Incorporation of Lutein into Wholegrain Bread as a Functional Ingredient and Antioxidant

Transcription

1 Incorportion of Lutein into Wholegrin Bred s Functionl Ingredient nd Antioxidnt By Hndi Alhwswi A Thesis presented to The University of Guelph In prtil fulfillment of requirements for the degree of Mster of Science in Food Science Guelph, Ontrio, Cnd Hndi Alhwswi, Mrch 2015

2 ABSTRACT INCORPORATION OF LUTEIN INTO WHOLEGRAIN BREAD AS A FUNCTIONAL INGREDIENT AND ANTIOXIDANT Hndi Alhwswi University of Guelph, 2015 Co-Advisor: Dr. Elsyed Abdel-Al Co-Advisor: Dr. Mssimo Mrcone The lutein crotenoid plys significnt roles in humn helth but its consumption is low worldwide. The current study ws imed to investigte the effect of using different bking formuls nd lutein forms on lutein distribution nd stbility in wholegrin bred to improve its lutein content nd ntioxidnt properties. One bound wholegrin breds were mde using three lutein forms (lutein powder, lutein in oil emulsion, nd lutein in ethnol suspension) with two bking formuls (bsic nd enriched). Lutein nd other crotenoids were mesured in bred lof, crust, top crumb nd center crumb. Lutein-enriched breds hd significntly higher lutein compred with the non-enriched respective ones. The lutein powder with bsic formul ws more effective in preserving lutein during bking process. The lutein content remined firly stble during bred storge t room temperture up to 7 dys. Enrichment of wholegrin bred with lutein resulted in significnt increses in ntioxidnt properties s mesured by three ssys DPPH, ABTS nd ORAC, prticulrly for breds mde from the enriched formul. The study provides insights into the production of wholegrin bred enriched with lutein to boost lutein consumption nd its nticipted positive helth effects. More reserch is needed to investigte lutein biovilbility nd helth benefits of the developed products.

3 DEDICATION I would like to dedicte this thesis to my wonderful fmily; my beloved prents- my mother Emn, my fther Abdulhfidh, my uncle Amir, my sisters nd my brothers for their unconditionl, selfless love, support nd understnding. iii

4 ACKNOWLEDGEMENTS First nd foremost I would like to thnk Allh the Grcious, the Merciful. I would like to express my sincere grtitude to my dvisors Dr. Elsyed Abdel-Al nd Dr. Sn Rgee for the continuous support of my MSc study nd reserch, for their guidnce, ptience, motivtion, nd immense knowledge. They hve helped me from the fundmentl steps to complete my thesis. Their guidnce helped me during the period of my reserch nd writing. Thnk you for vluble effort supporting nd helping me to improve my thesis writing. I could not hve imgined hving better dvisors for my MSc study. They were lwys there when I need their help during my study. I would lso like to thnk Dr. Mssimo Mrcone for his dvisory role. I would lso like to extend my sincerest thnks nd pprecition to Ms. Iwon Rblski t Guelph Food Reserch Centre, Agriculture nd Agri-Food Cnd, Guelph. I relly pprecite her ssistnce during my lb work. I would like to thnk everyone tht I hve shred the lb with, for ll their friendship, enthusism nd good opportunity to know them. Lstly nd most importntly, I would like to thnk my fmily- my prents, my sisters Abeer, Aish, Sf, Do, Ftimh nd Mnr, my brothers Abdullh nd Ryn, my uncle Amir nd my untie Nejood nd the rest of my fmily for their, prying, ptience, encourgement nd love during this process. A big thnks lso for my best friend Hjr nd Sehm for being beside me when I need their help nd for their supporting during my life in Cnd. I would like lso to thnk King Abdull for giving us s Sudi students this chnce to study brod. iv

5 TABLE OF CONTENT Dediction. iii Acknowledgements...iv Tble of Contents.. v List of Tbles.. viii List of Figures... ix 1. CHAPTER 1: Introduction CHAPTER 2: Literture Review Wholegrin nd Wholegrin Foods Bioctive Compounds in Wholegrin Crotenoids Structure Occurrence of Lutein in Cerel Grins Functionlity Role of Lutein in Humn Helth Helth of Eye Helth of Skin Lutein nd Cncer Effects of Processing nd Storge on Lutein Milling Bking Steming Extrusion Storge..19 v

6 2.6. Effect of Storge nd Processing on Antioxidnt Properties High-Lutein Functionl Foods CHAPTER 3: Effect of Lutein Enrichment on its Stbility nd Distribution in Wholegrin Bred Abstrct Introduction Mteril nd Methods Mterils Methods Preprtion of Lutein Formuls Preprtion of Wholegrin Bred Qulity of Breds nd Bred Preprtion for Anlysis Anlysis of Totl Crotenoid Content nd Individul Crotenoids Sttisticl Anlysis Results nd Discussion Bred Qulity Effect of Bking on Lutein nd Other Crotenoids Effect of Bred Storge on Lutein nd Other Crotenoids Conclusion CHAPTER 4: Antioxidnt Prosperities of Wholegrin Bred Enriched with Lutein Abstrct Introduction Mteril nd Methods Mterils Methods 50 vi

7 Preprtion of Lutein Formultions Preprtion of Wholegrin Bred Bred Preprtion for Anlysis DPPH Rdicl Scvenging Cpcity ABTS Ction Rdicl Scvenging Cpcity Assy ORAC Assy Sttisticl Anlysis Results nd Discussion Antioxidnt Properties Mesured by DPPH Assy Antioxidnt Properties Mesured by ABTS Assy Antioxidnt Properties Mesured by ORAC Assy Conclusion CHAPTER 5: Conclusions nd Future Work References Cited vii

8 LIST OF TABLES Tble 3.1: Lof volume, weight nd specific volume of non-enriched nd lutein-enriched wholegrin breds bked from bsic nd enriched formuls...38 Tble 3.2: Totl crotenoid content of non-enriched nd lutein-enriched enriched wholegrin bred bked from bsic nd enriched formuls Tble 3.3: Concentrtions of ll-trns-lutein nd zexnthin of non-enriched nd luteinenriched wholegrin breds bked from bsic nd enriched formuls Tble 4.1: The inflection points of wholegrin bred smples using ABTS nd DPPH ssys...59 viii

9 LIST OF FIGURES Figure 2.1: Structure of ll-trns-lutein, zexnthin, ß-cryptoxnthin, ß-crotene, nd 13-cislutein found in whet Figure 3.1: Effect of bking on totl crotenoid content in crust (A), top crumb (B) nd center crumb (C) of breds mde from bsic nd enriched formuls Figure 3.2: Effect of bking on lutein content in crust (A), top crumb (B) nd center crumb (C) of breds mde from bsic nd enriched formuls Figure 3.3: High-performnce liquid chromtogrm of crotenoids extrcted from nonenriched bred (control) (A), enriched bred with lutein (bsic formul) (B) nd enriched bred with lutein (enriched formul) (C).43 Figure 3.4: Amount per serving (mg/30g) of totl crotenoids (A), lutein (B) nd zexnthin (C) Figure 3.5: Effect of storge t mbient temperture on totl crotenoids in crust (A), top crumb (B) nd center crumb (C) of breds mde from bsic nd enriched formuls..45 Figure 3.6: Effect of storge t mbient temperture on lutein in crust (A), top crumb (B) nd center crumb (C) of breds mde from bsic nd enriched formuls Figure 4.1: Scvenge cpcity of DPPH rdicl crust (A), top crumb (B) nd center crumb (C) of breds mde from bsic nd enriched formuls by using DPPH ssy.60 Figure 4.2: Scvenge cpcity of ABTS rdicl of crust (A), top crumb (B) nd center crumb (C) of breds mde from bsic nd enriched formuls by using ABTS ssy.61 Figure 4.3: Scvenge cpcity using ORAC of crust (A), top crumb (B) nd center crumb (C) of breds mde from bsic nd enriched formuls by using ORAC ssy..62 Figure 4.4: The kinetics of non-enriched nd enriched wholegrin bred crust (A), top crumb (B) nd center crumb (C) in both bsic nd enriched formul by using DPPH ssy.63 Figure 4.5: The kinetics of non-enriched nd enriched wholegrin bred crust (A), top ix

