University of Alberta

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1 University of Alert Physiochemicl nd Rheologicl Properties of Alkline Isolted Poultry Proteins y Vid Moyedi Mmghni A thesis sumitted to the Fculty of Grdute Studies nd Reserch in prtil fulfillment of the requirements for the degree of Mster of Science in Food Science nd Technology Deprtment of Agriculturl, Food nd Nutritionl Science Vid Moyedi Mmghni Spring 2010 Edmonton, Alert Permission is herey grnted to the University of Alert Lirries to reproduce single copies of this thesis nd to lend or sell such copies for privte, scholrly or scientific reserch purposes only. Where the thesis is converted to, or otherwise mde ville in digitl form, the University of Alert will dvise potentil users of the thesis of these terms. The uthor reserves ll other puliction nd other rights in ssocition with the copyright in the thesis nd, except s herein efore provided, neither the thesis nor ny sustntil portion thereof my e printed or otherwise reproduced in ny mteril form whtsoever without the uthor's prior written permission.

2 Exmining Committee Dr. Mirko Betti, Agriculturl, Food nd Nutritionl Science Dr. Jinping Wu, Agriculturl, Food nd Nutritionl Science Dr. Wendy Wismer, Agriculturl, Food nd Nutritionl Science Dr. Ellen Goddrd, Rurl Economy

3 DEDICATION To my fther, Ali Moyedi Mmghni, who lwys wtches over me from the sky with his specil smile. To my mother, Sorour, who hs lwys supported me in ll wys without you I would never e t this stge. To Pouy, Lid, nd Alirez, my motivtors nd supporters, without your gret help, this would not hve een possile.

4 ABSTRACT Chicken drk met hs een considered s mjor underutilized commodity due to the incresing demnd for further processed rest met products. Alkli ided protein extrction is n option to increse the utiliztion of chicken drk met. First, the effect of ph ( ) on lkline extrction of chicken drk met hs een studied, nd protein yield, composition, color, nd TBARs of the extrcted met hve een determined. Second, texturl nd rheologicl properties nd wter holding cpcity (WHC) of lkli extrcted chicken drk met hve een evluted. The highest protein yield (94.2%) ws otined t ph Lipid content of the extrcted met decresed y 50% compred to chicken drk met. WHC, hrdness nd chewiness of extrcted met were greter t higher ph. The gel from recovered met with dded cryoprotectnts showed more stility. This process my offer the possiility to use the underutilized poultry resources for preprtion of functionl foods. Keywords: Chicken drk met, lkli ided protein extrction, composition, TBARs, color, texture, rheology, wter holding cpcity, cryoprotectnt.

5 ACKNOWLEDGEMENTS Mirko Betti for giving me this opportunity, nd for guiding me to higher pths of knowledge. Without him, I would not e here. Wendy Wismer, Jinping Wu, nd Ellen Goddrd for their wise input throughout the whole project. George Britton for eing unique techer, motivtor, nd supporter. Dileep Omn for his gret help nd dvice for cdemic writing nd for his ptience nswering my questions. Tuli Perez for eing such specil friend nd for helping me throughout the project, especilly with sttistics. Jcky Chn, Sndeep Singl, nd Yuliy Hrynets for eing such nice friends nd co-workers, nd for their willingness to help others. Yn Xu for his technicl expertise. Kelvin A. Lien for helping me with chromtogrphy nd for eing ptient to explin ll the procedure for me nd nswering my endless questions. Mrym nd Leil Zrgrzdeh for eing my second fmily. Shr Nvidghsemizd for eing such wonderful friend, for listening to me nd for lwys eing there when I needed her. Srh Goomeshi Nory for eing my gret friend who lwys helped me to rek up my dily routine nd to do something exciting. Most importntly, my mother, Sorour, for her gret help nd support, for listening to me nd giving me dvice. Also, for understnding my reserch. Lid nd Pouy Moyedi Mmghni, nd Alirez Dnesh for their gret help nd support. Grnt, Ptti, nd Crly Henley for ll their endless kindness nd cring, which is rrely found nowdys. All the students nd stff t the Poultry Reserch Centre for their ssistnce nd expertise.

6 1 LITERATURE REVIEW Low Vlue Poultry Drk Met Structure nd Protein Composition of Muscle Skeletl Muscle Muscle Proteins Muscle Fier Types Alterntives to Increse Low Vlue Drk Met Utiliztion Conventionl (Trditionl) Surimi Processing Technology ph Shifting Processing: A New Technology for the Recovery of Functionl Proteins Fctors Influencing Protein Recovery using ph shifting Processing nd their Comprison with Trditionl Surimi Production Functionl Properties of Muscle Proteins nd their Link to ph Shifting Process nd Trditionl Surimi Production Protein Soluility Protein Geltion Rheologicl Properties Dynmic Mechnicl Anlysis (DMA) Texturl Properties Texturl Profile Anlysis (TPA) Wter Holding Cpcity Cooking Loss Emulsifiction Cpcity Protein Denturtion The Effect of Surimi nd ph Shifting Process on Functionl, Texturl, nd Rheologicl Properties of the Recovered Proteins Color nd its Link to the Recovered Proteins from Surimi nd ph Shifting Process The Effect of Surimi nd ph Shifting Process on the Recovered Proteins Stility during Storge Potentil Applictions of the Recovered Proteins y ph Shifting Methods Reserch Ojectives Min Ojective Specific Ojectives Approch TABLES FIGURES REFERENCES... 32

7 2 ALKALI AIDED PROTEIN EXTRACTION OF CHICKEN DARK MEAT: COMPOSITION AND STABILITY TO LIPID OXIDATION OF THE RECOVERED PROTEINS INTRODUCTION MATERIALS AND METHODS Mterils Protein Soluility Curve Protein Isoltion Determintion of Totl Protein Content Extrctility of Recovered Proteins Determintion of Totl Ft Content Seprtion of the Min Lipid Clsses Ftty Acid Anlysis Susceptiility to Oxidtion Color Mesurements Totl Heme Pigments Content Sttisticl Anlysis RESULTS AND DISCUSSION Protein Soluility Protein Recovery nd Protein Content Extrctility of Recovered Proteins Lipid Profile Totl Lipid Content Neutrl nd Polr Lipids Content Ftty Acid Profile Neutrl Lipids Frction Polr Lipids Frction Susceptiility to Oxidtion Color Mesurements nd Totl Heme Pigments CONCLUSIONS TABLES FIGURES REFERENCES ALKALI AIDED PROTEIN EXTRACTION FROM CHICKEN DARK MEAT: TEXTURAL AND RHEOLOGICAL CHARACTERISTICS OF RECOVERED PROTEINS INTRODUCTION MATERIALS AND METHODS Mterils Methods Extrction Method Cooking Loss... 79

8 Expressile Moisture Texturl Profile Anlysis Dynmic Viscoelstic Behviour of Recovered Proteins Sttisticl Anlysis RESULTS AND DISCUSSION Cooking Loss Expressile Moisture Texturl Profile Anlysis/Dynmic Viscoelstic Behviour CONCLUSIONS TABLES FIGURES REFERENCES PROJECT SUMMARY AND IMPLICATIONS... 95

9 LIST OF TABLES Tle 1.1. Protein (%), lipid (%), moisture (%), nd iron (mg/100 g of met) content of skinless chicken drk nd white met...28 Tle 1.2. Color ttriutes (L*, *, nd *) of chicken drk nd white met 29 Tle 2.1. Protein (%) nd lipid (%) composition of chicken drk nd extrcted met (wet sis).61 Tle 2.2. Level (mg ftty cid / 100 g of met) of ftty cids in the neutrl lipids of chicken drk nd extrcted met...62 Tle 2.3. Level (mg ftty cid / 100 g of met) of ftty cids in the polr lipids of chicken drk nd extrcted met...63 Tle 2.4. Color mesurements nd totl heme pigments of chicken drk nd extrcted met...64 Tle 3.1. Texturl profile nlysis of recovered proteins fter protein extrction y ph-shift method...86

10 LIST OF FIGURES Figure 1.1. Schemtic digrm of muscle structure, strting from cross section of whole muscle (), including the lyers of connective tissue, the muscle undle (), fire (c), myofiril (d) nd myofilments (e)..30 Figure 1.2. Flow chrt of surimi mnufcturing 31 Figure 2.1. Soluility profile of chicken drk met proteins s function of ph. Protein homogentes were djusted to ph rnge of 1.5 to 12.0 using HCl or NOH. The vlues presented re mens of 4 replicte determintions 65 Figure 2.2. Effect of extrction ph on protein yield of extrcted chicken drk met. Dissimilr letters in the grph represent significnt (P < 0.05) difference. The vlues presented re mens of 4 replicte determintions..66 Figure 2.3. Regression nlysis showing the reltion etween protein yield (%) nd ft (% on dry sis) of extrcted chicken drk met s function of extrction ph..67 Figure 2.4. Regression nlysis showing the reltion etween protein yield (%) nd neutrl lipids (% on dry sis) of extrcted chicken drk met s function of extrction ph..68 Figure 2.5. Extrctility of recovered proteins fter protein extrction y phshift method. Srcoplsmic proteins were soluilized in phosphte uffer, while totl proteins were soluilized in phosphte uffer (ph 7.4) contining potssium iodide. Dissimilr letters for respective prmeters in the grph represent significnt (P < 0.05) difference.69 Figure 2.6. Effect of time nd extrction ph on oxidtive stility of chicken drk nd extrcted met mesured s induced TBA rective sustnces (TBARS). The vlues presented re mens of 4 replicte determintions..70 Figure 3.1. Cooking loss of different tretments during lkli extrction of chicken drk met. Dissimilr letters (,, c) in the figure denotes significnt difference (P < 0.05)..87 Figure 3.2. Expressile moisture of different tretments during lkli extrction of chicken drk met. Dissimilr letters (, ) in the figure denotes significnt difference (P < 0.05)..88 Figure 3.3. Dynmic viscoelstic ehviour (DVB) of recovered proteins from chicken drk met fter lkli extrction (without cryoprotectnts)..89

11 Figure 3.4. Dynmic viscoelstic ehviour (DVB) of recovered proteins from chicken drk met fter lkli extrction, stored t -30 o C for one month with dded cryoprotectnts 90

12 LIST OF ABBREVIATIONS AA: Archidonic cid DHA: Docoshexenoic cid DMA: Dynmic mechnicl Anlysis DMCM: Drk minced chicken met EM: Extrcted met EPA: Eicospentenoic cid FA: Ftty cids GC: Gs chromtogrphy LA: Linoleic cid LC ω-3 PUFA: Long chin omeg-3 polyunsturted ftty cids LNA: lph-linolenic cid MDA: Mlonldehydes MT: Metric tons MUFA: Monounsturted ftty cids PL: Phospholipids PUFA: Polyunsturted ftty cids SFA: Sturted ftty cids TBA: Thiorituric cid TBARs: Thiorituric cid rective sustnces TG: Tricylglycerols TPA: Texture profile nlysis WHC: Wter holding cpcity

13 1 LITERATURE REVIEW 1.1 Low Vlue Poultry Drk Met Poultry met production worldwide pproched 94.7 million metric tons (MT) in 2009 (FAO, 2009). Currently the US, Chin, Brzil nd Europen Union (EU) with 19.4, 12.1, 11.3, nd 8.5 million MT respectively, re the primry roiler producers (USDA, 2009,,c,d). In 2008 the mount of poultry production in Cnd reched 1.2 million MT (Sttistics Cnd, 2009). According to the US Livestock Mrketing Informtion Centre, estimted per cpit totl red met nd poultry consumption in the US in 2007 ws kg (World Poultry, 2008). Chicken nd turkey met consumption in the US hd drmtic increse from 12 kg in 1950, to 52 kg per cpit in 2007 (USDA/ERS, 2007). Chicken met consumption in Cnd ws 21.5 kg per cpit in 1989 while in 2008 it reched the level of 31.8 kg (CFC, 2008). The mjor fctors which contriuted to the increse in Cndin poultry consumption were popultion growth, demnd for non red met species, wreness of perceived helth enefits of poultry met, nd continuing consumer preference for convenient nd cretive vlue-dded chicken products with reduced mel preprtion time (AAFC, 2006; Goddrd et l., 2007). Poultry met cn e ctegorized into high-vlue (mostly white met) nd lowvlue (mostly drk met) (Peterson nd Orden, 2005). Although, chicken met minly consists of roilers (high vlue), spent reeder hens nd mles, nd spent tle egg lyers re lso ctegorized in the group of low vlue poultry met (Bilgili, 2000). Due to the low qulity nd yield of the met from roiler reeders nd spent lyers, they re regrded s Low Vlue met, nd there is little demnd for them in food processing (AFAC, 2003). In the United Sttes, white met is preferred over drk met (USDA, 2000). Therefore, with the incresing demnd for further processed rest met products (i.e. white met), chicken drk met hs een regrded s y-product (USDA, 2004). The mjor concerns with chicken drk met which ffect consumers selection nd stisfction re color (due to heme pigments contining iron), high ft content nd poor shelf stility (Froning, 1

14 1995; Fletcher, 1997; Betti nd Fletcher, 2005). The differences in protein, lipid, moisture nd iron content of chicken white nd drk met re presented in Tle 1.1. Low vlue drk poultry met should not e considered only s muscle tissue which is processed into trditionl forms of food, ut rther s source of functionl ingredients for food product development. Agriculturl commodities which re frctionted into components hve een widely used s ingredients in other food products to improve functionl properties. For exmple, isolted soyen proteins re used s inders, extenders, nd fillers. In ddition, isolted soy proteins cn improve wterholding cpcity, nd lighten drk met (Owens, 2001). Therefore, this ide cn e lso pplicle to poultry drk met. Since met qulity is ffected y the muscle structure in the living niml, it is importnt to overview chemicl nd physicl composition of muscle proteins, which is provided in the following sections. 1.2 Structure nd Protein Composition of Muscle Skeletl Muscle A schemtic digrm of the muscle structure is shown in Figure 1.1. As cn e seen in the digrm, skeletl muscle is formed y mny muscle undles. Epimysium is the cover for muscle undles. Perimysium is connective tissue which seprtes muscle undles from one nother. Endomysium is thin lyer y which muscle fiers re seprted from ech other. Muscle fiers re the units which form muscle undle. Ech muscle fier consists of myofirils nd ech myofirill contins myofilments. The Srcomere is smll muscle unit which is involved in muscle contrction. There is drk re in which thick filment (myosin) nd thin filment (ctin) overlp. This re is known s the A-nd. There is light re in the A-nd which contins no thin filments clled the H-zone. The Z-line is the re which determines the orders of srcomere, nd it is composed of nrrow drk nds of proteins (Strsurg et l., 2008). Muscle contrction occurs when thick filments slide towrd the Z-line (Brut, 2002). There is gret vrition in the size of the skeletl muscles from rest muscle to the muscle which control the movement of the eyes. 2

