rothy flvor (Russell et l. 2006). References Adchi M, Knmori J, Msud T, Ygski K, Kitmur K, Mikmi B, Utsumi S (2003)

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