10 crumb (B) nd center crumb (C) in both bsic nd enriched formul by using ABTS ssy.64 x

11 CHAPTER 1: INTRODUCTION Lutein is yellow pigment from the crotenoid fmily found in fruits, drk green vegetbles nd grins. It plys significnt roles in humn helth prticulrly the helth of eyes nd skin nd is linked with reduced risk of ge-relted mculr degenertion (AMD), ctrcts, cncer nd crdiovsculr disese. Lutein long with zexnthin constitute the min pigments found in the yellow spot of the humn retin providing severl protective functions such s protection of the mcul from dmge by blue light nd scvenging hrmful rective oxygen species. They lso reduce or inhibit the oxidtion of vulnerble molecules in food products or humn body. Since crotenoids cn t be produced in humns, they must be provided in the diet. Wholegrin bred is considered helthy food becuse it is good source of mny bioctive components such s dietry fiber, ntioxidnts, polyphenols, crotenoids nd tocols. Lutein is the min crotenoid found in whet but its concentrtion is not enough to meet the suggested physiologicl dose (e.g. 5-6 mg per dy). Severl pproches cn be used to boost lutein content in whet such s bio-fortifiction (e.g. developing high-lutein whet) or fortifiction with food-grde lutein products. Fortifiction of whet with lutein supplement ws found to be prcticl nd economic wy to boost lutein content in whet products prticulrly for high-ft bked products such s cookie nd muffin (Red et l. 2015, Abdel- Al et l. 2010). However, incorportion of lutein in low-ft food system such s bred is big chllenge. Lutein is lipophilic compound nd is difficult to uniformly distribute in n queous food system. Thus specil cre should be tken when incorporting lutein into food system tht is low in its ft content. Lutein stbility in wholegrin bkery products is significnt considertion. Lutein is 1

12 sensitive to oxidiztion nd isomeriztion due to het, storge, temperture nd bking conditions which could ffect its content nd ntioxidnt properties. A high ft content in bked products cuses greter loss of lutein becuse crotenoids re soluble in ft nd cn be exposed to isomeriztion nd oxidtion during bking process. In low-ft bkery product lutein is less prone to oxidiztion nd isomeriztion due to its lipophilic nture. On the other hnd, incorportion of lutein in n queous or low-ft food system could pose technologicl chllenge for the sme reson. This could result in uneven distribution of lutein in bred products (Red et l. 2015). To this end, the bred formul needs to be modified when fortified with lutein supplement to improve lutein content nd its distribution in the end product. There hs been interest in bkery products due to their populrity nd widespred consumption. To this end, previous work hs been done showing tht bkery products (bred, muffin nd cookie) cn be enriched with lutein to produce high-lutein foods (Abdel-Al et l. 2010). The bked products were ssessed for stbility of lutein nd in terms of ntioxidnt properties (Abdel-Al nd Rblski 2013). More reserch is needed to improve uniformity nd stbility of lutein in bked products especilly the low-ft ones. The overll gol of the current study ws to improve incorportion nd stbility of lutein in wholegrin bred using different forms of lutein. It ws hypothesized tht the form of lutein (e.g. powder versus liquid) nd the method of ddition of lutein (e.g. dry mix, queous ethnol suspension nd oil emulsion) could ffect incorportion nd stbility of lutein in wholegrin bred. Additionlly, bking ingredients (e.g. enriched formul versus len formul) could lso ffect incorportion nd stbility of lutein in wholegrin bred. Therefore, the following objectives were investigted: 1) to investigte impct of lutein enrichment in vrious forms on lutein distribution in wholegrin bred; 2) to study effect of lutein incorportion in vrious forms on compositionl nd structurl chnges of lutein; 3) to investigte impct of lutein 2

13 incorportion in vrious forms on ntioxidnt properties of wholegrin bred; 4) to determine differences between bsic (len) nd enriched bking formul on incorportion of lutein; 5) to investigte stbility of lutein in wholegrin bred during storge. Enhncing stbility of lutein in wholegrin bred is of interest to reserchers nd food industry becuse bkery products re the most pproprite vehicles to trnsfer the recommended dietry intke of essentil nutrients. Fortified wholegrin bred with lutein would hold promise for the development of high-lutein functionl foods. The current study is directed t chieving better understnding of the fctors influencing lutein stbility nd distribution in bred s they re linked with helth effects. The outcome of this reserch would help boost the dily intke of lutein nd increse consumption of wholegrin products through the development of wholegrin bred enriched with lutein supplement. 3

14 CHAPTER 2: LITERATURE REVIEW 2.1. Wholegrin nd wholegrin foods The Americn Assocition of Cerel Chemists Interntionl (AACCI) defines wholegrin s wholegrins shll consist of the intct, ground, crcked, or flked cryopsis (kernel or seed), whose principl ntomicl components the strchy endosperm, germ, nd brn-re present in the sme reltive proportion s they exist in the intct cryopsis (AACCI 2010). The grin or kernel contins three min prts: endosperm (80-85% of the grin), germ (2-3%) nd outer lyers or brn (13-17%). The endosperm is composed of cells hving strch grnules embedded in protein mtrix. The germ is rich in oil nd lipid-soluble vitmins, nd the brn contins high concentrtions of minerls, cellulose nd hemicelluloses. In ddition, the outer lyers re rich in bioctive components such s phenolic compounds, nthocynins, β-glucn nd dietry fiber (Rgee et l. 2013; Abdel-Al et l. 2006). Phenolic cids nd flvonoids re the most common phenolic compounds in wholegrin (Abdel-Al nd Rblski 2013; Abdel-Al et l. 2012; Liu 2007). Ferulic cid is the dominnt phenolic cid found profusely in whet brn (Abdel-Al t l. 2001; 2011). Crotenoids re very wide spred pigments in plnts, nd re found in whet prticulrly einkorn, durum nd Kumt t reltively high levels (Abdel-Al et l. 2002; 2007). They re present t exceptionl high concentrtion in corn (Abdel-Al et l. 2007). Lutein is the min crotenoid found in whet, followed by zexnthin nd β-cryptoxnthin (Abdel-Al et l. 2007). AACCI defined wholegrin food s A whole grin food must contin 8 grms or more of wholegrin per 30 grms of product (AACCI 2013). Wholegrin foods such s wholegrin bred nd pst re unique source of dietry fiber (Liu 2003). They re 4

15 lso rich in vitmins E nd B, nd minerls such s selenium, copper, zinc, proteins, iron nd mgnesium. They re lso contining phytochemicls such s ntioxidnts tht ply significnt roles in humn helth nd disese prevention. They deliver severl nutrients to protect humn body from crdiovsculr disese (CAD), type II dibetes, cncer nd other chronic diseses (Slvin et l. 2001). Wholegrin whet products contin diverse rry of bioctive compounds such s dietry fiber, β-glucn, tocopherols, tocotrienols, phenolic cids, nthocynins, crotenoids nd phytosterols. Mny of these compounds hve demonstrted humn helth benefits Bioctive compounds in wholegrin Cerel grins re rich sources of mny helth-enhncing nd/or disese-preventing components known s bioctive compounds. These components mke wholegrin products helthier thn their corresponding refined ones. Mny of the bioctive compounds re phytochemicls produced by plnts primrily for protection ginst predtors nd diseses. Phytochemicls hve lso been found to protect humns ginst certin chronic diseses. In generl, phytochemicls re nturl nd non-nutritive bioctive compounds produced by plnts tht ct s protective gents ginst externl stress nd pthogenic ttck (Chew et l. 2009). They re secondry metbolites tht re crucil for plnt defense which enble plnts to overcome temporry or continuous threts integrl to their environment. Phytochemicls could exhibit severl bio-ctivities such s ntimutgenic, nticrcinogenic, ntioxidnt, ntimicrobil, nd nti-inflmmtory properties (Okrter nd Liu 2010). The type nd concentrtion of phytochemicls vry mong grins, nd grin species nd genotypes (Adom et l. 2003). For exmple, totl phenolic content significntly vried between whet species nd cultivrs rnging from 881 µg/g to 2382 µg/g (Abdel-Al nd Rblski 2008). Ferulic cid concentrtion ( µg/g) rnged brodly in the whet species due to their environmentl nd genetic diversity used in the study. Four tocols including α-tocopherol, 5