15 1.2.2 Muscle Proteins Bsed on their soluility function, proteins in skeletl muscle hve een ctegorized into srcoplsmic, myofirillr, nd stroml proteins (Strsurg et l., 2008). Srcoplsmic proteins include proteins locted in the srcoplsm (cellulr fluid) of the myofier including myogloin, hemogloin, cytochromes, glycolytic enzymes nd cretine kinse. These proteins re lso clled wter solule proteins. This frction constitutes out 30% of the totl muscle protein content (Scopes, 1970). Myofirillr proteins include 50-60% of muscle proteins. These proteins re slt solule nd thus they re clled slt solule proteins. Myosin nd ctin which re ctegorized in this group, re thick nd thin filments, respectively (Strsurg et l., 2008). They ply the min role in muscle contrction nd consist of out 65% of the totl myofirillr proteins nd round 40% of the totl muscle protein content (Ytes nd Greser, 1983). Other proteins included in this group re tropomyosin, troponin, C- Protein, α-actinin, nd β-actinin (Brut, 2002). According to the fct tht ctin nd myosin re the most undnt proteins mong the myofirillr proteins, the soluility of ll other proteins in this group will e ffected y these two proteins, nd gretly influence the functionl properties of the processed met products. Myosin which constitutes out 45% of the myofirillr proteins is the min skeletl muscle protein involved in muscle contrction. Myosin forms the thick filments nd is the undnt protein present in the A-nd re (Ytes nd Greser, 1983). Myosin with moleculr weight of pproximtely 540,000 dltons, is long rod-shped protein composed of six suunits including two hevy chins nd four light chins. The hevy chins consist of the myosin hed. Movement occurs when the myosin heds connect with ctin (Brut, 2002). Thin filments re composed of three min myofirillr proteins including ctin, tropomyosin nd troponin. Actin with moleculr weight of 42,000 dltons, mkes up the thin filments. The ctin molecule is composed of two chins which re twisted together. Tropomyosin, nother protein of the thin filments, surrounds the ctin molecule. It consists of 5% of the myofirillr proteins nd is rod shped protein. Troponin, nother protein of the thin filments, is composed of 5% of the myofirillr proteins nd is in gloulr shpe. There re three types of troponin which include troponin-c, troponin-i, nd troponin-t. They re ll locted long the ctin filment. 3

16 When Troponin-C inds with clcium ions, the tropomyosin moves nd s result, the myosin inding site in ctin molecule ecomes exposed which leds to the connection of ctin nd myosin (Jckson et l., 1975). Stroml proteins, which comprise 10-20% of totl muscle protein content, provide strength nd protection for muscle tissue. The composition nd undnce of these kinds of proteins gretly ffect the qulity of met products. The mjor protein of this group is collgen (Strsurg et l., 2008). Other proteins of this group re elstin nd mitochondril proteins (Brut, 2002). Collgen contriutes to met toughness nd increses with the ge of the niml. Thus, the met from older nimls is of lower qulity ecuse it is more tough compred to the met from the younger ones (McCormick, 1999; Purslow, 2005). During met processing, cooking leds to reking nd prtil soluiliztion of collgen which will increse met tenderness (Resurreccion, 1994) Muscle Fier Types According to muscle ppernce, it is possile to clssify the muscle fiers into red nd white. The poultry drk met (e.g. thigh) is minly composed of red fiers, nd due to this reson its color is drk compred to white met (i.e. rest). The myogloin content of red fiers is higher thn those in white fiers leding to the color difference (Brut, 2002). On the other hnd, ccording to the type of metolism, muscle fiers cn e divided into two groups of oxidtive nd neroic (glycolytic). Thus, sed on this clssifiction, muscle fiers cn e grouped into type I (slow oxidtive), type II (fst oxidtive glycolytic), nd type II (fst glycolytic) (Strsurg et l., 2008). Type I fiers, which re red fiers, (e.g. in thigh muscle) hve high myogloin content which supplies the oxygen for the oxidtive metolism, nd ftigue occurs slowly. Mitochondri re the orgnelles which re responsile for eroic respirtion of the cell. The rections during eroic respirtion require oxygen for ATP production, which is the energy source for ll the cell ctivities. Since the metolism of type I fiers is oxidtive, they hve higher numer of mitochondri nd the size of mitochondri is lrger compred to the white fiers. Furthermore, the lipid content of the fiers is higher. These chrcteristics enle type I fiers to contrct for longer time. The speed of contrction for these fiers is lower, 4

17 nd their dimeter is smller thn the white fiers. In contrst, type II fiers (e.g. in rest muscle) hve low myogloin content nd their metolism is glycolytic. Thus, ftigue occurs fster. Glycolytic metolism cn hppen in the presence or sence of oxygen. Due to this fct, type II fiers hve the lowest numer of mitochondri, nd lso the size of their mitochondri is smller compred to the red fiers. The lipid content of these fiers is lso lower thn the red fiers. All these chrcteristics led ftigue to hppen fster in these muscles compred to the red muscles. In ddition, the contrction speed of type II fiers is higher thn the red fiers nd their dimeter is lrger compred to the red fiers (Brut, 2002). Type II fiers re red, hve medium mount of myogloin nd ftigue occurs on medium speed compred to type I nd type II fiers (Strsurg et l., 2008). According to the reserch y Smith et l. (1993) chicken Pectorlis muscle (i. e. rest) contined 100% Type II muscle fiers. According to Bez nd Brillrd (1999), on verge, chicken Srtorius muscle (i. e. thigh) ws composed of type I (16%), type II (54%), nd type II (30%) fiers. 1.3 Alterntives to Increse Low Vlue Drk Met Utiliztion The low vlue met includes oth drk met fishes nd poultry drk met. The most importnt options to increse the utiliztion of low vlue drk met include trditionl surimi technology nd ph shifting processing. These technologies im to concentrte nd isolte muscle proteins from this type of rw mterils Conventionl (Trditionl) Surimi Processing Technology Surimi is Jpnese word which literlly mens minced met nd refers to frozen concentrte of fish myofirillr proteins which hs een stilized y ntidenturnts (known s cryoprotectnts). Surimi preprtion ws discovered 900 yers go y Jpnese fishermen. The ncient surimi process involved mincing the fish with slt nd spices, grounding it into pste nd cooking it to chieve shelf stle gelled product. With the ddition of cryoprotectnts, which re sugrs, surimi will e protected ginst freeze denturtion (Zmul, 1985; Vieir, 1996). As result, surimi hs longer stility when stored in frozen conditions. For the first time cryoprotectnts were used y Nishiy et l. (1960). In their study, fter dewtering of fish myofirillr proteins, they dded crohydrtes (sucrose nd soritol) nd froze the product fterwrds. The results 5

18 indicted tht freeze denturtion of proteins ws inhiited. The discovery revolutionized the surimi industry. Figure 1.2 shows n outline of the surimi mnufcturing process. As the figure shows, surimi production includes severl steps which involve prepring the fish for deoning, deoning process, mincing, wshing nd refining processes, dewtering, ddition of cryoprotectnts, nd finlly freezing the finl surimi product. The detils of ech step re descried in the following prgrph. Due to its resonle price nd good sensory chrcteristics, surimi sefood is well-known product in the mrket (Shie nd Prk, 1999). The im of deoning process is to seprte the flesh from the fish; usully crried out using mechnicl met seprtors. Before proceeding to deoning, fish should e prepred to get deoned. One method for fish preprtion is removing hed, gut nd clen the elly wlls (Pigott, 1986). The min concern t this step is the complete removl of viscer from the met. Otherwise, the stility of the met will e considerly decresed due to microil infection (Prk nd Lin, 2005). Deoning nd mincing processes re oth using the sme procedure, which is utiliztion of roll-type met seprtor. During these opertions, the prepred fish is pressed through perforted drum. While met psses through the perfortions nd is conducted to the inner drum, one, skin, etc. will e left in the outer drum. The optimum size of the perfortions is suggested to e 3-4 mm in dimeter (Tked, 1971; Lee, 1984). The minced met thus otined is then wter wshed. During the wshing process, the minced met is wshed with iced wter with the rtio of 1:4 or 1:5, nd this process is repeted twice or three times. Slt cn e dded to the minced met t the level of 0.2 to 0.3% to help in removing the wter from the met (Southest Asin Fisheries Development Center, 1988). Srcoplsmic proteins nd lipids re removed y the wshing process. Srcoplsmic proteins include myogloin, hemogloin nd other components, nd lipids include neutrl lipids (tricylglycerols) nd polr lipids (minly phospholipids). The presence of lipids (especilly polr lipids) nd srcoplsmic proteins put the storge stility of myofirillr proteins t risk (Scopes, 1970; Prk nd Lin, 2005; Betti et l., 2009). On the other hnd, the mount of wter used for wshing is of gret importnce 6

19 due to economic issues. Therefore, choosing suitle wshing procedure which uses resonle mount of wter nd t the sme time efficiently removes the dispersed ft nd wter-solule proteins is highly importnt. According to Adu et l. (1983) some fctors such s freshness of fish or the structure of wshing unit ffect the wshing process. The im of the refining process is to remove stroml proteins (minly connective tissues components such s collgen) from the met using refiner. The qulity of the refining process vries with the size of the refiner screen size nd speed. For exmple, lrger screen size nd fst speed leds to higher yields s well s greter mount of impurities in the isolted proteins. The composition of the frction seprted from the myofirillr proteins y the refiner ws reported s protein (15.4%), moisture (81.4%), lipid (1.9%) nd sh (1.0%) (Kim nd Prk, 2003) in which the composition of the protein prt ws minly stroml proteins. After the wshing process, the wter content of the met is incresed y pproximtely 10% (from 82% to 92%). The wshed minced met is further pssed through screw press. The gol of this process is to dewter the met which mens to remove the extr wter gined from the wshing process y pplying mechnicl force. According to Prk nd Lin (2005) y the use of slts, such s NCl nd CCl 2 t the level of 0.1 to 0.3%, seprtion of the moisture from the screw press will e improved. As mentioned efore, cryoprotectnts ply n importnt role in surimi production, ecuse they prevent muscle protein denturtion during frozen storge. Cryoprotectnts such s sucrose nd soritol (9% w/w) re used for this purpose. A numer of studies performed to present new cryoprotectnts to the industry. For exmple, trehlose ( discchride) nd short-chin glucose polymer hve een introduced y Crgill Corportion nd Roquette Corportion respectively (Hunt et l., 2001, 2002). Although the mechnism of cryoprotectnts re not fully understood yet, the ddition of cryoprotectnts leds to improved wter holding cpcity of the myofirillr proteins, which further leds to smoother pste in the finl product. The lst step in surimi production is freezing; usully done t -30 C. The temperture for trnsporttion of the frozen surimi is pproximtely -20 C (Southest Asin Fisheries Development Center, 7

20 1988). A qulity control test for the surimi product is the detection of metls. In order to prevent lipid oxidtion nd incresing the surimi shelf life, it is of gret importnce to detect metls in surimi finl product, which is prt of surimi HACCP progrm. The common metls detected re ferrous, copper, luminum, nd stinless steel, nd ccording to the FDA s Helth Hzrd Evlution Bord metl frgments which hve length of 7-25 mm re considered hzrdous (FDA, 2001) ph Shifting Processing: A New Technology for the Recovery of Functionl Proteins Trditionl surimi processing hs een revolutionized y the introduction of new technique. The new technology which is nmed ph shifting processing is sed on shifting ph vlues, nd is utilized in cid nd lkline soluiliztion processes (Hultin nd Kelleher, 1999, 2000). This process consists of different phses. The first phse is homogeniztion of minced muscle with wter. The rtio of met to wter vries etween 1:5 to 1:9 (Nolsøe nd Undelnd, 2009). Homogeniztion is followed y the ddition of cid or lkli solution to ring the ph ove 10.5 or elow 3.5 where proteins hve the mximum soluility (see chpter 2). In order to remove lipids nd stroml proteins from the muscle, centrifugtion is performed. During centrifugtion, mterils re seprted sed on their density. Thus, ft (with lest density) will e present s the top lyer, while other impurities which hve more density (skin, one nd cellulr memrnes) will e precipitted t the ottom of the centrifuge tue. Therefore, the middle frction contins soluilized proteins which will e collected nd then djusted to the isoelectric point of the myofirillr proteins, which is ph of 5.2 to 5.5. At this ph vlue, ecuse myofirillr proteins hve no net chrge, they will precipitte. Another centrifugtion step is used to collect the sedimented proteins. After collection of the functionl proteins the ph vlue cn e re-djusted to the nturl ph of the muscle. The finl step is dding cryoprotectnts nd freezing the finl product. ph shifting processing is n idel method for the utiliztion of the low-vlue rw met, especilly ecuse this kind of rw mteril hs high mount of ft which cn e difficult to seprte y conventionl surimi process. The dvntges of ph shifting processing over trditionl surimi production include higher protein yields (85% vs. 70%) due to more recovery of srcoplsmic 8

21 proteins, nd more efficient removl of lipids, connective tissue nd other impurities thnks to the use of high-speed centrifugtion (Kristinsson nd Hultin, 2003; Hultin et l., 2005; Snmrtin et l., 2009). Also y utilizing ph shifting processing, there is no need for deoning the rw fish mteril (Nolsøe nd Undelnd, 2009). Using optimum mount of wter, oth neutrl nd polr frctions of lipids cn e seprted from the met nd thus, the shelf life of the finl product will e incresed due to reduction in lipid oxidtion. Also, the wste wter otined from the ph shifting processing contins less solid prts due to the use of centrifugtion (Prk et l., 2003) Fctors Influencing Protein Recovery using ph shifting Processing nd their Comprison with Trditionl Surimi Production Due to the fct tht trditionl surimi production ws not efficient enough, ph shifting processing ws introduced s new technology to improve nd overcome prolems in surimi processing. Most of the studies in this field re focused on fish s rw mteril. Therefore, in this section, comprisons etween trditionl surimi production nd cid / lkline soluiliztion process for fish hve een reviewed, nd relevnt studies on poultry sources hve lso een included. A vriety of processes were used for seprtion of myofirillr proteins from ft nd pigments, nd to increse oxidtive stility of the finl poultry products. One of the methods ws the use of centrifugtion (Froning nd Johnson, 1973; Dhillon nd Murer, 1975). Young (1975) performed n queous extrction using solution hving ph of 7.0 followed y precipittion t ph of 4.5, for the protein recovery from mechniclly deoned chicken met (MDCM). Hernndez et l. (1986) used phosphte uffer solution t ph of 8.0 for protein extrction, nd efficiently removed ft nd pigments from mechniclly deoned turkey met (MDTM). An queous wshing with dded sodium icronte (ph: 8.0) ws used y Dwson et l. (1989) for myofirillr proteins isoltion from mechniclly deoned chicken met. Some reserchers such s Jelen et l. (1982), McCurdy et l. (1986), Ling nd Hultin (2003), nd Betti nd Fletcher (2005) extrcted myofirillr proteins from poultry drk met nd poultry residues y the use of high extrction ph nd low precipittion ph. 9