16 β-tocopherol, α-tocotrienol nd β-tocotrienol were the predominnt compounds in ll the whet species exmined. All-trns lutein is the min crotenoid found in whet species (Abdel-Al et l. 2002; 2007). This polr dihydroxylted crotenoid constituted bout 77-83% of the totl crotenoids in high lutein whet species (Abdel-Al et l. 2007). Since the study gol is to improve incorportion of lutein in wholegrin bred, in the following section emphses will be put on crotenoids in cerel grins nd their role in humn helth nd nutrition Crotenoids Structure Crotenoids, in generl, re collection of tetrterpenoid compounds, with the bsic crotenoid structurl bckbone consisting of isoprenoid units formed either by hed-to-til or by til-to-til biosynthesis. The primry groups of crotenoids re crotenes nd xnthophylls. Crotenes re crotenoids tht contin only hydrocrbon mde up from isoprene units, such s α-, ß-, nd γ-crotene nd lycopene. Xnthophylls re crotenoids tht contin oxygen s hydroxyl, keto, crboxyl, methoxyl, nd epoxy group, such s lutein nd zexnthin (Figure 2. 1) (Abdel-Al nd Akhtr 2006). Lutein is defined structurlly s long crbon chin with conjugted double bonds, nd t the ends of the crbon bckbone, the molecule includes construction of cyclic hexenyl with n ttched hydroxyl group. The zexnthin hs similr structure s lutein, the only structurl differences between them is in the loction of the double bonds in the hexenyl ring or the ionone ring. Zexnthin hs 2 β-ionone rings (symmetric molecule), while lutein contin β-ionone ring nd ε-ionone ring (symmetric molecule). Lutein nd zexnthin re distinguished from other crotenoids becuse of the presence of hydroxyl group t the ends of the molecule. The bsorbnce of specific light wvelength nd the relese of other wvelengths crete chrcteristic color properties in the molecules s result of the structure 6

17 of the nine double bonds. The yellow or ornge color of lutein cn be identified bsed on its concentrtion, which ffects the mount of blue light bsorption. Lutein is lso present in severl isomers nd forms such s trns, cis, nd epoxy lutein. However, it is still uncler whether biovilbility or other physiologicl functions re ffected by lutein isomers. The trns form of lutein is the primry isomer in plnts nd vegetbles, nd processing my cuse chnges in this significnt isomer. In nture, lutein exists s ftty ester in which one or two of the hydroxyl groups re bound to ftty cid (Kijlstr et l. 2012) but is lso found in free form (Abdel-Al et l. 2007). There re 17 vrious esters detected in commercil lutein supplements, the level of monoesters ws very low (1.5%), mixed diesters were present t bout the sme level s the homogeneous diesters (50.2 vs 48.0%, respectively), nd plmitic cid ws the predominnt ftty cid (57.2%) followed by myristic cid (30.2%), steric cid (10.2%), nd luric cid (2.4%) (Piccgli et l. 1998). Abdel-Al nd Young (2009) observed low levels of two pirs of regioisomeric monoesters nd nerly equl levels of three homogeneous diesters nd five pirs of mixed diesters in commercil lutein supplement. Identifying lutein esters would help in improving qulity of lutein-bsed products by fortifying the whet species to deliver the physiologicl dose (Young et l. 2007). Lutein is redily bsorbed from foods nd dietry supplements, wheres to enter the bloodstrem, the esters require prior de-esterifiction by intestinl enzymes (Alves-Rodrigues nd Sho 2004). 7

18 Figure 2.1: Structure of ll-trns-lutein, ll-trns-zexnthin, ll-trns-ß-cryptoxnthin, lltrns-ß-crotene, nd 13-cis-lutein found in whet (source: Abdel-Al et l. 2007). 8

19 Occurrence of lutein in cerel grins Crotenoids, specificlly lutein, re the min yellow pigment in whet (Abdel-Al et l. 2007). They re effective ntioxidnts due to the long series of lternting double nd single bonds (Okrter nd Liu 2010). The concentrtion of crotenoids in cerel grins exhibited wide rnge from very low in white nd red whet to reltively high in einkorn nd durum whet (Abdel-Al et l. 2002; 2007). The lutein in whet is the min crotenoid existing in high concentrtion rnging from 26.4 to µg/100 g grin, followed by zexnthin rnging from 8.7 to 27.1 µg/100 g grin, nd then β-cryptoxnthin rnging from 1.1 to 13.3 µg/100 g grin (Adom et l. 2003). In corn flour, there is 11.5 µg/g of lutein nd 17.5 µg/g of zexnthin content nd 3.7 µg/g of β-cryptoxnthin, (Brenn nd Berrdo 2004). In study by Abdel-Al et l. (2007) showed tht corn is exceptionlly high in lutein t concentrtion of 21.9 µg/g showing good potentil s blending flour in the development of high-lutein whet-bsed functionl foods. It lso hs high concentrtion of ll-trnszexnthin 10.3 µg/g nd smll concentrtions of ll trns β-cryptoxnthin nd ll-trns-β crotene 0.95 nd 0.31 µg/g, respectively. Severl studies hve shown tht einkorn is rich source of crotenoids (Abdel-Al et l. 2002; 2007; 2010; Hidlgo et l. 2006). High levels of ll-trns-lutein (7.41 µg/g) were identified in einkorn whet with smll mounts of ll-trns-zexnthin, cis-lutein isomers, nd α-crotene (Abdel-Al et l. 2007). The lutein content is distributed in the endosperm of einkorn nd bred whet (74.6 % nd 69.4 %, respectively) (Hidlgo nd Brndolini 2008). Adom et l. (2003) reported tht the lutein content of the brn plus germ-milled frction of whet ws bout 4-fold higher thn tht of the flour frction. In the United Sttes, the verge consumption of lutein is below the recommended dily intke which is 1.5 mg to 2 mg per dy (Fullmer nd Sho 2001), 2.2 mg per dy in Europe (O Neill et l. 2001) nd 1 mg to 2 mg per dy in Cnd (Lyle et l. 1999; Wright et l. 2003). In humn diet, in ddition to 9

20 cerels, fruits nd vegetbles, such s drk green vegetbles (e.g., green lettuce nd Brussels sprouts) re lso considered mjor source of crotenoids, nd their consumption is recommended s wy to increse dietry intke of lutein nd zexnthin (Sommerburg t l. 1998). Egg yolk is lso good source of highly biovilble lutein nd zexnthin (Hndelmn et l. 1999) Functionlity Plnt crotenoids provide the pigments for photosynthesis s n importnt role in the protection ginst photo-oxidtion. They ply fundmentl role in plstid pigments nd in provision of substrte for biosynthesis of plnt growth regultor (Tnk et l. 2008). The function of crotenoids pigments is determined by whether the tissue is photosynthesis or non-photosynthesis. In photosynthesis tissue, the most significnt function is to be photoprotective in plnt life ginst hrmful oxygen species while the pigments in nonphotosynthesis tissue provide the color for fruits nd flowers (Brtley nd Scolnik 1995; Tnk et l. 2008). Crotenoids hve the bility to bsorb the blue light of the spectrum in which bsorbed energy cn be trnsformed to chlorophylls. Then, they function s light hrvesting of rdint light in region of the spectrum tht not covered by the chlorophylls. Crotenoids re needed for photo-protection. Plnts would suffer photo-oxidtive dmge, in the bsence of crotenoids which cn led to the deth of the orgnism. Chromoplsts, which re plstids tht responsible for pigment synthesis, contin crotenoids tht imprt the ornge, red nd yellow pigments to roots, flower nd fruits. Crotenoids re stored t very high level in chromoplsts to provide the severe color of plnts (Brtley nd Scolnik 1995). Crotenoids s phytochemicls cn lso function biologiclly. They re effective ntioxidnts due to their bility of reducing free electrons nd scvenging hrmful free rdicls from biologicl system resulting in inhbittion of oxidtive rections. 10

21 Phytochemicls cn modulte cellulr physiology t the moleculr, biochemicl nd physiologicl levels. They lso mnge infectious diseses s ntibcteril, ntiulcer, ntivirl nd ntifungl (Vttem nd Shetty 2005). Crotenoids imprt foods their yellow nd ornge colors nd provide severl beneficil effects s dietry ntioxidnts, helth-enhncing nd disese-prevention components Role of lutein in humn helth Helth of eye The structure of the eyes is reltively more complex thn tht of ny other prt of the humn body, nd vision loss is considered widespred problem, especilly mong the elderly (Stringhm nd Hmmond 2005). Mres-Perlmn et l. (2002) hve suggested tht lutein nd zexnthin help to decrese the dnger of developing eye diseses, such s ctrcts nd ge-relted mculr degenertion (AMD), which re the most common eye diseses in elderly people. More thn 20% of these popultions might hve this disorder (Lim et l. 2012). Abdel-Al et l. (2013) lso reported the prevlence of mjor eye diseses nd the role of lutein nd zexnthin in reducing AMD nd ctrcts. AMD leds to legl blindness nd numerous visul losses, nd involves complex interction of metbolic nd functionl fctors (Nowk 2006; Klein et l. 2004; Kuehn 2005). Thus, to protect ginst AMD, diet with low glycemic index such s high intke of wholegrin products nd low intke of sturted nd polyunsturted fts might be needed to help moderte the risk (Mres nd Moeller 2006). Epidemiologicl studies hve proven tht lutein plys protective role with respect to the development of mculr degenertion, the primry cuse of blindness in the elderly in Western countries, nd the consumption of mel contining significnt mounts of crotenoids is ssocited with lower risk of mculr degenertion. Stringhm nd Hmmond (2005) hve dded tht giving ptients who suffered from ctrcts 15 mg of lutein three times per week for two yers resulted in improved visul cuity. In ddition, the 11

22 mculr pigments, lutein nd zexnthin, cn decrese both longitudinl nd lterl chromtic berrtions tht dmge the retinl imge due to the wvelength tht is concentrted in the retin. This mculr pigment cn lso enhnce visul cuity (Stringhm nd Hmmond 2005). High mculr pigment density (MPD), mculr pigment opticl density (MPOD) nd mculr pigment (MP) hve been ssocited with reduced AMD (Abdel-Al et l. 2013) Helth of skin The skin is n orgn of humn body tht requires protection from environmentl dmge by severl mechnisms. First, the presence of nturl ntioxidnt system cn protect skin cells by neutrlizing the free rdicls produced by sunlight exposure. Second, some cells in the skin clled melnocytes produce melnin tht results in the tnning of the skin. This tn provides protection to the skin by filtering sunlight becuse melnin hs the bility to bsorb the wvelengths of sunlight. Lstly, hyperplsi process reduces the dmge tht the light my cuse in the vible cells of the skin. The ntioxidnts present in the skin t the time of light exposure provide protection ginst the dmge cused by sunlight in the three mechnisms. However, ntioxidnt cpcity cn be significntly reduced by sunlight exposure. Lutein my hve the bility to bsorb visible blue-light wvelengths in the skin (Roberts et l. 2009). Lutein nd zexnthin, s result of dietry intke, re found in humn skin. Both components help to protect the skin ginst dmge cused by UV light, ginst skin swelling nd ginst hyperplsi (González et l. 2003). Roberts (2013) hs proved tht lutein nd zexnthin supplementtion helps to reduce the effect of UV rdition exposure nd help to provide skin protection ginst cutneous effects by UV. The ccumultion of lutein nd zexnthin my lso help to decrese the formtion of rective oxygen species (ROS) Lutein nd cncer The bsorption of totl provitmin A crotenoids such s α-crotene, β-crotene nd 12

23 β-cryptoxnthin hve helped to reduce the risk of lung cncer (Rock 2009). There ws n inverse ssocition between serum concentrtions of α-crotene, β-crotene, lutein, lycopene nd β-crypoxnthin, nd the risk of lung cncer exist (Woggon nd Kundu 2004). Crotenoids, single or in combintion form, hve the bility to remove free rdicls nd enhnce the immune response ginst tumor development. A study by Freudenheim et l. (1996) proved tht the intke of crotenoids especilly lutein nd zexnthin is correlted with reduced risk of premenopusl brest cncer. According to Mres-Perlmn et l. (2002) mong women who hve history of brest cncer, the defensive effect of zexnthin nd lutein is very strong Effects of processing nd storge on lutein Milling The milling process entils trnsforming rw mterils into finer nd primry products for subsequent processing. With respect to cerel grins, milling seprtes the brn nd germ from the strchy endosperm to produce white flours for use in mking bkery products. Becuse of the removl of the kernel outer lyers (pericrp, test, nd leurone), milling hs significnt influence on the helth-promoting components found in grins, such s phytochemicls, so the concentrtion of grin bioctive compounds is significntly reduced through this process (Rgee et l. 2012). Whet milling consists of controlled breking, reduction, nd seprtion to produce vriety of milled products for vrious end uses. The objective of whet milling is to seprte the pericrp nd germ of the whet kernel from the endosperm to produce white flours. On the other hnd durum whet is milled into grnulr product clled semolin for pst production. Significnt differences in the composition nd concentrtion of phenolic cids in eight durum whet smples were observed between kernel prts (strchy endosperm, leurone lyer nd pericrp) (Peyron et 13

24 l. 2002). The strchy endosperm ws chrcterized by low content of ferulic cid, the leurone lyer ws rich in trns-sinpic cid, while the pericrp exhibited high content of ferulic cid dehydrodimer. In study on two Cndin whets, durum nd bred whet, bioctive constituents were minly concentrted in the outer lyers of grins with the brn frction hving the highest ntioxidnt cpcity compred with shorts nd flours (Liyn- Pthirn nd Shhidi 2007). Corn cn lso be frctionted into its components either through dry milling process, in which the kernel is seprted into endosperm, brn, nd germ, or through wet milling process, in which the corn is seprted into strch, protein, fiber, nd oil. Qulittively, the phenolic composition of corn kernels is similr to tht of other cerel grins (McDonough et l. 1983). The crotenoids in milled corn hve been extrcted nd nlyzed using high-performnce liquid chromtogrphy (HPLC). The results indicted tht the level of lutein nd zexnthin in milled corn were 1.60 nd 7.83 µg/g respectively (Mmth et l. 2012). Reducing the prticle size of corn grins through processing opertion such s milling or grinding could enhnce the extrctbility of the crotenoids. For exmple, the levels of lutein nd zexnthin were 1.60 nd 7.83 µg/g in milled corn respectively) nd they reduced by 5.2% milled corn fter over drying of (Mmth et l. 2012). Therefore, to reduce the loss of bioctive compounds nd possibly increse the helth benefits of end products, removl of the outer lyers of cerel grins during the milling process should be considered (Rgee et l. 2012) Bking Bking is trditionl process for mking ssorted types of products such s breds, ckes, pstries, pies, trts, quiches, cookies, nd crckers. Abdel-Al et l. (2010) studied the stbility of lutein nd zexnthin in unfortified or fortified bked products (pn bred, flt 14