22 Protein recovery, which is n importnt issue in oth surimi nd ph shifting processes, is ffected y some other fctors. One of these fctors is protein soluility t lkli nd cid ph rnges; which should e high to otin good protein yields. Another fctor is the size of the sediments formed fter the centrifugtions which should e low to increse the recovered proteins. The lst fctor is the soluility of the proteins t the precipittion ph, which should e low in order to increse the proteins precipittion fter the centrifugtion process. Kristinsson nd Ingdottir (2006) who worked on tilpi fish using lkline nd cid soluiliztion processes indicted protein yields of 61-68% for the lkline process, while the yields for the cid process ws reported s 56-61%. Therefore, in their study higher yields were otined from the lkline soluiliztion process. On the other hnd, study performed in 2002 y Undelnd et l. who worked on white muscle of herring fish indicted protein yields of 68% nd 74% for lkline nd cid protein extrction, respectively. The reson for lower yield for lkline soluiliztion ws due to the fct tht during the first centrifugtion lrger sediment ws formed, which reduced the yield. Btist (1999) tested lkli process on hke nd monkfish nd otined the verge protein yield of 71.8%. To study the reltionship of protein yield with soluiliztion ph, Kim et l. (2003) used 1:10 fish met to wter rtio, nd cidic nd lkline ph tretments. The results showed tht t ph of 12.0, the yield of proteins ws pproximtely 70% (mximum level), while the yield ws round 60% (minimum level) when ph of 10.5 ws used. However, due to economicl issues, the mount of wter used in the process is of high importnce. Thus, n efficient mount of wter should e chosen in wy to meet oth yield nd economicl mtters. Froning nd Johnson (1973) studied the effect of centrifugtion on the qulity of mechniclly deoned fowl met. The centrifugtion process led to the formtion of three phses including ft, queous, nd met frctions. The results indicted tht the protein content of the finl product ws significntly incresed from 14.2% to 22.7 % using centrifugtion. In study conducted y Young (1975), protein extrction from mechniclly deoned poultry met ws crried out. Protein extrction ws performed using solvents 10

23 with ph rnge of 5.5 to 7.5, nd precipittion ph rnge of 4.0 to 7.5. Results suggested the use of extrction ph vlues of ove 6.5, with precipittion ph of 4.5 for otining mximum yield of 83.7%. In Study y Ling nd Hultin (2003) lkline soluiliztion (ph: 10.8) nd cid precipittion (ph: 5.2) ws used for protein isoltion from mechniclly deoned turkey met. The rtio of met to wter ws 1:6 (w:v), nd dewtering ws performed y centrifugtion t 10,000 g for 25 min t 0 C to 10 C. The moisture content of the isolted proteins ws djusted to pproximtely 80%, nd cryoprotectnts (soritol, sucrose, sodium tripolyphosphte, nd sodium icronte) were dded efore freezing the finl product. The results showed tht protein content of the finl product ws incresed from 12.0% to 17.0% compred to the rw met. Betti nd Fletcher (2005) studied the effect of extrction nd precipittion ph on the yield of roiler drk met. They studied extrction ph rnge of 8.0 to 12.0, followed y precipittion ph rnge of 3.8 to 5.2. The etter dry yields (ove 70%) were otined t extrction ph vlues of ove 10.5 nd precipittion ph vlues of ove 4.4. Due to the prolems ssocited with lipid oxidtion, it is of gret importnce to otin low level of lipid content in the finl product. Severl studies hve een performed on lipid reduction using cid or lkline protein extrctions. Lipid removl depends on some fctors such s the speed which is chosen for the first step of centrifugtion process, the lipid content of the rw met nd the wter mount used in the process. Since wter is polr solvent, using high mounts of wter helps to seprte the polr frction of lipids from the muscle proteins. More detils re given on this topic in chpter 2. During the first step of centrifugtion process, the memrne lipids precipitte, which reduce the risk of lipid oxidtion in the finl product. Richrds nd Hultin (2001) studied the effect of lipid content on lipid oxidtion in minced wshed cod fish contining 0.1% ft. The results indicted high level of lipid oxidtion for the finl product. This result could emphsize the role of hemogloin s strong prooxidnt, which cuses lipid oxidtion even when the mount of ft in the minced wshed cod fish ws s low s 0.1%. Thus, in order to prevent lipid oxidtion in the presence of strong prooxidnts like hemogloin, the lipid content of the finl product should e very low. The following 11

24 studies were conducted on lipid reduction using cid, lkli nd trditionl surimi processing. The im of reserch conducted on severl fish species including ctfish, Spnish mckerel, mullet, nd croker y Kristinsson nd Demir in 2003, ws to compre lipid removl using surimi processing with cid nd lkli ided process. The results indicted tht the highest mount of lipid loss ws found using lkline soluiliztion followed y cid ided nd trditionl surimi process. A lipid removl of 68.4% (croker), 81.4% (mullet), 79.1% (mckerel), nd 88.6% (ctfish) ws reported using lkline process. The lipid reduction vlues for cid process for the mentioned fish species were 38.1%, 58.0%, 76.9%, nd 85.4%, respectively. Finlly, the vlues for trditionl surimi process were 16.7%, 10.4%, 72.1%, nd 58.3%, respectively. The min difference etween the processing of surimi nd the cid or lkline process ws the dewtering step. In cid nd lkli processes, centrifugtion is used to remove the excess wter from the recovered proteins, while in surimi technology cheesecloth is used for this mtter, which proly ffected removl of lipids. A study conducted y Cortes-Ruiz et l. (2001) on lipid reduction of fish (Bristly srdine) led to 67.4% lipid reduction in surimi process, nd 88.3% for the protein recovered y cid soluiliztion process, while the totl lipid content of the fish ws 3.3%. Another study on lipid reduction ws conducted y Kristinsson nd Ling (2006). In this study Atlntic croker fillets contining 3.1% lipid, ws used for cid, lkline, nd surimi processing. The dewtering step ws performed using centrifugtion (10,000 g) for 20 min for cid nd lkli process, nd y cheesecloth for the surimi process. The results showed ft reduction of 68% for the cid process, 38% for the lkli process, nd 17% for the surimi process. A study conducted y Kristinsson et l. (2005) on chnnel ctfish met compred lipid reduction using three processes of cid nd lkli protein extrction with trditionl surimi processing. The results showed tht the highest lipid reduction (88.6%) for lkline processing, while the vlue for cid nd surimi processing were 85% nd 58%, respectively. The ctfish met ws skinless fillets with lipid content of 4.7% to 9.8%. While the dewtering step for surimi process involved squeezing y cheesecloth, this step 12

25 for cid or lkli process ws operted y centrifugtion (10,000 g) for 20 min in 2 steps. The reson for the noticele difference in lipid reduction etween the conventionl surimi process nd the cid nd lkline processes ws due to the first centrifugtion step in cid nd lkli processes (Kristinsson nd Demir, 2003). Lipid removl ws greter in cid nd the lkline processing thn the trditionl surimi processing due to the use of centrifugtion. In most of the comprtive studies on the cid nd lkline soluiliztion processes in fish, lipid reduction ws higher y lkline soluiliztion. A study y Froning nd Johnson (1973) ws performed to improve the qulity of mechniclly deoned fowl met y centrifugtion. The results showed tht centrifugtion process (20,000 rpm for 15 min t 5 C) hd significnt effect on ft reduction from mechniclly deoned poultry met. Totl ft content of the recovered proteins using centrifugtion ws decresed y 62.8%. The finl product of n lkline extrction followed y cid precipittion from mechniclly seprted poultry residues showed 51.3% decrese in totl ft content (Jelen et l., 1982). In study y Dwson et l. (1989) n queous wshing process ws used to remove ft nd pigments from mechniclly deoned chicken met. MDCM ws mixed with solution of wter with dded sodium icronte (ph: 8.0) to soluilize myofirillr proteins which re slt solule. The rtio of met to wter ws 1:4 (w:w), ph of 6.8 ws used to precipitte myofirillr proteins, nd seprte them from pigments, nd dewtering ws performed y centrifugtion. The results of this study showed tht ft content of the extrcted MDCM ws decresed y 88.3% compred to the rw MDCM. The initil ft content of the MDCM ws 12.8%. Totl ft content of the finl product ws decresed y 93.3% in study using lkline soluiliztion (ph: 10.8) nd cid precipittion (ph: 5.2) which ws performed on mechniclly deoned turkey met. The rtio of met to wter ws 1:6 (w:v), nd centrifugtion (10,000 g for 25 min, t 0-10 C) ws used for dewtering (Ling nd Hultin, 2003). 13

26 Another fctor for the qulity of met products is lipid oxidtive stility which is relted to the product storge stility. Minced met products s well s products contining greter mount of prooxidnts re those which re t high risk of lipid oxidtion. This could e explined y the fct tht fter mincing process with the incresed lipid surfce eing exposed to oxygen nd prooxidnts, lipid oxidtion increses. Becuse of the ctivtion of heme proteins in cidic conditions leding them to ct s prooxidnt, the cid soluiliztion process cused more lipid oxidtion thn the lkline process. In ddition, the exposure of minced wshed cod fish to cidic conditions (ph: 2.5) for 20 minutes mkes the muscle more susceptile to oxidtion (cused y hemogloin) compred to ntive wshed minced fish met (Kristinsson, 2001). Acidic conditions exist in oth cid nd lkli processes. However during the cid soluiliztion process there is greter exposure to cidic conditions. As hs een mentioned efore, there is n exposure to low ph (isoelectric point i.e. ph: ) for myofirillr proteins precipittion, which would led to lipid oxidtion. Kristinsson nd Ling (2006) reported more lipid oxidtion for the recovered proteins from Atlntic croker fish y cid process compred to the lkline process. In study conducted y Kristinsson nd Demir (2003) on some fish species, lipid oxidtion of the protein isoltes using cid, lkli, nd surimi process ws evluted. The species of fish used in this study included ctfish, croker, mullet, nd Spnish mckerel, nd the temperture of the wter used in the process ws 4 C. After the process, the protein isoltes otined from ll three processing methods were put in freezer gs, nd kept in the fridge (4 C). Afterwrds, every third dy smples were chosen nd were put in the freezer (-70 C) to prevent lipid oxidtion. After this tretment, thiorituric cid rective sustnces (TBARs) were mesured s method for lipid oxidtion evlution. The results indicted tht the lowest mounts of lipid oxidtion were found with surimi process compred to cid nd lkli processing. The highest level of lipid oxidtion ws found with the cid soluiliztion process followed y the lkli ided process. The reson for the more lipid oxidtive stility of surimi process would e tht during this process, the minced met is not sujected to low ph, which prevents hemogloin nd myogloin ctivtion leding to reduction in lipid oxidtion. 14

27 A potentil solution to reduce lipid oxidtion is the ddition of compounds such s metl cheltors which cn ct like ntioxidnts nd improve lipid oxidtive stility of the recovered proteins. A study y Undelnd et l. (2005) indicted tht chelting gents such s ethylenediminetetrcetic cid (EDTA) (0.04%) nd sodium tripolyphosphte (0.2%), nd lso reducing gents such s erythorte (0.2%), could ct s ntioxidnts nd improve lipid oxidtive stility of cid-processed fish nd lso during the storge of the protein isolte. The results of this work showed tht while the ddition of only erythorte or in comintion with EDTA nd sodium tripolyphosphte hd significnt effect in decresing lipid oxidtion in the finl protein isolte, without using these ntioxidnts, severe lipid oxidtion occurred in the protein isolte. Lipid oxidtive stility of mechniclly deoned poultry met which ws centrifuged ws higher thn those without centrifugtion (Froning nd Johnson, 1973). This finding indicted tht ft reduction cused y centrifugtion led to higher stility to lipid oxidtion for the finl product. In study conducted y Dwson et l. (1990) myofirillr proteins from mechniclly deoned chicken met were extrcted y phosphte solution (ph: 8.0), followed y precipittion ph of 6.8, nd dewtered y centrifugtion. The results of the study indicted tht even though totl ft content decresed y 88.3%, extrcted met ws more susceptile to lipid oxidtion thn the rw met. The lower lipid oxidtive stility of extrcted met compred to the rw met ws due to unsuccessful removl of phospholipids which re more susceptile to lipid oxidtion Functionl Properties of Muscle Proteins nd their Link to ph Shifting Process nd Trditionl Surimi Production Protein Soluility The definition of soluility or extrctility of proteins is the percent of the totl proteins extrcted y wter or suitle solvent in specific conditions (Sikorski, 2007). One of the mjor fctors ffecting protein soluility in queous solutions is the ph of the 15

28 solution. In lkline nd cidic conditions, proteins crry negtive nd positive chrge, respectively. This leds to repulsion etween the proteins which finlly cuse proteins to e soluilized. On the other hnd, ecuse of the fct tht proteins crry no net chrge t the isoelectric point, they precipitte t this ph. For the isoltion of different kinds of proteins from food, soluility difference is considered s n importnt issue (Kristinsson, 2001). Another fctor which ffects soluility is denturtion of the proteins. Soluility my decrese when proteins re dentured. According to Sikorski (2007) during heting, denturtion which is followed y ggregtion of the proteins leds to n increse in hydrophoicity, nd s result, soluility reduces. The other criticl fctor which ffects protein soluility is the ionic strength of solution. Ionic strength determines the concentrtion of ions in solution, nd is clculted y the following formul: µ = 1 2 C i Z i 2 In this formul C i is the concentrtion of n ion nd Z i is the net chrge of the ion (Fox nd Foster, 1957). When ionic strength of slt solution is low (i. e. lower thn 0.5) protein soluility cn increse or decrese depending on the mino cid composition on the protein surfce. For exmple, for soy proteins, ecuse hydrophoic (non-polr) mino cids re locted on the surfce of the protein molecule, they re unle to ind to the ions of the slt solution, nd therefore, soluility of the proteins is reduced. In contrst, β-lctogloulin nd myosin hve hydrophilic (polr) mino cids locted on the surfce of the protein molecule, thus they cn ind to the ions in the slt solution, nd increse protein soluility. With the increse in ionic strength of slt solution (i. e. μ >1), different ions ffect soluility in different wys. For exmple, for slt solutions 2- contining ions of SO 4 nd F, soluility decreses with the increse in the slt concentrtion (Dmodrn, 2008). It mens tht slt solutions contining these ions re le to precipitte proteins from the solution. This concept is nmed s slting out. On the other hnd, when slt solutions contining ions of Br, I, SCN, nd ClO 4 re dded to the protein solution, soluility increses with the increse in the slt concentrtion. This phenomenon is nmed slting in (Sikorski, 2007). When ionic strength of solution is kept t constnt mount, protein soluility cn either increse or decrese 16