25 bred, cookies, nd muffins) using different bking recipes nd bking conditions. Fortified flt bred contined bout mg of lutein/serving (serving=30g), wheres the unfortified einkorn hd <0.2 mg/serving. The unfortified pn bred hd reltively smll mounts of lutein, bout mg/serving. In ddition to lutein, einkorn/corn pn bred contined zexnthin t bout 0.1mg/serving. Bking of flt bred resulted in significnt reduction in ll-trns-lutein, being bout 37-41% for the unfortified breds nd 29-33% for the fortified breds. Hidlgo et l. (2010) lso showed crotenoids loss during processing. Bred crumbs lost 21% of the crotenoid content, while 47% of the crotenoids were lost in bred crusts due to mnufcturing. A miniml mount of crotenoids were lost in wter biscuits (19%) due to levening. During bking, ll crotenoids were reduced except ß-crypotoxnthin, which is concentrted in bred crusts nd wter biscuits (Hidlgo et l. 2010). Only smll effect on crotenoids ws evident s result of the bred levening, wheres significnt losses occurred during bking (Abdel-Al et l. 2010). In bred levening, insignificnt crotenoid losses of only 3% were observed (Hidlgo et l. 2010). Moreover, vritions in the dough fermenttion time from 0 h to 48 h resulted in no significnt effect on ntioxidnt properties. Longer dough fermenttion times nd incresed bking times or tempertures could be considered potentil pproch for enhncing the ntioxidnt properties of whole whet pizz crust (Moore et l. 2009). The long kneding time involved in pst production lso decresed the mount of crotenoids significntly, but the degrdtion ws not significnt during the drying step (Hidlgo et l. 2010). Kneding cused 9% degrdtion in bred nd 6% in wter biscuits. Kneding step mixes oxygen nd wter in the dough, incresing lipoxygense (Hidlgo et l. 2010) nd peroxidse (Leenhrdt et l. 2006), nd thus oxidizing crotenoids (Hidlgo et l. 2010). Abdel-Al et l. (2010) lso investigted stbility of lutein nd zexnthin in cookies 15

26 produced from einkorn flour lone or in blend with corn flour, either fortified with lutein or unfortified. Lutein content in fortified cookies ws lower thn tht in flt bred, rnging from 0.5 to 0.6 mg/serving. The results indicted tht the fortified einkorn nd control cookies hd significnt losses of lutein concentrtions, but tht only moderte decline ws evident in the unfortified einkorn cookies. The decline in lutein ws bout 62% in unfortified einkorn, 65 % in fortified einkorn, nd 63% in the control cookies. These losses were considered to be due to the bking recipe nd the concentrtion of lutein. A high ft content cused greter loss of lutein in the cookies thn in the bred becuse crotenoids re soluble in ft nd cn be exposed to isomeriztion nd oxidtion during bking. The degrdtion rte of zexnthin during bking ws lower thn tht of lutein becuse of its low concentrtion in the bking formul. The muffin recipe is mde of einkorn nd corn four, either fortified with lutein or unfortified nd high ft, so the ft could help cuse degrdtion of the lutein during the bking process becuse of its solubility in ft, which destroys the lutein through oxidtion nd isomeriztion (Hidlgo et l. 2010). Muffins hd resonble mount of lutein t 0.8 mg/serving. The unfortified cookies nd muffins contined smll mounts of lutein, bout 0.1 mg/serving. The decline in their lutein content ws 64% in the unfortified muffins nd 55% in the fortified muffins (Abdel-Al et l. 2010). Abdel-Al et l. (2010) hs ssessed the formtion of cis-lutein nd cis-zexnthin isomers in bred, cookie, nd muffin products during bking nd subsequent storge. When lutein-contining products undergo therml processing or long-term storge (up to few yers), lutein my prtilly convert into cis-isomers. In cookie, the formtion of 13- nd 13'- cis-isomers ws dominnt nd higher compred to the other products. In the unfortified flt bred, smll mounts of cis-isomers were formed wheres cis-isomer concentrtions in fortified flt breds were lower thn tht of their respective flours. On bking of fortified cookies, significnt mounts ( 57%) of cis isomers were formed. However, the unfortified 16

27 cookies exhibited only slight reduction ( 21%) in cis-isomer concentrtions. As in cookies, fortified muffins hd significnt mounts of cis-isomers formed during bking, in prticulr 13- nd 13'-cis-lutein, wheres the concentrtion of cis-isomers in the unfortified cookies dropped slightly during bking Steming Hidlgo et l. (2008) studied the effect of vriety of steming tretment conditions on crotenoids in hulled einkorn nd bred whet. Their experiment reveled tht steming hs considerble influences on the concentrtion of crotenoids. Extreme steming conditions led to significnt degrdtion in ntioxidnts. For exmple, lutein tht exhibited the lest stbility of the crotenoids lost more thn 25 % of its content. The chemicl nd technologicl chrcteristics of wholegrin flours, which mde up with germ, endosperm nd brn, were strongly ffected by the steming tretments (Hidlgo et l. 2008). Updike nd Schwrtz (2003) lso reported tht therml processing using microwve ffects the isomeriztion of lutein nd zexnthin in vegetbles. A recent study by Junptiw et l. (2013) exmined the effect of steming of sweet corn cultivrs on lutein, zexnthin, ß-crotene, nd ß-cryptoxnthin, demonstrting increses in ll crotenoids concentrtions, with the highest for lutein, zexnthin nd ß-cryptoxnthin t 232%, 457% nd 405%, respectively, while the increse in ß-crotene ws only 88%. This is might be becuse of the difference in tissue structure of both smples nd cooking time for the steming process. Steming is lso not very effective technique for trnsferring het compring with boiling, but the results of both domestic methods indicted tht therml tretment enhnced vilbility of the crotenoid. The crotenoid content in sweet corn cultivrs could thus be preserved nd enhnced through pproprite cooking methods nd conditions tht ssocited with n incresing in tissue brekdown during heting (Junptiw et l. 2013). Bengtsson et l. (2008) found tht steming of ornge-fleshed potto resulted in 17

28 decrese in ll trns-ß-crotene nd the retention ws between 69% nd 81% of ll trns-ßcrotene Extrusion Since 1930 extrusion hs been one of the most significnt cooking technologies employed in food processing. It is used extensively for processing redy-to-et food nd brekfst cerel products (Brennn et l. 2011; Riz et l. 2009; Cheftel 1986). It is preferred over other processing technologies becuse of the short cooking time involved nd the vriety of products mde by extrusion (Brennn et l. 2011). Severl studies hve demonstrted the significnt reduction in bioctive compounds in food products fter extrusion processing (Korus et l. 2007; Delgdo-Licon et l. 2009; Shih et l. 2009). The conditions ssocited with this process would cuse reduction in bioctive compounds in generl, nd in prticulr, significnt decrese in ß-crotene hs been shown fter the extrusion of sweet potto nd ornge (Delgdo-Licon et l. 2009; Shih et l. 2009). The reduction in phenolic compounds occurs during extrusion becuse of the high temperture of the brrel nd the high moisture content, which encourge the polymeriztion of phenols nd finlly led to decrboxyltion (Dlmini et l. 2007; Repo-Crrsco-Vlenci et l. 2009). Fonsec et l. (2008) compred the mount of crotenoids lost in dehydrted nd extruded products. During dehydrtion, the totl mount of crotenoids in sweet potto flour cultivrs did not significntly decresed, but extrusion cooking cused significnt degrdtion. The totl crotenoid reductions in sweet potto flour, ornge sweet potto flour nd crem sweet potto flour were 24.3% nd 50.5%, respectively. When sweet potto flour is mixed with rice flour, the reduction in crotenoids in the mixed flours (ornge nd crem sweet potto flours) is smller thn for the unmixed sweet potto flours, the losses were 2.6%, 3.8%, nd 16.2% for sweet potto, ornge, nd crem flour, respectively. The mixed 18