29 sed on the nions nd ctions which re present in the solution. In this condition, while nions which most increse protein soluility involve SCN, ClO 4, I, Br, Cl, F, nd SO 4 2- respectively, ctions which most decrese protein soluility include C 2+, Mg 2+, Li +, N +, K + +, nd NH 4, respectively. This rnking of nions nd ctions is nmed the Hofmeister series (Dmodrn, 2008) Protein Geltion Proteins from different sources of niml or plnt hve the gel forming ility. In cse of myofirillr proteins, when they re extrcted from muscle nd exposed to het, they form n irreversile semisolid, three-dimensionl structure, which is clled gel. Since myofirillr proteins form the mjor component of processed met products, geltion is n importnt fctor in met products industry (Acton et l., 1978; Brut, 1995). Myofirillr protein geltion process involves severl steps. During the first step, muscle fiers re extrcted y the use of slt. Afterwrds, with the ppliction of het, protein denturtion occurs. The next step is ggregtion, nd it hppens when unfolded proteins connect with the surrounding proteins. The lst step occurs when ggregtes cross-link which leds to the formtion of three-dimensionl gel network nd wter entrpment within the network (Trte nd Amundson, 2006) Rheologicl Properties Dynmic Mechnicl Anlysis (DMA) Dynmic mechnicl nlysis (DMA) is method which is used to study dynmic viscoelstic ehvior (DVB) of mteril. For this purpose, dynmic or oscilltory tests re performed. During DMA, dynmic stress t given frequency is pplied to mteril. Viscoelstic mterils show oth elstic (solid) nd viscous (liquid) ehviors. The rheologicl fctors which show DVB of the mteril, includes the storge modulus (G'), the loss modulus (G''), nd loss ngle (Tn delt). The storge modulus (G') is relted to the elstic (solid) chrcter of the mteril which indictes the energy which is stored during deformtion (Peleg et l., 1989; Hmnn et l., 1990). On the other hnd, the loss modulus (G'') is relted to the viscous (liquid) ehvior of the mteril which indictes 17

30 the energy which is dissipted s the mteril is deformed (Vodovotz et l., 2001). For exmple if we ssume solid which is idelly elstic, ll the energy is stored (i.e. G''= 0) ut for viscous mteril in which ll the energy is dissipted, G' is equl to zero. Tn delt which is defined s the rtio of G''/G' (Dogn nd Kokini, 2007), shows the degree of viscoelsticity of smple. If tn delt is low vlue for mteril, it mens tht the mteril hs higher elsticity (higher G' vlue) nd is more solid-like mteril Texturl Properties Texture of foods is n ttriute which is strongly relted to consumer preference, selection, nd stisfction of food products. Food industry efforts re to develop the proper texture for food products (Rohm, 1990; Gunsekrn nd Ak, 2003). According to the Interntionl Orgniztion for Stndrdiztion, texture of food is defined s the rheologicl nd structurl ttriutes of food product which is perceived y humn senses (ISO, 1992). Texture of met is n ttriute tht is determined y severl fctors such s hrdness, springiness, chewiness, nd cohesiveness. Differences in met texture re relted to the composition nd structure of the met including different kinds of proteins s well s ft nd connective tissue. Some other fctors such s cooking lso ffect met texture (Biley, 1972; Solomon et l., 2009) Texturl Profile Anlysis (TPA) Texturl properties of mteril re mesured y technique clled texturl profile nlysis. According to Mllikrjunn (2006), cohesiveness, springiness, chewiness, resilience nd hrdness re the texturl ttriutes which re mesured y the utiliztion of TPA technique. Definition of these prmeters is importnt ecuse ech fctor dels with different spects of mteril s texture. While the mount of work needed to chew solid smple until the swllowing stte is determined y chewiness vlue, the ility of mteril to recover its originl shpe fter the removl of the force is known s springiness. Resilience, on the other hnd, determines n extent to which the smple cn fight to get ck to its originl position. Cohesiveness determines the mount of deformtion for mteril efore it reks, nd finlly, hrdness is the mximum force needed to compress mteril. By determining ech of the ove fctors, some informtion could e otined out the texture of mteril. 18

31 Wter Holding Cpcity Wter holding cpcity (WHC) which is one of the importnt ttriutes of oth fresh met or processed met products indictes the extent to which met cn keep its moisture inside when some conditions pply (Swtlnd, 2002). These conditions cn e the ppliction of temperture (e.g. cooking), or compression. A numer of fctors ffect WHC of met such s ph, niml species, nd ge. The most importnt fctor is the ph of the met. In order to study the effect of ph on WHC of the met, study hs een performed on eef (Gru et l., 1953). The eef smples were immersed in uffer solutions with different ph vlues nd their weight gin were mesured fterwrds. The results indicted the lowest weight gin for the smples which were immersed in the uffer solution with the ph vlue of 5.2. The reson could e explined due to the fct tht ph of 5.2 is the isoelectric point for met proteins. At this ph the net chrge of the proteins is equl to zero, hence they re ttrcted to ech other, nd s result they cnnot hold wter inside them. Thus, their WHC ws t its minimum level (Honikel, 2009). The reson for WHC to e such n importnt fctor for the met is tht it is ssocited with the ppernce of the met when exposed to het tretment (i.e. cooking), nd contriutes to the juiciness of the met. When it comes to comminuted met products (e.g. susges), WHC is prticulrly n importnt qulity ttriute. During the production of comminuted met products, due to mincing process of the met, structure of the met proteins which hold the wter is destroyed, nd therefore, the ility of the met to hold the wter will e decresed (Lwrie nd Ledwrd, 2006). Therefore, WHC gretly ffects the qulity of the comminuted met products Cooking Loss One of the importnt fctors which determine the qulity of the met product is cooking loss. Cooking loss indictes the mount of wter which is lost during cooking, nd therefore, it is ssocited with wter holding cpcity of the met. When met is heted, if the met hs lower cooking loss, it will e understood tht the met hd higher ility to hold wter, nd therefore, it hs greter WHC. If the wter holding cpcity of met product is low, the met will lose more mount of wter during cooking leding to production of drier product, which cn e uncceptle to consumers (Brut, 2002). 19

32 Cooking loss occurs with the denturtion of met proteins. During cooking, the structure of met proteins chnge y shrinkge of muscle fiers nd connective tissues, nd ggregtion of proteins occurs leding to wter relese from the cells (i.e. decrese of WHC). The temperture t which the met proteins denture is t the rnge of 37 C to 75 C, nd the recommended internl temperture for cooking is 75 C (Honikel, 2009) Emulsifiction Cpcity Emulsifiction cpcity, which is n importnt qulity fctor especilly in comminuted met products, is defined s the ility of the proteins to emulsify oil. In order to mesure emulsifiction cpcity, liquid oil is grdully dded to met protein solution, while using homogenizer to mix the oil. The im is to determine the point t which proteins cnnot emulsify more oil. This point is known s the rekdown point, nd is the stte when oil nd protein phses seprte (Brut, 2002). The rekdown point cn e mesured y sudden decrese in viscosity or conductivity of the solution, since the ft phse hs lower conductivity. The method is used to mesure the emulsifiction cpcity for proteins from chicken met (Murer et l., 1969) Protein Denturtion In food processing, denturtion is defined s n irreversile chnge which occurs when proteins unfold nd hydrophoic mino cids, which re originlly locted in the interior of the protein molecule, re exposed. A numer of fctors cuse protein denturtion such s chnge in ph (y the ddition of lkli or cid) nd temperture (i.e. cooking), or ddition of ions (e.g. NCl). Denturtion ffects numer of fctors such s soluility nd hydrophoicity of the proteins (Sikorski, 2007). There will e decrese in soluility s well s n increse in hydrophoicity of the dentured proteins compred to the ntive stte. Denturtion of met proteins occurs t the temperture rnge of C. For exmple, the denturtion temperture for myosin is C (Lesiow nd Xiong, 2001; Trte nd Amundson, 2006). According to Wng nd Smith (1994) nd Smyth et l. (1999) the denturtion temperture of myosin vries with ph nd ionic conditions. A ph of nd ionic strength of 0.6 M NCl re the typicl conditions used in processed met industry. 20

33 The Effect of Surimi nd ph Shifting Process on Functionl, Texturl, nd Rheologicl Properties of the Recovered Proteins The geltion properties of the recovered proteins ply n importnt role in the preprtion of processed muscle food products. Two importnt fctors which influence the qulity of the gel formed y the recovered proteins re temperture nd the qulity of rw mteril used for processing. In n investigtion conducted y Kristinsson nd Demir (2003), surimi nd cid / lkli process were tested on four different fish species, nd the gel qulity mesurements were performed y oscilltory tests. The est gel ws otined for the protein isolte prepred using lkli process, nd the gel otined from the cid processed mince ws of the lowest qulity. The qulity of the surimi gel ws etween the lkli nd cid processed ones. These results could e explined y the lower protese ctivity during lkli process, which cused less protein rekdown. Thus, when the protein isolte otined from the lkli process ws exposed to geltion, due to the higher mount of protein-protein interction nd ggregtion, the resultnt gel hd higher qulity. According to Nolsøe nd Undelnd (2009) the results of severl comprtive studies on fish species out the qulity of gel for the recovered proteins during cid nd lkli process, indicted etter gel formtion for protein isoltes otined y lkline ided process. Regrding the qulity of rw mteril s well s comprison of cid nd surimi process, study ws conducted on fresh srdine fish to compre different gels otined from surimi nd cid process sed on texturl profile nlysis (hrdness nd cohesiveness, nd elsticity) (Cortes-Ruiz et l., 2001). The rw mteril for cid process included fresh srdines nd srdines which were stored on ice for 5 dys. The highest hrdness vlue ws reported for the proteins recovered y the cid process which used the 5-dy stored srdines s the strting mteril. This could e explined y the more protein content (+ 15%) in the finl product prepred using cid process. Higher hrdness vlues were proly ecuse of more protein-protein interction nd ggregtion which might hve led to hrder ut less elstic gels. Regrding texturl properties, mximum vlues were reported for cohesiveness nd elsticity of the gels otined from the surimi process, nd minimum vlues were ssocited with the proteins recovered y the cid process using the stored fish s the rw mteril. 21

34 In study conducted y Li (2006) isolted myofirillr proteins from spent hens were dded to chicken rest nd pork hm to evlute texturl properties. A solution contining 6% myofirillr proteins ws injected into chicken rest nd pork hm t rtio of 20% of the met weight. The results showed higher hrdness for chicken rest s well s higher hrdness nd chewiness for pork hm with dded myofirillr proteins compred to those without the dded proteins. Therefore, the use of isolted proteins from low vlue poultry met sources for functionl properties improvement of met products ws indicted in the study. According to Froning nd Johnson (1973) cooking loss of the recovered proteins from mechniclly deoned poultry met fter centrifugtion tretment ws lower compred to those with no centrifugtion tretment. The lower cooking loss for the recovered met might e explined y the higher protein content of it, which led to higher wter holding cpcity for the recovered proteins. An lkline soluiliztion with n cid precipittion ws performed on mechniclly deoned turkey met (Ling nd Hultin, 2003). The uthors found tht cooking loss of the cooked protein isolte decresed significntly compred to tht of cooked rw met. This finding indicted tht higher wter holding cpcity for the protein isolte ws due to its higher protein content. In ddition, good gel forming ility ws reported for the protein isolte Color nd its Link to the Recovered Proteins from Surimi nd ph Shifting Process Color is n importnt met qulity which gretly ffects consumers preference (Froning, 1995). Totl heme pigments including myogloin nd hemogloin re responsile for the color of met. The met pigment is minly myogloin ecuse hemogloin, which is the lood pigment, will e mostly removed fter the slughter. Therefore, myogloin is the determining fctor for the met color, nd vritions in met color indicte the differences in myogloin content. For exmple, poultry rest nd thigh which re known s white nd drk met respectively, esily cn e differentited from ech other (Schwrtz et l., 2009). Since myogloin nd hemogloin contin iron, the 22

35 difference in iron content of white nd drk chicken met is the result of vritions in totl heme pigments (see Tle 1.1). The CIE L color scle which is sed on the vlues of redness (*), yellowness (*), nd lightness (L*) is well-known method for mesuring the color of smple. In this regrd, the moisture content of the smple is n importnt fctor influencing the color mesurements. If the smple hs more wter content, ecuse of the greter light reflection, it will hve more lightness vlue. The CIELAB color ttriutes for chicken drk nd white met re presented in Tle 1.2. The fctors influencing the color of the recovered proteins re those ssocited with the rw strting mteril such s the mount of pigments, lood, nd drk muscle. Therefore, different processes should e pplied to different fish species for otining the lightest (in color) possile protein isoltes, ecuse consumers preferred the whitest color for the protein isoltes (Tilo-Munizg nd Bros-Cnovs, 2004). This shows tht color is n importnt fctor which ffects consumers selection for the products mde from isolted proteins. The results from comprtive studies performed on surimi, cid nd lkli process indicted the mximum whiteness vlue for the protein isoltes otined from the surimi process, followed y lkli nd cid processes. According to Nolsøe nd Undelnd (2009) the greter whiteness vlue in surimi production could e explined y the wshing process which might hve helped to remove higher mounts of heme pigments from the protein isolte. The higher whiteness vlue for the proteins recovered y lkline soluiliztion processes compred to the ones otined from the cid soluiliztion process indicted tht lkli ided process ws more efficient in the heme pigments seprtion from the fish met. A study y Hernndez et l. (1986) on mechniclly deoned turkey met indicted tht protein extrction using phosphte uffer with ph of 8.0 could efficiently remove heme pigments, incresed lightness of the finl product y 51%, nd reduced its redness y 64%. There ws significnt increse in lightness vlue of the extrcted met compred to the rw met from mechniclly deoned chicken met using n queous wshing with 23

36 dded sodium icronte (ph: 8.0). Redness vlue of the finl product ws lso significntly decresed (Dwson et l., 1989) The Effect of Surimi nd ph Shifting Process on the Recovered Proteins Stility during Storge One of the importnt prmeters which determines the qulity of the protein isolte is the stility of the isolted proteins during frozen storge which is considered s n dvntge for surimi production. As hs een mentioned previously, in surimi technology cryoprotectnts re compounds such s sugrs (sucrose nd soritol) which re le to reduce the protein denturtion during freezing nd thwing process s well s prevent ggregtion (Mtsumoto, 1980). The reson is tht cryoprotectnts void proteins ecoming unfolded (Crpenter nd Crowe, 1988). Thus, in this wy they increse the stility of protein products (e.g. surimi) when stored frozen. Chemicls which hve een used s cryoprotectnts in muscle proteins included sucrose, soritol, phosphtes, nd sodium tripolyphosphte (Nishiy et l., 1961; Mhon, 1964; Mtsumoto, 1978, 1979; Prk nd Lnier, 1987; Prk et l., 1988). One ction which hs een reported for phosphtes (e.g. sodium tripolyphosphte) s cryoprotectnt is tht they decrese the negtive effect of frozen storge on myofirillr protein soluility (Jittinndn et l., 2003). A study ws conducted on the recovered proteins (Thwornchinsomut nd Prk, 2006) from Pcific whiting fish regrding the effect of frozen storge on texturl properties. In order to determine the effect of cryoprotectnts on the stility of proteins during storge, they divided ech smple into two prts, nd used cryoprotectnts in one prt ut not in the other. Afterwrds, the smples were exposed to freezing nd thwing steps which were repeted three times. The tempertures for freezing nd thwing were - 18 C nd 4 C, respectively. The results indicted tht the est qulity gels were otined from the recovered proteins using n lkli process contining cryoprotectnts. In contrst, the lowest gel qulity ws ssocited with the gels otined from the protein smples without cryoprotectnts. From this study, it cn e concluded tht using cryoprotectnts for the protein isoltes otined from lkli process is of high importnce to void freeze denturtion. 24

37 On the other hnd, the ddition of cryoprotectnts is not the only wy of incresing the stility of protein product during frozen storge. Jczynski et l. (2006) reported tht y seprtion of memrne lipids from the protein isolte, even without using cryoprotectnts, the stility of freeze-dried fish protein could e improved. 1.4 Potentil Applictions of the Recovered Proteins y ph Shifting Methods The gol of ph shifting technology is the utiliztion of low vlue met nd development of new helthy vlue-dded products. By the dvntges which were mentioned for ph shifting technology, nd y the comprison of different chemicl nd functionl properties of the proteins recovered y this method, it cn e concluded tht ph shifting technology could etter utilize low vlue drk met thn the trditionl surimi processing. The US. Food nd Drug Administrtion (2004) hs confirmed the isolted proteins from sefood nd poultry sources using ph shifting processing s Generlly Regrded As Sfe (GRAS). The finl product of the recovered proteins using ph shifting processing cn e in the form of liquid or powder. Proteus Industries introduced isolted proteins s coting for ttered nd reded products ( Other pplictions include using recovered proteins s ingredients to improve functionl properties of food products. For exmple, isolted proteins cn e used s emulsifiers (Petursson et l., 2004) nd to improve wter holding cpcity of whole-muscle met products (Li, 2006). The coting product Nutrilen which is in the form of powder hs een produced y Proteus Industries using cid process on oth chicken nd fish sources. Nutrilen is pure protein isolte, nd it cn e used s tter ingredient which cn e spryed on to ny kind of met efore frying. Nutrilen forms thin rrier outside the coted product which prevents moisture from escping. Thus, during frying, less oil cn e sored into the product. This wy, the ft content of the finl fried product ws reported to decrese y 25% to 75%. Wter holding cpcity of the finl cooked product ws lso improved. As consequence, moisture loss from drying out the product under the het lmps in resturnts or fst foods ws reduced leding to more juicy products. Even the color of the finl product hs een reported to improve to richer ronze color, which is more 25

38 nturl color nd is usully found in home-mde products. According to the Proteus Industries the technology cn e pplied to met from ll sources. The food industries which re currently using Nutrilen technology involve Comfort Creek, Good Hror, nd Sunrise Foods. 1.5 Reserch Ojectives Min Ojective The ojective of this thesis project ws to exmine the use of high ph extrctions nd low precipittion ph on poultry drk met in order to produce functionl proteins extrcts suitle for the preprtion of further processed met products Specific Ojectives Determine the effect of ph, in the rnge of 10.5 to 12.0, on lkline soluiliztion process of chicken drk met Evlute the effect of ph on soluility nd extrctility of the proteins from chicken drk met Investigte the effect of lkli ided process on protein yield nd composition of chicken drk met Estlish the effect of lkli ided extrction on the distriution of ftty cids in etween neutrl nd polr lipid frctions of chicken drk met Study the effect of lkli ided extrction on color chrcteristics of the chicken drk met. Evlute the effect of lkli ided process on lipid oxidtive stility of chicken drk met Determine the effect of lkline soluiliztion process on the texturl nd rheologicl chrcteristics of chicken drk met 26

39 Evlute the influence of cooking on the wter holding cpcity of lkli extrcted chicken drk met 1.6 Approch The ojectives of this thesis were ssessed in two phses followed y lortory nlysis. Phse 1. Four extrction ph vlues (10.5, 11.0, 11.5, nd 12.0) were chosen for lkli protein extrction from chicken drk met. Ech experiment nd ech ssy ws done t lest in triplicte. Protein yield, protein nd lipid composition, ftty cid profile, color chrcteristics nd TBARS were studied in chicken drk nd extrcted met. Phse 2. Four extrction ph vlues (10.5, 11.0, 11.5, nd 12.0) were chosen for lkli protein extrction from chicken drk met. Ech experiment nd ech ssy ws done t lest in triplicte. Texturl nd rheologicl chrcteristics, s well s cooking loss nd expressile moisture hve een studied. 27

40 1.7 TABLES Tle 1.1. Protein (%), lipid (%), moisture (%), nd iron (mg/100 g of met) content of skinless chicken drk nd white met Chicken Protein Lipid Moisture Iron content Met content content content (mg/100g met) Drk Met White Met Modified from: USDA,

41 Tle 1.2. Color ttriutes (L*, *, nd *) of chicken drk nd white met Chicken Met Lightness (L*) Redness (*) Yellowness (*) Drk Met White Met Modified from: Perez-Alvrez, nd Fernndez-Lopez,

42 1.8 FIGURES Figure 1.1. Schemtic digrm of muscle structure, strting from cross section of whole muscle (), including the lyers of connective tissue, the muscle undle (), fire (c), myofiril (d) nd myofilments (e). Reprinted from POULTRY PRODUCTS PROCESSING: AN INDUSTRY GUIDE. EBOOK y Shi Brut. Copyright 2001 y Tylor & Frncis Group LLC - BOOKS. Reproduced with permission of Tylor & Frncis Group LLC BOOKS in the formt Disserttion vi Copyright Clernce Center. 30

43 Figure 1.2. Flow chrt of surimi mnufcturing. Reprinted from SURIMI AND SURIMI SEAFOOD, SECOND EDITION y J. W. Prk nd T.M. JOHN LIN. Copyright 2005 y Tylor & Frncis Group LLC - BOOKS. Reproduced with permission of Tylor & Frncis Group LLC - BOOKS in the formt Disserttion vi Copyright Clernce Center. 31

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47 Gunsekrn, S., nd M. M. Ak Cheese texture in Cheese Rheology nd Texture. S. Gunsekrn, nd Ak, M. M., eds. CRC Press, Boc Rton, FL ch7 Accessed Fe Hmnn, D. D., S. Purkysth, nd T. C. Lnier Applictions of therml scnning rheology to the study of food gels. Pge 306 in Therml Anlysis of Foods. V. R. Hrwlkr, nd C. Y. M, eds. Elsevier, New York, NY. Hernndez, A., R. C. Bker, nd J. H. Hotchkiss Extrction of pigments from mechniclly deoned turkey met. J. Food Sci. 51: Honikel, K. O Moisture nd wter-holding cpcity. Pges in Hndook of Muscle Foods Anlysis. L. M. L. Nollet, nd F. Toldr, eds. CPC Press, Boc Rton, FL. Hultin, H. O., nd S. D. Kelleher Process for isolting protein composition from muscle source nd protein composition. US Pt. No. 6,005,073. Hultin, H. O., nd S. D. Kelleher High efficiency lkline protein extrction. US Pt. No. 6,136,959. Hultin, H. O., H. G. Kristinsson, T. C. Lnier, nd J. W. Prk Process for recovery of functionl proteins y ph shifts. Pges in Surimi nd Surimi Sefood. 2nd ed. J. W. Prk, ed. Tylor & Frncis, Boc Rton, FL. Hunt, A., J. W. Prk, nd C. Jundoo Cryoprotection of Pcific whiting surimi using non-sweet glucose polymer. Proc. IFT Annul Meet (Astr.) Hunt, A., J. W. Prk, nd H. Zoer Trehlose s functionl cryoprotectnt for fish proteins. Proc. IFT Annul Meet (Astr.) ISO (Interntionl Orgniztion for Stndrdiztion) Sensory Anlysis- Voculry. ISO

48 Jckson, P., G. W. Amphlett, nd S. V. Perry Primry structure of troponin-t nd interction with tropomyosin. J. Biochem. 151: Jczynski, J., W. Hunt, nd J. W. Prk Sfety nd qulity of frozen fish, shellfish, nd relted products. Pges in Hndook of Frozen Food Processing nd Pckging. D. W. Sun, ed. CRC Press, Boc Rton, FL. Jelen, P., R. A. Lwrence, nd M. Cerrone Evlution of lkli extrcted chicken protein for use in luncheon mets. Cn. Inst. Food Sci. Technol. J. 5: Jittinndn, S., P. B. Kenney, nd S. D. Slider Cryoprotection ffects physiochemicl ttriutes of rinow trout fillets. J. Food Sci. 68: Kim, Y. S., J. W. Prk, nd Y. J. Choi New pproches for the effective recovery of fish proteins nd their physicochemicl chrcteristics. Fish. Sci. 69: Kim, J. S., nd J. W. Prk Geltin from solid y-products of Pcific whiting surimi processing. IFT Annul Meet. 76A-11. (Astr.) Kristinsson, H. G Conformtionl nd functionl chnges of hemogloin nd myosin induced y ph: Functionl role in fish qulity. PhD Diss. Univ. Msschusetts, Amherst. Kristinsson, H., nd N. Demir Functionl fish protein ingredients from fish species of wrm nd temperte wters: comprison of cid- nd lkli-ided processing vs. conventionl surimi processing. Pges in Advnces in Sefood Byproducts. P. J. Bechtel, ed. Univ. Alsk, Anchorge. Kristinsson, H. G., nd H. O. Hultin Effect of low nd high ph tretment on the functionl properties of cod muscle proteins. J. Agric. Food Chem. 51: Kristinsson, H. G., A. E. Theodore, N. Demir, nd B. Ingdottir A comprtive study etween cid- nd lkli-ided processing nd surimi processing for the recovery of proteins from chnnel ctfish muscle. J. Food Sci. 70:C298-C

49 Kristinsson H. G., nd B. Ingdottir Recovery nd properties of muscle proteins extrcted from tilpi (Oreochromis niloticus) light muscle y ph shift processing. J. Food Sci. 71:E132-E141. Kristinsson, H. G., nd Y. Ling Effect of ph-shift processing nd surimi processing on Atlntic croker (Micropogonis undultes) muscle proteins. J. Food Sci. 71:C304-C312. Lwrie, R. A., nd D. A. Ledwrd The eting qulity of met. Pge 290 in Lwrie s Met Science. 7th ed. R. A. Lwrie, nd Ledwrd, D. A., eds. CPC Press, Boc Rton, FL. Lee, C. M Surimi process technology. Food Technol. 38: Li, C. T Myofirillr protein extrcts from spent hen met to improve whole muscle processed mets. Met Sci. 72: Ling, Y., nd H. O. Hultin Functionl protein isoltes from mechniclly deoned turkey y lkline soluiliztion with isoelectric precipittion. J. Muscle Foods. 14: Mhon, J. H., nd C. G. Schneider Minimizing freezing dmge nd thwing drip in fish fillets. Food Technol. 18: Mllikrjunn, P Physicl mesurements. Pge 528 in Hndook of Frozen Food Processing nd Pckging. D. W. Sun, ed. CPC Press, Boc Rton, FL. Mtsumoto, J. J Minched fish technology nd its potentil for developing countries. Proc. Symp. Fish Utiliztion Technology nd Mrketing in the IPFC Region, Mnil, Philippines. Mtsumoto, J. J Denturtion of fish muscle proteins during frozen storge. Pges in Proc. Am. Chem. Soc. Symp. Wshington, DC. Mtsumoto, J. J Chemicl deteriortion of muscle proteins during frozen storge. Pges in Proc. Am. Chem. Soc. Symp. Wshington, DC. 37

50 Murer, A. J., R. C. Bker, nd D. V. Vdehr Kind nd concentrtion of solule protein extrct nd their effect on the emulsifying cpcity of poultry met. Food Technol. 23: McCormick, R. J Extrcellulr modifictions to muscle collgen: implictions for met qulity. Poult. Sci. 78: McCurdy, S., P. Jelen, P. Fedec, nd D. F. Wood Lortory nd pilot scle recovery of protein from mechniclly seprted chicken residues. J. Food Sci. 51: Nishiy, K., F. Tked, K. Tmoto, O. Tnk, nd T. Kuo Studies on freezing of surimi (fish pste) nd its ppliction. III. Influence of slts on qulity of fish met. Mon. Rep. Hokkido Fish. Res. L. Fisheries Agency. Jpn. 17: Nishiy, K., F. Tked, K. Tmoto, O. Tnk, T. Fukumi, T. Kityshit, nd S. Aizw Studies on freezing of surimi (fish pste) nd its ppliction: studies on mnufcture of frozen surimi for the mteril of kmoko nd susge. Hokkido Munic. Fish Exp. Stn. 18: Nolsøe, H., nd I. Undelnd The cid nd lkline soluiliztion process for the isoltion of muscle proteins: stte of the rt. Food Bioprocess Technol. 2:1-27. Owens, C. M Coted poultry products. Pge 229 in Poultry Met Processing, A. R. Shms, ed. CRC Press, Boc Rton, FL. Prk, J. W., nd T. C. Lnier Comined effects of phosphtes nd sugr or polyol on protein stiliztion of fish myofirils. J. Food Sci. 52: Prk, J. W., T. C. Lnier, nd D. P. Green Cryoprotective effects of sugrs, polyols, nd phosphtes on Alsk pollock surimi. J. Food Sci. 53:1-3. Prk, J. D., C. H. Jung, J. S. Kim, D. M. Cho, M. S. Cho, nd Y. J. Choi Surimi processing using cid nd lkli soluiliztion of fish muscle protein. J. Koren Food Sci. Nutr. 32:

51 Prk, J. W., nd T. M. J. Lin Surimi: mnufcturing nd evlution. Pges 35-47, nd in Surimi nd Surimi Sefood. J. W. Prk, ed. CRC Press, Boc Rton, FL. Peleg, M., I. Roy, O. H. Cmpnell, nd M. D. Normnd Mthemticl chrcteriztion of the compressive stress-strin reltionships of spongy ked goods. J. Food Sci. 54: Perez-Alvrez J. A., nd J. Fernndez-Lopez Color mesurements on musclesed foods. Pge 470 in Hndook of Muscle Foods Anlysis. L. M. L. Nollet nd F. Toldrà, eds. CRC Press, Boc Rton, FL. Peterson, E. B., nd D. Orden Effects of triffs nd snitry rriers on high- nd low-vlue poultry trde. J. Agric. Resour. Econ. 30: Petursson, S., E. A. Decker, nd D. J. McClements Stiliztion of oil-in-wter emulsions y cod protein extrcts. J. Agric. Food Chem. 52: Pigott, G. M Surimi: the high tech rw mterils from minced fish flesh. Food Rev. Int. 2: Purslow, P. P Intrmusculr connective tissue nd its role in met qulity. Met Sci. 70: Resurreccion, A. V. A Cookery of muscle foods. Pge in Muscle Foods. D. M. Kinsmn, A. W. Kotul, nd B. C. Breidenstein, eds. Chpmn nd Hll, New York, NY. Richrds, M. P., nd H. O. Hultin Rncidity development in fish muscle model system s ffected y phospholipids. J. Food Lipids. 8: Rohm, H Consumer wreness of food texture in Austri. J. Texture Stud. 21: Snmrtin, E., J. C. Aroley, M. Villmiel, nd F. J. Moreno Recent dvnces in the recovery nd improvement of functionl proteins from fish processing y- 39

52 products: use of protein glyction s n lterntive method. Comprehensive Rev. Food Sci. Food Sfety. 8: Schwrtz, S. J., J. H. von Ele, nd M. M. Giusti Colornts. Pge 537 in Fennem s Food Chemistry. S. Dmodrn, K. L. Prkin, nd O. R. Fennem, eds. CRC Press, Boc Rton, FL. Scopes, R. K Chrcteriztion nd study of srcoplsmic proteins. Pges in The Physiology nd Biochemistry of Muscle s Food. 2 nd ed. E. J. Briskey, R.G. Cssens, nd B. B. Mrsh, eds. Univ. of Wisconsin Press, Mdison, WI. Shie J. S., nd J. W. Prk, Physicl chrcteristics of surimi sefood s ffected y therml processing conditions. J. Food Sci. 64: Sikorski, Z. E The role of proteins in food. Pges in Chemicl nd Functionl Properties of Food Components. 3 rd ed. Z. E. Sikorski, ed. CRC Press, Boc Rton, FL. Smith, D. P., D. L. Fletcher, R. J. Buhr, nd R. S. Beyer Pekin ducklings nd roiler chicken pectorlis muscle structure nd composition. Poult. Sci. 72: Smyth, A. B., E. O. O Neill, nd D. M. Smith Functionl properties of muscle proteins in processed poultry products. Pges in Poultry Met Science. R. I. Richrdson, nd G. C. Med, eds. CABI Pulishing, New York, NY. Solomon, M. B., J. S. Estridge, E. W. Proczy, nd B. C. Bowker Mesuring met texture. Pge 480 in Hndook of Muscle Foods Anlysis. CPC Press, Boc Rton, FL. Southest Asin Fisheries Development Center A Hndook on the Processing of Frozen Surimi nd Fish Jelly Products in Southest Asi. Mrine Fish. Res. Dep. Sttistics Cnd Poultry nd Egg Sttistics X:12. Accessed Sep

53 Strsurg, G., Y. L. Xiong, nd W. Ching Physiology nd chemistry of edile muscle tissues. Pges nd in Fennem's Food Chemistry. 4th ed. S. Dmodrn, K. L. Prkin, nd O. R. Fenem, eds. CRC Press, Boc Rton, FL. Swtlnd, H. J On-line monitoring of met qulity in Met Processing: Improving Qulity J. Kerry, Kerry, J., nd Ledwrd, D., eds. CRC Press, Boc Rton, FL. Accessed Sep Tilo-Munizg, G., nd G. V. Bros-Cnovs Color nd texturl prmeters of pressurized nd het-treted surimi gels s ffected y potto strch nd egg white. Food Res. Int. 37: Tked, F Technologicl history of frozen surimi industry. New Food Ind. 13: Trte, R., nd C. M. Amundson Protein interctions in muscle foods. Pges in Ingredient Interctions: Effects on Food Qulity. 2nd ed. A. G. Gonkr, nd A. McPherson, eds. CRC Press, Boc Rton, FL. Thwornchinsomut, S., nd J. W. Prk Frozen stility of fish protein isolte under vrious storge conditions. J. Food Sci. 71:C227-C232. Undelnd, I., S. D. Kelleher, nd H. O. Hultin Recovery of functionl proteins from herring (Clupe hrengus) light muscle y n cid or lkline soluiliztion process. J. Agric. Food Chem. 50: Undelnd, I., G. Hll, K. Wendin, I. Gngy, nd A. Rutgersson Preventing lipid oxidtion during recovery of functionl proteins from herring (Clupe hrengus) fillets y n cid soluiliztion process. J. Agric. Food Chem. 53: USDA (U.S. Food nd Drug Administrtion) Nutrient dtse for stndrd references - Relese 13. United Sttes Dept. of Agriculture, Wshington, DC. USDA (U.S. Food nd Drug Administrtion) USDA Bseline Projections. United Sttes Deprtment of Agriculture. 41

54 Accessed Sep USDA (U.S. Food nd Drug Administrtion) Sensory nd physicochemicl property reltionships in food tht define nd predict end-use qulity. Accessed June USDA/ERS Frm niml sttistics: met consumption. Fctory frming. Accessed Sep USDA (U.S. Food nd Drug Administrtion) Livestock, diry, nd poultry outlook. Economic Reserch Service Report LDP-M-183:1. USDA (U.S. Food nd Drug Administrtion) Chin poultry nd products. USDA Foreign Agriculturl Service GIAN Report CH9014:3. USDA (U.S. Food nd Drug Administrtion). 2009c. Brzil poultry nd products. USDA Foreign Agriculturl Service GIAN Report BR9602:3. USDA (U.S. Food nd Drug Administrtion). 2009d. EU-27 poultry nd products. USDA Foreign Agriculturl Service GIAN Report E49020:3. Vieir, E. R Fish nd shelfish. Pges in Elementry Food Science, 4th ed. Chpmn & Hll, New York, NY. Vodovotz, Y., M. Bik, E. Vittdini, nd P. Chinchoti Instrumentl techniques used in red stling nlysis in Bred Stling. P. Chinchoti, nd Y. Vodovotz, eds. CRC Press, Boc Rton, FL. Wng, S. F., nd D. M. Smith Poultry muscle proteins nd het-induced geltion. Poult. Sci. Rev. 5: World Poultry US: poultry consumption lowest in 7 yers. Accessed Sep

55 Ytes, L. D., nd M. L. Greser Quntittive-determintion of myosin nd ctin in rit skeletl-muscle. J. Mol. Biol. 168: Young, L. L Aqueous extrction of protein isolte from mechniclly deoned poultry met. J. Food Sci. 40: Zmul, E Getting hooked on surimi. FDA Consumer. Accessed Jnury

56 2 ALKALI AIDED PROTEIN EXTRACTION OF CHICKEN DARK MEAT: COMPOSITION AND STABILITY TO LIPID OXIDATION OF THE RECOVERED PROTEINS INTRODUCTION Currently, with the incresing demnd for further processed rest met products, chicken drk met hs een considered mjor underutilized commodity (USDA, 2004). The mjor concerns with chicken drk met which ffect consumers selection nd stisfction re color, high ft content nd poor shelf life stility (Froning, 1995; Fletcher, 1997). One of the lterntives to increse the utiliztion of chicken drk met is to extrct myofirillr proteins nd seprte them from ft nd pigments to enhnce their ppliction, which is required for the preprtion of further processed met products (Betti nd Fletcher, 2005). The use of lkline extrction followed y cid precipittion hs een investigted for protein recovery from vegetle sources (Fletcher nd Ahmed, 1977). Vrious reserchers hve exmined potentil extrction techniques for mechniclly seprted poultry met (MSPM), met offl, eef ones, nd fish met (Meinke et l., 1972; Young nd Lwrie, 1974; Jelen et l., 1979; Lwrence et l., 1982). Jpnese reserchers developed process for the recovery of proteins from fish resulted in Surimi, which is functionl protein ingredient. Surimi preprtion involves wshing the minced rw fish mteril in 5-10 C wter or slightly lkline solution, with the im of removing undesirle flvor components, pigments, lood nd ft, leding to concentrted functionl myofirillr proteins (Lnier, 1986; Kristinsson nd Ingdottir, 2006). An evolution of the conventionl surimi preprtion which is more suitle to extrct proteins from pelgic ftty fishes is the lkline soluiliztion technique developed y Hultin et l. (2000). In this new process, soluilized fish myofirillr proteins t ph ove 10.0 cn e seprted from ft nd pigments y high speed 1 This Chpter ws ccepted for puliction in the Journl of Poultry Science, 2010 (uthors: Vid Moyedi, Dileep Omn, Jcky Chn, Yn Xu nd Mirko Betti) 44

57 centrifugtion nd recovered y precipittion t the isoelectric point (Hultin nd Kelleher, 1999; Hultin et l., 2003; Kristinsson nd Ingdottir, 2006). The extrction mechnism increses the chrge on myofirillr proteins y ph-shifting processing technique. The recent over-production of leg qurters nd drk met hs resulted in drmticlly low mrket for these two rw mterils. Thus, the production of lightcolored, low-ft protein isolte from the drk met products, which cn e used for the preprtion of mny processed food products, hs received incresed ttention. It could e used, for exmple, s n lterntive to phosphtes for met enhncement (Vnn nd DeWitt, 2007) nd especilly s functionl food ingredient for further processed met products. The current study ws performed to develop n efficient technology for extrction of myofirillr proteins from chicken drk met. Hence, the ojective of the present investigtion ws to determine the effect of ph, in the rnge of 10.5 to 12.0, on the lkline soluiliztion of chicken drk met. Aspects studied were the effect of lkli ided process on protein nd lipid composition, lipid oxidtion nd color chrcteristics of the extrcted met. 2.2 MATERIALS AND METHODS Mterils Skinless frozen chicken thigh met ws otined from Lilydle (Edmonton, AB, Cnd). The chicken thigh met ws thwed (4 C) overnight. The thwed chicken thigh met ws then ground using met grinder (Wring Pro, MG100NC, Tiwn), vcuum pcked in 400 g plstic gs nd ws stored t -30 C prior to nlysis. The resulting drk minced chicken met (DMCM) ws used for this study Protein Soluility Curve The protein soluility curve ws prepred using method y Kim et l. (2003). 6.0 g drk minced rw met nd 300 ml refrigerted distilled wter were mixed in homogenizer (Fisher Scientific, Power Gen 1000 S1, Schwerte, Germny) t setting 3 for 1 minute. Aliquots of homogente (30 ml) were djusted to rnge of ph vlues (1.5 to 45

58 12.0) using 0.2 M nd 1 M HCl or NOH. The smple solutions were centrifuged t 25,900 g t 4 C for 20 minutes. The middle liquid lyer ws used for protein nlysis. Protein soluility ws mesured y the Biuret method (Gornll et l., 1949) nd expressed s mg/g of protein. Bovine serum lumin (Sigm-Aldrich, St Louis, MO, USA) ws used s the stndrd Protein Isoltion Protein isoltion ws performed s modifiction of the methods of Ling nd Hultin (2003), nd Betti nd Fletcher (2005). To prepre protein isolte, 400 g of frozen minced chicken thigh met ws prtilly thwed (4 C) overnight it ws then mixed with ice-distilled wter (1:2.5, w:v) using 900 Wtt Food Processor (Wolfgng Puck, WPMFP15, W.P. Applinces, Inc., Hollywood, FL, USA) for 15 min. The homogente ws djusted to ph 10.5 to 12.0 in 0.5 increments with ph meter (UB10, Denver Instrument, Denver, CO, USA) using 2N NOH over 5 min period. After ph djustment, the homogente ws llowed to stnd still (4 C for 30 min) nd centrifuged (Beckmn Coulter, Avnti J-E centrifuge, CA, USA) t 25,900 g for 20 min (4 C). Three lyers were formed fter centrifugtion: n upper ft lyer, middle queous lyer of solule proteins nd ottom sediment lyer. The middle protein superntnt lyer ws crefully removed nd the other two lyers were discrded. The resultnt protein superntnt ws djusted to ph 5.2 using 2N HCl over 5 min period. The precipitted proteins were then centrifuged t 25,900 g for 20 min (4 C). The sedimented proteins were mixed with 700 g of ice-distilled wter using the food processor for 7 min nd djusted to ph of 6.2, followed y centrifugtion t 25,900 g for 20 min (4 C). The protein isolte were then weighed nd protein yield ws determined using the formul elow. Moisture content of the resulting protein isolte ws djusted to 80% nd stored t -30 C. The resulting protein isolte ws clled extrcted met (EM). Protein yield (%) = Protein content of EM (g) x 100 Protein content of DMCM (g) Determintion of Totl Protein Content Totl protein content ws determined using the iuret procedure (Gornll et l., 1949; Torten nd Whitker, 1964). The iuret regent ws prepred s descried y 46

59 Lyne (1957). The prtilly dispersed met smple (1.0 g) in 20 ml of 0.5M NOH ws heted in oiling wter th for 10 min nd cooled in n ice-wter th. The solution ws filtered to remove the elstin prticles nd some of the ft. Anhydrous ether (15 ml) ws dded to 15 ml of the filtrte, which ws then centrifuged (J-6B/P Beckmn, Beckmn Instruments, Inc, CA, USA) t 2,278 g for 10 min fter shking. After centrifugtion, 1 ml of the lower queous lyer ws tken nd mixed with 4 ml of iuret regent. Opticl density ws mesured t 540 nm fter 30 min using UV/VIS spectrophotometer (V-530, Jsco Corportion, Tokyo, Jpn). Bovine serum lumin (HyClone, UT, USA) ws used for preprtion of the stndrd curve. Totl protein content ws mesured on the DMCM s well s the EM Extrctility of Recovered Proteins The extrctility of srcoplsmic proteins in the protein isolte ws mesured y vortexing 2 g of smple with 20 ml of 30 mm phosphte uffer (ph 7.4) to crete homogente solution. This solution ws llowed to stnd still overnight t 4 o C, nd ws then filtered using Whtmn No. 1 filter pper. The protein content of the filtrte ws estimted using the Biuret method (Gornll et l., 1949). For mesuring totl protein extrctility, 50 mm phosphte uffer contining 0.55 M potssium iodide (ph 7.4) ws dded to dissolve the proteins. Anlysis ws crried out in triplicte Determintion of Totl Ft Content Totl ft content ws determined using method descried y Folch et l. (1957) g of EM nd 5.0 g of DMCM were seprtely mixed with 2:1 chloroform-methnol (v/v) solution (120 ml) using vortex mixer for 10 min nd stood still for 30 min. The slurry ws filtered nd the filtrte ws trnsferred to seprtion funnel, followed y ddition of 40 ml of 0.88% NCl solution. The funnel ws then left for 30 min for phse seprtion. The ottom chloroform phse ws filtered on n nhydrous sodium sulphte ed (Fisher Scientific, NJ, USA). 60 ml of the cler chloroform phse ws tken out into pre-weighed flsk followed y rotry evportion (Rotvpor, RE 121, Buchi, Switzerlnd) for 15 min t 40 C. The flsk ws then put in 60 C oven for 30 min, followed y plcing in desicctor for 30 min efore weighing it gin to otin the totl 47