29 flour hd smll losses of totl crotenoids becuse of the composition of rice flour. Borrelli et l. (2003) reported tht composition of rice flour (9% of protein, 0.75% of crude fiber nd 1% of lipid) could crete crotene-lipid-protein net to protect crotenoids from thermic denturtion. The pproprite processing conditions re importnt to reduce or prevent the loss of constituents during extrusion process (Cmire et l. 1990). Altn et l. (2009) found significnt reduction in both ntioxidnt cpcity (60%-68%) nd totl phenolics (46%-60%) in brley extrdites compred with those reported for unprocessed brley flour Storge Hidlgo nd Brndolni (2008) studied the effect of storge temperture on degrdtion of crotenoids in white nd whole mel flour of einkorn (cv. Monlis) nd bred whet (cv. Serio) t rnge of temperture (-20, 5, 20, 30, nd 38 C) for more thn 239 dys. The temperture degree hd substntil effect on the mount of the crotenoids decresed in stored einkorn whet flour, which cn lso be ffected by temperture nd time ccording to the first-order kinetics. The reduction of lutein nd totl crotenoids in both white nd whole mel flour ws similr. However, losses of lutein were fster in bred whet thn in einkorn. The temperture 20 C or less hs been considered to be n pproprite for preserving crotenoids for long-term storge. Storing flt bred t the mbient temperture for more thn 5 dys lso hd little effect on the ll-trns-lutein in unfortified products, nd liner losses tht dhered to first-order kinetics occurred in lutein-fortified products (Abdel- Al et l. 2010). Crotenoid compounds were destroyed t higher storge tempertures. At 38 C, the crotenoids content in einkorn flour hd greter stbility thn in bred whet (Hidlgo nd Brndolini 2008). The difference could be due to the significnt initil concentrtion of these compounds in both the whole mel nd white flour (Trono et l. 1999), which subsequently decrese the ntioxidnt ctivity of crotenoids (El-Agmey et l. 2004). It cn 19

30 lso be due to the lower lipoxygense ctivity in einkorn (Leenhrdt et l. 2006). In einkorn, becuse of the higher concentrtion of lipoxygense in the brn portion, with the exception of ß-crypotoxnthin, crotenoids re more stble in white flour thn in whole mel flour (Rni et l. 2001). In study on white nd golden corn, lutein nd zexnthin did not relly chnge in cnned corn tht ws kept in sugr/slt brine solution for 12 min t C, but, - crotene decresed to 62 % (Scott nd Eldridge 2005). The study did not mesure cis-isomers of lutein nd zexnthin, which were found to increse in cnned vegetbles (Updike nd Schwrtz 2003). Junptiw et l. (2013) hve studied the crotenoid content in sweet corn fter freezing in domestic freezer for one month. They found tht the crotenoid content in frozen corn ws higher thn fresh corn, which would mke it greter dietry source of crotenoids thn fresh corn. This improvement in crotenoid content fter freezing is the result of the relese of the bound crotenoids from the structurl mtrix (Dewnto et l. 2002) Effect of storge nd processing on ntioxidnt properties Wholegrin cerels, fruits nd vegetbles re the most importnt dietry sources of ntioxidnts. Storge of brekfst cerels t room temperture slightly chnged ntioxidnt cpcity, but storing brekfst cerels t 100 F for two months, which equivlent to eight months t room temperture, resulted in bout 10% loss of ntioxidnt (Miller et l. 2000). Nicoli et l. (1999) hve reported tht the ntioxidnt properties of polyphenol products cn be incresed or decresed becuse of the oxidtive rections. Moore et l. (2009) hve studied the effect of processing conditions (fermenttion, bking time nd temperture) nd brn size on ntioxidnt properties in whole whet pizz crust using 2,2 zinobis-3-ethylbenzothizoline-6-sulfonic cid (ABTS), 2,2-diphenyl-1- picrylhydrzyl (DPPH) nd Oxygen rdicl bsorbnce cpcity (ORAC) methods. The 20

31 results of ABTS, ORAC nd DPPH rnged from 16.5 to 18.5, 16.1 to 21.2 nd 1.6 to 1.9 µmol trolox equivlents/g, respectively. The study indictes tht decresing whet brn prticle size did not significntly ffect ntioxidnt cpcities of pizz crust during processing. In ddition, microniztion of whet brn improved the extrction of ntioxidnt components, but it s not cler, in the study, if the improvement due to n increse in the extrct surfce re or the therml processing (Zhou et l. 2004). In terms of fermenttion for 0, 18 nd 48 h t 4 C, the three ssys ABTS, DPPH nd ORAC indicte tht there is no significnt effect on ntioxidnt properties of pizz dough. However, bking time nd temperture showed significnt chnges in ntioxidnt properties. Two bking conditions of the pizz crust (from 7 to 14 min t 204 C) nd (for 7 min t 204 C nd 288 C) incresed ABTS scvenging cpcity from 42% to 47% with no significnt different in smples bked t 204 C for 7 min. The DPPH scvenging cpcity lso incresed from 50% to 82%. The ORAC vlue significntly incresed from 47% to 51% when bking conditions chnged from 204 C to 288 C for 7 min (Moore et l. 2009). Millrd rections, tht contin high moleculr weight brown compounds from therml tretments help to increse the ntioxidnt cpcity of the crust (Lindenmeier nd Hofmnn 2004). Therml processing t 115 C decresed totl ntioxidnt cpcity of bound phytochemicls from sweet corn smples by 21.9, 27.9 nd 49.8% (fter 10, 25 nd 50 min, respectively) when compred with the rw smples. However, the ntioxidnt cpcity of free phytochemicls from sweet corn smples t 115 C for 10, 25 nd 50 min significntly incresed by 35.4, 44.0 nd 94.0%, respectively, compred with the rw smples (Dewnto et l. 2002). Relesing bound phenolic from food mtrix with therml processes could explin the improved in ntioxidnt cpcity (Eberhrdt et l. 2000). In generl, there re severl possible effects on ntioxidnt properties of grin products during storge nd processing. First, in some cses insignificnt or slight chnges 21

32 could occur during storge nd processing such s chnges in crotenoids like lycopene nd β-crotene, which re stble even in intense het tretments. Second, nturlly occurring ntioxidnts cn be lost becuse mny compounds re reltively not stble or they cn be incresed due to storge nd/or processing conditions nd type of ntioxidnt compounds. Third, new compounds such s Millrd rection products cn be formed. Finlly, interctions mong different food components (e.g. between lipid nd nturl ntioxidnts, nd between lipids nd Millrd rection products) hve uncertin effects on ntioxidnt properties nd stbility of food (Nicoli et l. 1999) High-lutein functionl foods The Food nd Nutrition Bord of the Ntionl Acdemy of Sciences in USA defines functionl food s ny modified food or food ingredient tht my provide helth benefit beyond the trditionl nutrients it contins (Hsler 2002). The Interntionl Life Sciences Institute in the United Stte suggests slightly different definition: foods tht, by virtue of the presence of physiologiclly ctive components, provide helth benefit beyond bsic nutrition (Hsler 2002). Heth Cnd defines functionl foods s functionl food is similr in ppernce to, or my be, conventionl food, is consumed s prt of usul diet, nd is demonstrted to hve physiologicl benefits nd/or reduce the risk of chronic disese beyond bsic nturl functions. Functionl foods re ttrctive to consumers nd my be preferred to pure bioctive compounds, dietry supplements or drugs s evidenced by the incredible growth of the functionl foods mrket in North Americ in recent yers (Lewis 2008). Sheeshk nd Lcroix (2008) conducted n officil survey indicting tht Cndin dietitins encourge the improvement nd intke of functionl foods s well s foods with lbels stting helth clims on products. Becuse of the increse in the vilbility of functionl foods in the mrket, pproprite legisltion should be encted with respect to some 22

33 products in order to protect consumers. An dditionl considertion is tht functionl bked products re very ttrctive wy to deliver bioctive compounds such s lutein becuse they cn be produced esily (Red 2011). Functionlizing bked products lso increse the profitbility nd mrketbility of grins nd cerels (Sheeshk nd Lcroix 2008). In terms of grins, whet species such s einkorn (ncient whet) nd durum (pst whet) nd corn were identified s promising ingredients for the development of high-lutein functionl foods bsed on their reltively higher levels of lutein compred with other whet species such s spelt, soft nd hrd whet (Abdel-Al nd Akhthr 2006; Fregeu-Reid nd Abdel-Al 2005; Abdel-Al et l. 2002; 2013; Abdel-Al nd Hucl 2014). The use of grin with high endogenous lutein content hs been shown to improve crotenoid content in food products (Red 2011). Approximtely 70% of the totl crotenoids re ccumulted in cornmilled frctions which mkes corn promising blending flour ingredient in the development of high-lutein functionl foods (Abdel-Al et l. 2007). The high-lutein wholegrin bred, cookie nd muffin were found to be ble to scvenge peroxyl, ABTS nd DPPH rdicls (Abdel-Al nd Rblski 2013). Unbound nd bound phenolic extrcts were found to contribute to the ntioxidnt properties of the products with higher shre given by the bound phenolic compounds (Abdel-Al nd Rblski 2013). Three wholegrin foods with high level of lutein (bout 1 mg per 30 g serving) were developed nd evluted in terms of lutein stbility during bking process (Abdel-Al et l. 2010) nd ntioxidnt properties (Abdel-Al nd Rblski 2013). The wholegrin bkery products include high-lutein flt bred, high-lutein cookie nd high-lutein muffin. Lutein ws found to drop significntly during bking process (28% to 64% loss) due to oxidtion nd isomeriztion. The isomers 13- nd 13 -cis-lutein were found s dominnt cis-isomers. Different pproches hve been used to protect the lutein during processing nd storge nd to compenste for the losses of lutein. For exmple, functionl food products hve been 23

34 fortified with lutein. In ddition, whet nd corn verities, with higher lutein content thn the existing ones, hve been developed or under development. The fortified bked products were found to contin resonble concentrtions (up to 1 mg/serving) of lutein despite of its significnt losses. The wholegrin bkery products re lso considered good sources of phenolic ntioxidnts (Abdel-Al et l. 2013). 24

35 3.1. Abstrct CHAPTER 3: Effect of Lutein Enrichment on its Stbility nd Distribution in Wholegrin Bred The crotenoid lutein plys significnt roles in humn helth but its dily intke is low. Bred is stple food which could be good vehicle for lutein delivery. This study ws designed to investigte the effect of two different bred formuls (bsic nd enriched) nd three forms of lutein (e.g. lutein powder, lutein in oil emulsion nd lutein in ethnol suspension) on stbility nd distribution of lutein in wholegrin bred. Totl crotenoid content nd lutein concentrtion were mesured in three different prts of bred lof (crust, top crumb, nd center crumb) using spectrophotometry nd liquid chromtogrphy. The stbility of lutein during storge of bred t room temperture up to 7 dys ws lso investigted. There were no significnt differences in totl crotenoid content between the bsic nd enriched formul breds. While the concentrtion of lutein in the bred prts (crust, top crumb nd center crumb) vried by the bking formul nd form of lutein used. The crust hd significntly lower crotenoids thn the crumb, with no significnt differences between the top crumb nd center crumb. The ddition of lutein in powder form ws more stble in both formuls thn the other two forms with lutein in oil emulsion hd the lowest stbility in both formuls. The presence of ft in the formul would mke lutein more ccessible during bking process cusing more degrdtion. Higher levels of lutein were found in center nd top crumb for breds mde from bsic formul thn their counterprts mde from the enriched formul. Slight reductions in lutein content were observed with storge up to 7 dys, but in generl, the content remined firly stble. The results suggest tht enrichment of bred, especilly bred mde from bsic formul, with lutein powder would be promising food to boost the consumption of this importnt crotenoid. 25

36 3.2. Introduction Crotenoids re group of pigments tht imprt the red, yellow, nd ornge colors in fruits, vegetbles, nd cerel grins (Borneo nd León 2012). The concentrtion of crotenoids in cerel grins rnges from very low in white nd red whet to reltively high in einkorn nd durum whets (Abdel-Al et l. 2007). The primry crotenoid in whet is lutein present in high concentrtions rnging from 26.4 µg/100 g to µg/100 g of grin, followed by zexnthin, which rnges from 8.7 µg/100 g to 27.1 µg/100 g of grin, nd then β-cryptoxnthin, t 1.1 µg/100 g to 13.3 µg/100 g of grin (Adom et l. 2003). In humn diet, in ddition to cerels, drk green vegetbles such s spinch, green lettuce nd brussels sprouts re considered mjor source of lutein (Sommerburg t l. 1998). Lutein nd zexnthin re oxygented crotenoids contining two hydroxyl groups ttched to the two ionone rings. They hve similr chemicl structures with slight difference in their ionone ring wheres lutein hs β-ionone ring nd ε-ionone ring, while zexnthin hs two β-ionone rings (Abdel-Al et l. 2010). They re the primry crotenoids in whet, durum nd corn (Abdel-Al et l. 2002; 2007; Ken et l. 2008). Lutein nd zexnthin ply significnt roles in promoting the helth of eyes nd skin (Abdel-Al et l. 2013) s well s reducing the risk of ge-relted mculr degenertion (AMD) nd ctrcts (Mres-Perlmn et l. 2002) nd crdiovsculr disese (Slvin et l. 2001). They ccumulte in the mculr region of humn retin tht gives them curtive function (Hndelmn et l. 1999). β-crotene (nother crotenoid pigment) lso reduces proxy rdicls nd exhibit ntioxidnt properties tht defend ginst oxidtive hrm (Mres- Perlmn et l. 2002). Becuse of the significnt roles in humn helth, dily intke of lutein hs been boosted lthough it is low worldwide. For exmple, In the United Sttes, the verge consumption of lutein is mg per dy (Fullmer nd Sho 2001), 2.2 mg per dy in Europe (O Neill et l. 2001) nd 1 mg to 2 mg per dy in Cnd (Lyle et l. 1999; 26

37 Wright et l. 2003). These mounts re below the suggested dily intke of lutein (5-6 mg). Drk green vegetbles re considered s good source of crotenoids nd their consumption is recommended s wy to increse dietry intke of lutein nd zexnthin (Sommerburg et l. 1998). Bred is stple food tht hs been used s suitble vehicle to deliver vitmins nd minerls. The incorportion of lutein into bred products could be big chllenge due to the nture of lutein s lipophilic compound. Lutein is lso sensitive molecule to het, light nd oxygen which cn led to isomeriztion nd oxidtion during bking process. In previous study (Abdel-Al et l. 2010), the stbility of lutein in breds, cookies nd muffin bked by nturlly high-lutein nd lutein-enriched whole whet ws found to substntilly declined. The loss of lutein in cookies nd muffin ws greter t 64 nd 55% compred to flt bred t 31%. The use of einkorn s high lutein whet with or without enrichment of flours with lutein showed the potentil to boost lutein content in bked products (Red 2011). The current study is intended to find wy to improve incorportion of lutein into bred recipes. The study imed to investigte the effect of two different bred formuls (bsic nd enriched) nd three forms of lutein (lutein powder, lutein in oil emulsion nd lutein in ethnol suspension) on stbility nd distribution of lutein in wholegrin bred. Distribution of lutein in three prts of lof (e.g. crust, top crumb nd center crumb) ws evluted. The stbility of lutein during storge of bred t room temperture up to 7 dys ws lso investigted Mteril nd Methods Mterils Wholegrin flour ws obtined from P&H milling group (Cmbridge, ON, Cnd). The wholegrin flour contins protein, sh nd totl dietry fiber t verge of 13.7, 1.4 nd 11.5%, respectively. Crisco shortening nd oil, commercil dry yest, sugr, slt nd whey protein were purchsed from the retil mrket in Guelph, ON, Cnd. Stndrds ll-trns- 27