60 ft content. The residue ws dissolved in 10 ml of chloroform nd stored in the freezer (- 30 C) until required for nlysis Seprtion of the Min Lipid Clsses The min lipid clsses were seprted using method descried y Rmdn nd Mörsel (2003). A glss column (60 cm x 1.5 cm) ws pcked with ctivted silic gel ( mesh; Whtmn, NJ, USA) y pplying slurry of the silic gel in chloroform (1:5, w/v). Totl lipids were dissolved in 9.5 ml of chloroform nd pplied to the column, which ws then eluted sequentilly with 50 ml chloroform (for neutrl lipids), nd with 50 ml methnol (for polr lipids) therefter. The solvent from ech frction ws evported y rotry evportion nd the percentge of ech frction ws determined grvimetriclly. The residues were dissolved seprtely in 2 ml of chloroform nd stored in the freezer (-30 C) until required for nlysis Ftty Acid Anlysis Totl ftty cid profile nd composition of the neutrl nd polr lipid clsses for rw thigh met nd extrcted met were nlyzed y gs chromtogrphy (GC) on 4 replicte smples. Derivtiztion ws conducted using solution of hydrochloric cid in methnol (methnolic HCl, 3N; Supelco, PA, USA) nd ccording to mnufcture instructions provided y Supelco. Extrcted ft ws diluted with chloroform. 2mL of methnolic HCl were dded to 50µl of the extrcted ft chloroform mixture. In order to stimulte the rection, smples were put into 60 C wter th for 120 min. Ftty cid profile of the neutrl nd polr lipid clsses ws determined with GC Vrin (Vrin Wlnut Creek, Cliforni, USA) 3400 gs chromtogrph equipped with flme ioniztion detector nd SGE-BPX70 cpillry column (60m x 0.25 mm x 0.25 µm film thickness; SGE Anlyticl Science Pty Ltd, Victori, Austrli). Operting conditions for the GC for ftty cid composition of neutrl nd polr lipid clsses were s follows: initil temperture of 50 C ws held for 0.2 min; incresed t 20 C/min to 170 C, which ws mintined for 5 min. At rte of 10 C/min, temperture of 230 C ws reched, which ws mintined for 13 min. A Cool-on-Column injection method ws used, with n initil nd finl injector temperture of 60 C nd 230 C (30 min) 48

61 respectively, incresing t rte of 150 C/min. The temperture of the detector remined t 240 C nd the column hed pressure of the crrier gs (helium) ws 25 PSI. The ftty cid pek integrtion ws performed using the Glxie Chromtogrphy Dt System (Vrin). Ftty cids were quntified using methyl-heptdecnoic cid s n internl stndrd (Sigm-Aldrich Co., MO, USA) nd were identified y comprison of uthentic stndrds (GLC-463 NU-CHEK PREP, INC. Elysin, MN, USA). Sturted ftty cids (SFA) levels were clculted s 14:0 + 15:0 + 16:0 (plmitic cid) + 18:0 (streric cid) + 19:0 + 20:0 + 22:0. Monounsturted ftty cids (MUFA) levels were clculted s 14:1 + 16:1 ω-7 cis +17:1 + 18:1 ω-7 trns + 18:1 ω-7 +18:1 ω-9 (oleic cid) + 20:1. Polyunsturted ftty cids (PUFA) levels were clculted s 18:2 ω-6 (linoleic cid; LA) + 18:3 ω-3 (α-linolenic cid; LNA) + 18:3 ω :2 ω :3 ω :3 ω :4 ω-6 (rchidonic cid; AA) + 20:5 ω-3 (eicospentenoic cid; EPA) + 22:4 ω :5 ω-3 (docospentenoic cid; DPA) + 22:6 ω-3 (docoshexenoic cid; DHA). Totl ω-6 ftty cid levels were clculted s LA + 18:3 ω :2 ω :3 ω-6 + AA + 22:4 ω- 6. Totl ω-3 ftty cid levels were clculted s LNA + 20:3 ω-3 + EPA + DPA + DHA. Long chin ω-3 ftty cid (LC ω-3) levels were clculted s 20:3 ω-3 + EPA + DPA + DHA. Long chin ω-6 ftty cid (LC ω-6) levels were clculted s 20:2 ω :3 ω-6 + AA + 22:4 ω Susceptiility to Oxidtion Lipid susceptiility to oxidtion ws mesured y using the induced thiorituric cid rective sustnces (TBARs) test s modifiction of the method of Kornrust nd Mvis (1980) on rw thigh met s well s extrcted leg met. Four replicte mesurements were conducted. To prepre homogentes, 3 g of the met smple ws homogenized with 25 ml of 1.15% KCl solution for 45 s. A 200 µl liquot of the homogente ws incuted t 37 C in 80 mm Tris-mlete uffer (ph 7.4) with 2.5 mm scoric cid nd 50 µm ferrous sulphte in totl volume of 2 ml. At fixed intervls (0, 30, 60, 100 nd 150 min), liquots were removed for mesurement of TBARS. TBARs were expressed s nnomoles of mlondildehyde (MDA) per milligrm of met. 49

62 Color Mesurements The CIE (1978) system color profile of lightness (L*), redness (*) nd yellowness (*) ws mesured y HunterL colorimeter (LSXE/UNI, Virgini, USA) on EM smples immeditely fter extrction prior to frozen storge t -30 C. A white stndrd plte ws used to clirte the colorimeter. Three redings of L*, *, nd * vlues were tken from ech tch of the rw drk nd extrcted met smples, nd the vlues were verged. Whiteness, intensity of the red, sturtion nd HUE vlues were clculted ccording to the following formul ( 100 L) + Whiteness = Sturtion = + 2 HUE = rctn / Intensity of the red = * / * Hue ngle mesures the degree of deprture from the true red xis of the CIE color spce (Brewer et l., 2006). As hue ngle increses, visully perceived redness decreses (Little, 1975; Brewer et l., 2006). Chrom refers to the rightness/colorfulness of n oject (Firchild, 2004) Totl Heme Pigments Content Totl heme pigments were extrcted from 10.0 g of homogenized met smple within solution of 40 ml cetone, 1 ml HCl nd 2 ml wter ccording to Hornsey (1956). The solution ws filtered fter1-hour puse nd the sornce ws mesured using UV/VIS spectrophotometer (V-530, Jsco Corportion, Tokyo, Jpn) t 640 nm wvelength. The sornce vlue otined ws multiplied y the fctor vlue 17.18, nd the concentrtion of totl hemic pigments ws expressed in milligrms of myogloin per grm of met. 50

63 Sttisticl Anlysis Ech experiment nd ech ssy ws done t lest in triplicte. Reported results represent n verge of ech experimentl ssy. All dt were sujected to Anlysis of Vrince using the generl liner model procedure of SAS (SAS System, 2002). Differences etween lest squres mens were determined using HSD Tukey differences, nd were reported s significnt t the P < 0.05 level. Liner regression nlysis nd Krl Person s coefficient of correltion hs een determined to find the correltion etween vrious prmeters s function of ph tretments. 2.3 RESULTS AND DISCUSSION Protein Soluility The soluility of proteins from chicken thigh met hs een mesured in ph rnge of (Figure 2.1). The highest vlue for protein soluility ws determined t the extremes of ph in oth cidic nd lkline rnge. The incresed soluility t extreme ph vlues hs een relted to n incresed positive nd negtive chrge of the muscle proteins t low nd high ph, respectively, leding to electrosttic repulsion etween the proteins (Hmm, 1994). The protein soluility showed U-shped curve; however, unlike the typicl soluility curve for fish muscle protein homogentes, the soluility ws found to e mximum t ph 10.5 nd showed mrginl reduction up to ph The reson for this mrginl decrese in protein soluility my e due to the exposure of more hydrophoic groups t the higher ph vlues. ph-shift soluilizing processing is method which hs een rodly used for fish muscle; however in this study we hve shown tht this process cn lso e pplicle to underutilized chicken met sources for protein extrction. Investigtions y Kristinsson et l. (2005) on ctfish muscle indicted tht there is less protein denturtion in lkli ided process thn in cid ided process. As consequence, sed on the soluility curve, higher lkline ph, rnging from 10.5 to 12.0 ws considered for the present study. 51

64 2.3.2 Protein Recovery nd Protein Content Alkline ph tretments rnging from 10.5 to 12.0 were chosen for protein extrction ccording to the results otined from the protein soluility curve. The yield of the protein extrction ws significntly incresed s the ph of extrction incresed. The highest recovery of proteins which is equivlent to 94.2% ws otined t ph of 12.0 (Figure 2.2). This high level of protein yield could e explined y the denturtion of myofirillr proteins nd the exposure of hydrophoic groups (Omn et l., 2010). Denturtion of proteins t high ph vlues results in unfolding of proteins (Sikorski, 2007). As consequence, hydrophoic mino cids will e exposed to the surfce of the protein structure, which my led to more ggregtion nd precipittion of myofirillr proteins t the isoelectric point (Ingdottir, 2004; Gehring et l., 2009). The reson for the higher protein yields in this study compred to Kristinsson nd Ingdottir (2006), who recovered 68% of proteins from Tilpi fish muscle y lkli ided process, could e explined y the differences etween chicken nd fish proteins s strting mterils. As expected, protein yield ws negtively correlted with totl nd neutrl lipid content of EM (R = nd R = -0.81; P < ), respectively (Figures 2.3 nd 2.4). The protein content ws significntly incresed when ph of 12.0 ws reched compred with the one of 10.5 (P <.0001) (Tle 2.1). The highest level of protein content on wet sis, 19.4%, ws reched t ph of 12.0, however; it ws not significntly different s compred to the vlues of chicken drk met. It my indicte tht the mximum protein could e extrcted from chicken drk met using lkli-ided processing t ph of Extrctility of Recovered Proteins Extrctility of recovered proteins is of gret importnce for the mnufcture of processed muscle foods, including restructured mets. This is ecuse most of the functionl properties of muscle protein, such s emulsifiction nd geltion, re relted to this prmeter. Srcoplsmic protein extrctility from recovered proteins s function of vrious ph ws significntly decresed (P < ) compred to the extrctility of 52

65 srcoplsmic proteins in rw met (Figure 2.5). The reduction ws found to e most significnt t ph 11.5 nd ph This reduction my e explined y the instility of proteins due to ggregtion ehvior (Mohn et l., 2006) during the process of extrction. Totl protein extrctility showed similr trend. Approximtely 50% reduction ws found t ph tretments of 10.5 nd 11.0 compred to the initil rw mterils. The reduction ws even more evident for the proteins recovered t lkli t ph vlues of 11.5 nd 12.0 (Figure 2.5). The strong reduction of totl protein extrctility for smples extrcted t higher ph my e explined y unfolding of myofirillr proteins, leding to exposure of more hydrophoic groups to the exterior (Srm et l., 1999). However, the decrese in protein extrctility t higher ph vlues might e consequence of ltered protein conformtions induced y chemicl denturtion, which could not e fully restored fter ph redjustment. Kristinsson nd Hultin (2003) showed tht the conformtion of myosin hed could not revert to its ntive conformtion during lkli tretment (ph 11.0) followed y ph (7.5) redjustment Lipid Profile Totl Lipid Content The presence of high lipid content hs importnt implictions in the storge, processing, stility, nd nutritionl vlues of muscle food. It is lso reson for the rpid sensory qulity losses oserved. In order to increse the utiliztion of extrcted proteins, totl ft content from drk met must e reduced. Lipid content of the thigh met ws 6.3% (on wet sis). Ft content of the extrcted met y lkli tretments showed n verge of 50% reduction compred to chicken drk met with no significnt difference mong the ph tretments (Tle 2.1). This reduction is possile ecuse during the lkli soluiliztion process, when high ph vlues ( ) re used, n increse in negtive chrge of muscle proteins occur, leding to electrosttic repulsion etween the proteins (Hmm, 1994). As result, proteins soluilize in lkline solution. When this slurry is centrifuged, lipids, due to their low density, re seprted from the protein phse (Hultin nd Kelleher, 2000). 53

66 However, the low mount (~50%) of ft reduction found in the current investigtion compred to other studies (65-70%) conducted in fish (Hultin nd Kelleher, 2000; Nolsøe nd Undelnd, 2009) could e explined y the lower rtio of met to wter used for lkli extrction. In this study, the rtio of met to wter ws 1:2.5 (w: v) while other investigtions on mechniclly deoned turkey used rtio of 1:6 (w: v) (Ling nd Hultin, 2003). In ddition, reserch on fish lkli extrction indicted tht rtio of 1:5 is used in order to increse ft removl, especilly polr lipids (Nolsøe nd Undelnd, 2009). The low mount of wter used in this study ws chosen ecuse of the growing concern of wter usge during food processing Neutrl nd Polr Lipids Content In generl, tricylglycerol (neutrl lipid) content of the drk muscles, minly rich in sturted ftty cids, is relted to the sustinle energy source required y the nimls, while the highly unsturted nture of the polr lipids is necessry for the metolic functionl requirements of the memrne (Betti et l., 2009). However, ftty cids composition is not the only difference etween the neutrl oils nd the polr memrnes lipids. Becuse the polr lipids (minly phospholipids) re in the cell memrnes, which exist primrily s i-lyer, they hve very lrge surfce re exposed to the queous phse of the cell compred to tricylglycerol (Hultin, 1995). As consequence, even though the neutrl ft predomintes in drk met, polr lipids would hve ten times more exposure to pro-oxidnts thn the tricylglycerols t the surfce of the oil droplets. In ddition to their greter surfce re, memrne lipids re found in ssocition with components tht cn ccelerte their oxidtion, such s rective oxygen species (ROS) (Hultin nd Kelleher, 2000). For ll these resons, polr lipids must e strongly reduced during the extrction process. A significnt decrese (P < ) in neutrl lipids in extrcted met ws oserved compred to chicken drk met. The content of neutrl lipids in chicken drk met (% on wet sis) ws 5.4 % (Tle 2.1). On verge, neutrl lipid removl from chicken drk met using the lkli ided process ws 61.5%. In this study, in contrst to the neutrl lipids, polr lipids were not ffected y the lkli extrction (ph vlues ). The unsuccessful removl of polr lipids my result from the lower mount of 54

67 wter used for lkli extrction (2.5 vs. 5 (w: v)) (Nolsøe nd Undelnd, 2009). As previously discussed, polr lipids re difficult to remove ecuse they re strongly ound to memrne proteins nd hence, their extrction ws difficult (Nylnder, 2004). These results lso concur with Dwson et l. (1990) who did not successfully remove polr lipids from MSPM y using slightly lkline wshing tretments. By contrst, recent study conducted y Hrynets et l. (2009) using cid extrction in the presence of citric cid showed considerle reduction in the level of polr lipids of MSPM; it hs een suggested tht citric cid might ply role s inding gent to the sic mino cid residues of cytoskeletl proteins competing with the cidic phospholipids of memrnes (Ling nd Hultin, 2005) In light of this, low ph of extrction nd ddition of citric cid my e exploited to improved removl of polr lipids from chicken drk met Ftty Acid Profile The ftty cid profile of neutrl nd polr lipids for chicken drk met nd extrcted met re presented in Tles 2.2 nd Neutrl Lipids Frction The predominnt SFA ws plmitic cid (C16:0) nd mong MUFA, oleic cid (C18:1 ω-9) ws the most undnt ftty cid (Tle 2.2). According to Wood et l. (2008), the content of oleic cid is higher in neutrl lipids thn in polr lipids frction. The levels of SFA, MUFA nd PUFA significntly decresed y 68%, 66% nd 60%, respectively in the neutrl lipids frction of chicken drk met due to lkli ph tretments (P < ). The lowest mounts of ftty cids were found t ph of 11.5 (448, 978 nd 339 mg per 100 g of chicken drk met for SFA, MUFA nd PUFA, respectively) (Tle 2.3). The reduction in SFA fter ph tretment ws minly due to 68% decrese in plmitic cid. The reduction of MUFA ws explined y the decrese of oleic cid. In fct, there ws 72% reduction in this ftty cid compred to chicken drk met t ph of The decrese of LA ws responsile for the significnt reduction in PUFA levels in lkli ph tretments compred to chicken drk met (P < ). The highest removl (72%) of LA ws found t ph of In neutrl lipids, LNA ws the only ω-3 ftty cid found in chicken drk met. The highest reduction of LNA in lkli tretments ws lso 72% t ph of

68 Totl ω-6 ftty cids were significntly reduced y lkli extrction, with the mximum reduction t ph of 11.5 (297 mg/100 g of chicken drk met). The ω-6: ω-3 rtio for chicken drk met ws 7.20 nd due to the similr reduction in totl ω-3 nd ω-6 ftty cids in the lkli tretments, the rtio of ω -6: ω -3 did not chnge significntly compred to chicken drk met (Tle 2.2). However, there ws significnt improvement of the rtio (8.26) when ph of 12.0 ws used compred to the one t 10.5 (Tle 2.2) Polr Lipids Frction In chicken drk met, plmitic cid, steric cid, oleic cid nd LA were the predominnt ftty cids in polr lipids (87.8, 103, 155 nd 122 mg per 100 g of met for the ftty cids respectively) (Tle 2.3). SFA levels did not decrese due to the lkli tretments. There ws significnt increse in SFA of 47% t ph of 11.5 compred to chicken drk met (198 vs. 292 mg SFA/100 g of met; P = ). The MUFA nd PUFA levels in chicken drk met did not significntly chnge due to the lkli tretments (P = nd P = for MUFA nd PUFA respectively). Totl ω -3 nd LC ω -3 ftty cids tended to increse y lkli tretments while the increse ws only significnt t ph of 10.5 (P = nd P = for Totl ω - 3 nd LC ω -3 ftty cids respectively). Long chin polyunsturted ftty cids (LC PUFAs) including 22:4 ω -6, 20:3 ω -3, EPA, DPA nd DHA were not detected in the neutrl lipids frction, nd they were present only in polr lipids. These results re in greement with Betti et l. (2009), who studied the distriution of ftty cids of chicken thigh (drk) met in tricylglycerols nd phospholipids, nd found tht LC PUFAs were sent (or present in very low mount) in tricylglycerols compred to their level in phospholipids. LC ω-6 nd totl ω-6 ftty cids on the other hnd, were not significntly ffected y the tretments (P = nd P = for LC ω -6 nd totl ω-6 ftty cids respectively). Similrly, no significnt chnge in ω-6: ω-3 rtio ws oserved due to lkli extrction in reltion to chicken drk met (Tle 2.3). The rtio for chicken drk 56

69 met ws 7.47 nd it vried from 5.54 to 8.23 in the different ph tretments, reching its mximum t ph of 12.0 (8.23). As discussed previously, the unsuccessful removl of polr lipids did not decrese the concentrtion of LC PUFAs, which in some cses, (ph 10.5) they even incresed. If this is potentilly desirle from nutritionl point of view it does produce serious technologicl prolems due to lipid oxidtion during frozen storge (see elow) Susceptiility to Oxidtion Interctions etween incution time nd lkli ph tretments used for extrction were found in the induced TBARs mesured to test the oxidtive stility of chicken drk met nd extrcted met (Figure 2.6). The MDA formtion incresed s the time of incution incresed, s expected. The highest MDA vlue ws found fter n incution time of 150 min in chicken drk met, s well s, in extrcted met. There ws significnt increse (P < 0.05) in MDA formtion in extrcted met compred to chicken drk met, indicting tht extrcted met ws more susceptile to oxidtion. This denotes tht lthough totl ft of chicken drk met decresed y pproximtely 50% due to lkli extrction, polr lipids were not removed. These polr lipids contin more LC PUFAs, nd hence extrcted met is more susceptile to oxidtion thn chicken drk met. It confirms tht LC PUFAs deposited in polr lipids re responsile for lipid oxidtion (Erickson, 2002). This is further evidenced y the ftty cid dt. In fct, the unsuccessful removl nd concentrtion of LC PUFAs, including EPA, DPA, DHA, C20:3 ω-3 nd C22:4 ω-6 in the polr frction, which were not present in the neutrl lipids, my e the reson for the incresed lipid oxidtion susceptiility in extrcted met compred to chicken drk met Color Mesurements nd Totl Heme Pigments Color is n importnt ttriute which influences consumers selection of nd stisfction with met products (Froning, 1995; Fletcher, 1997). The color mesurements (L*, *, *, */*, sturtion, HUE nd whiteness) nd totl heme pigments for chicken drk nd extrcted met re descried in Tle 2.4. The individul color mesurements of * nd *, were significntly lower in extrcted met compred to chicken drk met. 57

70 The ppliction of lkli ided extrction to chicken drk met lightened the color of the drk met. This could e explined y the fct tht during the lkli ided process, the ph is djusted to the isoelectric point of myofirillr proteins (ph: 5.2), which led to precipittion. Thus, y further centrifugtion, srcoplsmic proteins (totl heme pigments included) which re wter-solule components (Scopes, 1970) will e esily seprted from the myofirillr proteins. The color vlues of redness (*) nd yellowness (*) in extrcted met significntly decresed (P < nd P = ) y 83% nd 11%, respectively compred to chicken drk met. The decrese in redness vlue could e explined y the fct tht the rw met presents red color ecuse of the myogloin (Terr et l., 2009), nd s result, y removing myogloin, the redness vlue decresed. The reduction in yellowness for extrcted met could e ecuse of lipid removl. According to Irie (2001), crotene concentrtion is the min fctor ffecting yellowness of the ft in met ut crotene levels were not mesured here. The vlue of lightness (L*) significntly incresed with lkli extrction compred to chicken drk met (P < ). Moreover, studies on turkey muscle color indicted tht totl pigments concentrtion is one of the fctors influencing met L* vlues (Boulinne nd King, 1995, 1998; Gsperlin et l., 2000). Thus, the reson for increse in lightness vlues in extrcted met could e ttriuted to the removl of totl heme pigments from chicken drk met y lkli ided process. Whiteness vlues of the extrcted met significntly incresed (P < ) compred to chicken drk met, minly ecuse of the considerle increse in L* vlue which is the min fctor ffecting whiteness (Tle 2.4). The intensity of the red (*/*) significntly decresed compred to chicken drk met, due to the reduction of redness. The sturtion vlue of extrcted met significntly decresed compred to chicken drk met (P < ) indicting tht extrcted met hs less color intensity compred to chicken drk met. This could e explined y the removl of totl heme pigments y lkli extrction which my ffect color intensity (Irie, 2001). The HUE vlue considerly incresed in extrcted met (P < ) compred to chicken drk met. This indictes tht the visully perceived redness ws reduced y the extrction process. 58

71 The vlues of *, * nd L* for chicken rest ws reported s 0.99, 3.55 nd 62.11, respectively (Perez-Alvrez nd Fernndez-Lopez, 2009) while the vlues otined in our study for extrcted met were 1.54, nd respectively. The comprison etween these vlues indictes tht L* nd * vlues for extrcted met re comprle with those in rest met. Yellowness vlue (*) in extrcted met is higher compred to chicken rest. The reson for this higher yellowness could e explined y the unsuccessful removl of polr lipids which contin higher mounts of LC PUFAs nd oxidtion of the remining pigments to some extent in the EM (Irie, 2001). Alkline soluiliztion considerly decresed (P < ) totl heme pigment content compred to chicken drk met. The vlues reched the verge of 0.60 mg/g which showed 53% reduction compred to chicken drk met (Tle 2.4). According to Pikul et l. (1982) nd Niewirowicz et l. (1986) the vlues otined in this study re similr to those found in chicken rest met. Despite ll the color chrcteristics tht were improved y lkli tretments, Omn et l. (2010) found different results when color ws mesured on EM smples stored for 30 d t -30 C; L* nd whiteness were lower in EM smples thn minced chicken drk met. The inefficient removl of polr lipids, which incresed the unsturtion level in EM, nd the pronounced oxidtion of the remining pigments proly resulted in slightly drker met s compred to the strting mteril lthough the index of red decresed. These results suggest tht immeditely fter extrction, EM smples re distinguished y n overll right white-yellow color nd fter 30 d of frozen storge, the formtion of oxidized pigments, minly met-myogloin isolted proteins resulted in more drk-yellow color. 2.4 CONCLUSIONS This first experiment demonstrted tht it is possile to recover protein yields of more thn 80% in the ph rnge of However, the isolted proteins were susceptile to lipid oxidtion s reveled y TBARs vlues, even though the totl ft content decresed to pproximtely 50%. The recovery process filed to remove polr 59

72 lipid nd in some cses (ph 10.5), they even incresed. However, neutrl lipids were removed y 61.51%. Alkli ided extrction helped to lighten the color of the drk met nd reduction of heme pigments y 53%. In spite of the susceptiility to lipid oxidtion of the recovered proteins, our erlier studies (Omn et l., 2010) reveled tht the proteins in the extrcted met otined fter tretment t ph 10.5 nd 11.0 could regin the originl conformtion fter ph redjustment to 6.2. Hence, it cn e concluded tht the met extrction t these ph vlues cn e utilized for product preprtions with desired functionl properties. The oxidtion issue my e overcome y modifying the process to remove more polr lipids (i.e. ddition of citric cid) or y dding stilizing gent such s vitmin E. 60

73 2.5 TABLES Tle 2.1. Protein (%) nd lipid (%) composition of chicken drk nd extrcted met (wet sis) Extrction ph Prmeters Chicken Drk Met SEM Pr > F Protein content c 17.1 c 18.2 c <.0001 Totl ft content <.0001 Neutrl lipids <.0001 Polr lipids c Lest squres mens in row corresponding to certin fctor with different letters re significntly different (P < 0.05). The vlues presented re mens of 4 replicte determintions. 61

74 Tle 2.2. Level (mg ftty cid / 100 g of met) of ftty cids in the neutrl lipids of chicken drk nd extrcted met Ftty cids 14: : : : :1 ω : :1 ω :1 ω :2 ω :3 ω :3 ω : :2 ω :3 ω :4 ω SFA* MUFA* PUFA* Totl ω-3* Totl ω-6* ω -6:ω Extrction ph Chicken Drk Met SEM Pr > F ND c c c c c c c c c c c c c ND c c c c c c c c c c c c c c c c c c c c c c c c c c c c <.0001 < <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 < < <.0001 <.0001 <.0001 <.0001 < ND = not detected. SEM= stndrd error of the men. -c Vlues within rw with no common superscript re significntly different (P < 0.05). The vlues presented re mens of 4 replicte determintions. * SFA levels were clculted s 14:0 + 15:0 + 16:0 + 18:0. MUFA levels were clculted s 14:1 + 16:1 ω :1ω-7 +18:1 ω :1. PUFA levels were clculted s 18:2 ω :3 ω :3 ω :2 ω :3 ω :4 ω-6. Totl ω-6 ws clculted s 18:2 ω :3 ω :2 ω :4 ω-6. Totl ω-3 ws clculted s 18:3 ω-3. 62

75 Tle 2.3. Level (mg ftty cid / 100 g of met) of ftty cids in the polr lipids of chicken drk nd extrcted met Ftty cids Chicken Drk Met 14: : Extrction ph SEM Pr > F :1 ω : :1 ω :1 ω :2 ω :3 ω : : :2 ω :3 ω ND :3 ω :4 ω :5 ω : ND :4 ω :5 ω :6 ω SFA* MUFA* PUFA* LC ω-3* LC ω-6* Totl ω-3* Totl ω-6* ω -6:ω ND = not detected. SEM= stndrd error of the men. - Vlues within rw with no common superscript re significntly different (P < 0.05). The vlues presented re mens of 4 replicte determintions. * SFA levels were clculted s 14:0 + 16:0 + 18:0 + 20:0 + 22:0. MUFA levels were clculted s 16:1 ω :1ω-7 +18:1 ω :1. PUFA levels were clculted s 18:2 ω :3 ω :2 ω-6 +20:3 ω :3 ω :4 ω :5 ω :4 ω :5 ω :6 ω-3. LC ω-3 levels were clculted s 20:3 ω :5 ω :5 ω :6 ω-3. LC ω-6 levels were clculted s 20:2 ω :3 ω :4 ω :4 ω-6. Totl ω-3 ws clculted s 18:3 ω :3 ω :5 ω :5 ω :6 ω-3. Totl ω-6 ws clculted s 18:2 ω :2 ω :3 ω :4 ω :4 ω-6. 63

76 Tle 2.4. Color mesurements nd totl heme pigments of chicken drk nd extrcted met Prmeters Chicken Drk Met Extrction ph SEM Pr > F * <.0001 * L* <.0001 */ * <.0001 Sturtion <.0001 HUE <.0001 Whiteness <.0001 Totl heme pigments (mg/g) < Lest squres mens in row corresponding to certin fctor with different letters re significntly different (P < 0.05). The vlues presented re mens of 4 replicte determintions. 64

77 2.6 FIGURES Soluility (mg/g) Tretment ph Figure 2.1. Soluility profile of chicken drk met proteins s function of ph. Protein homogentes were djusted to ph rnge of 1.5 to 12.0 using HCl or NOH. The vlues presented re mens of 4 replicte determintions. 65

78 Figure 2.2. Effect of extrction ph on protein yield of extrcted chicken drk met. Dissimilr letters in the grph represent significnt (P < 0.05) difference. The vlues presented re mens of 4 replicte determintions. 66