38 lutein (90%purity) nd Methyl tert-butyl ether nd butnol were obtined from Sigm (Sigm-Aldrich CndLtd., Okville, ON, Cnd), ll-trns zexnthin (95%purity), nd ll-trns β-cryptoxnthin (95%purity) were purchsed from ChromDex (Snt An, CA). Ethnol 95% (food grde) ws purchsed from LCBO (Guelph, ON, Cnd) Methods Preprtion of lutein formuls Lutein ws obtined in two forms, lutein powder (85%) nd lutein pste (20%) from (Lyc-O-Lutein, LycoRed Corp., Ornge, NJ). The concentrtion of lutein in ech product ws confirmed using HPLC s outlined lter. Lutein powder ws used s is, nd ws dded to the wholegrin flour in dry mix. Lutein pste ws used to prepre lutein oil emulsion nd lutein ethnol suspension. Lutein oil emulsion ws prepred by solubilizing 160 mg of the lutein pste in 5 ml oil in the presence of 160 mg whey protein s stbilizer. Lutein in ethnol suspension ws prepred by solubilizing 160 mg of the lutein pste in 5 ml ethnol in the presence of 160 mg whey protein. Lutein enrichment ws performed to chieve level of bout 1.0 mg of free lutein/serving of bked product (30 g) Preprtion of wholegrin bred The pproved methods, optimized stright dough (Method ) nd bsic stright dough (Method ) (AACCI 2011) were used to prepre one bound pn bred lof from wholegrin flour with nd without lutein enrichment. Wter bsorption of wholegrin flours ws previously determined using Frinogrph-E (C.W. Brbender Inc., Hckensck, NJ, USA). The verge wter bsorption of wholegrin flour ws pproximtely 70%. Wholegrin flour ws enriched with the three lutein forms (powder, oil emulsion, ethnol suspension) before or during mixing with other bking ingredients. The lutein powder were crefully nd evenly distributed with flour in dry mix, lutein in oil emulsion nd lutein in ethnol suspension were dded into flours with other ingredients (yest suspension, sugr 28

39 solution, wter) during mixing step Qulity of breds nd bred preprtion for nlysis Control nd lutein-enrichment breds were tken from oven nd left t room temperture for bout 2 hr to cool down. Breds were evluted bsed on lof volume, lof weight nd specific volume. Bred Loves were weighed in grms (g) using n nlyticl blnce nd lof volume ws mesured ccording to the rpeseed displcement method of mesuring volume in cubic centimeters (cm 3 ) s described by the AACC Intl. pproved method (AACCI 2011). Specific volume ws clculted s cm 3 /g by dividing the volume (cm 3 ) of the lof by its weight (g). For bred storge experiment, sub-smples of bred (6 loves per tretment) were stored in polyethylene Zip-lock bg (21 15 Uline, C) t room temperture for 7 dys. For bred nlysis bred loves were cut with n electric knife into crust 21.5% (crefully removed without crumb), top crumb 37.6% (the crumb tht under crust tken bout 1 cm lyer) nd center crumb 40.8% (the inside center of crumb). Ech prt ws cut into slices nd dried in oven t 35 o C. Dried bred smples (fresh nd stored) were milled using UDY Cyclone mill equipped with 0. 5 mm screen nd kept t - 20 C for further nlysis Anlysis of totl crotenoid content nd individul crotenoids Bred crust, top crumb nd center crumb were extrcted with wter-sturted 1- butnol for the determintion of totl crotenoid content s outlined by Abdel-Al el l. 2007). Totl crotenoid content ws determined ccording to the AACC Intl. pproved method (AACCI 2011). Crotenoid in extrcts were seprted nd quntified by high performnce liquid chromtogrphy (HPLC) using n 1100 series chromtogrph (Agilent, Mississug, ON, Cnd) s described by Abdel-Al et l. (2010). The HPLC system ws equipped with model G1311A quternry pump, G1329A temperturecontrolled injector, G1316A temperture- controlled column thermostt, G1322A 29

40 degsser, G1315B photodiode rry detector (PDA), nd Chem Sttion v.8.04 dt cquisition system with the cpbility of conducting isobsorbnce plotting nd threedimensionl grphic nlyses. The seprtion ws performed on 4.6 x 100 mm C30 reversephse Crotenoid column (Wters, Mississug, ON, Cnd). The column ws operted t 35 o C nd eluted with grdient mobile system consisting of (A) methnol/methyl tert-butyl ether/nno pure wter (81:15:4, v/v/v) nd (B) methyl tert-butyl ether/methnol (90:10, v/v) t 1mL/min. The grdient ws progrmmed s follows: 0-9 min, %A; 9-10 min, 75-0% A; min, hold t 0% A; min, 0-100% A; nd min, hold t 100% A for the short column. The seprted crotenoids were detected nd mesured t 450 nm, nd the identity of crotenoids ws bsed on the congruence of retention times nd UV/vis spectr with those of pure uthentic stndrds. Five concentrtions (0.0-5 mg/ml) were prepred for ech crotenoid stndrd in butnol nd used to check linerity, to optimize the nlyticl method, nd to generte regression equtions for quntifiction. The regression nlysis of response re nd injected mount within the bove rnge showed liner reltionship with coefficient of determintion (R 2 ) rnging from to The purity of ech compound in extrcts ws verified on the bsis of the spectroscopic properties of ech pek using isobsorbnce plot or three-dimensionl grphic nd pek purity nlyses provided with the ChemSttion softwre. Pek purity nlysis llows the spectrum of the identified compounds to be identified nd confirmed nd to determine whether interference occurs Sttisticl Anlysis All nlyses were performed t lest in duplicte for (crust, top crumb nd center crumb) nd the men vlues re reported. Anlysis of vrince ws performed using IBM SPSS Sttistics 21 softwre (Armonk, New York, USA). Significnt differences (P <0.05) mong mens were detected using Tukey s multiple rnge test t fixed level of α=

41 3.5. Results nd Discussion Bred qulity Wholegrin bked products re rich sources of nutrients nd helth-enhncing components such s dietry fiber nd phenolic ntioxidnts (Abdel-Al nd Rblski 2013). They hve lso been linked with reducing the risk of chronic diseses such s hert disese (Osgnin et l. 2003), cncer (Michud et l. 2000) nd dibetes (Slvin et l. 2001). In the current study wholegrin berd ws investigted in terms of lutein enrichment to determine the possibility of incorporting lutein (lipophilic compound) into bred product nd its stbility during bking nd subsequent storge. On the bsis of verge lof volume, lof weight nd lof specific volume slight differences were observed between control (nonenriched) nd enriched breds (Tble 3.1). The lof specific volume ws quite similr ( cm 3 /g) in breds bked from bsic or enriched formul. The ddition of lutein in different forms did not hve substntil effects on the overll qulity bsed on bred lof mesurements. Pn breds bked from blends of bred whet flour with einkorn flour (ncient whet) or with einkorn nd corn flours hd lower lof volume thn control bred (Abdel-Al et l. 2010). In the current study high qulity flour ws used to produce cceptble wholegrin breds since the use of wholegrin flour is nticipted to produce low qulity breds compred to tht mde from refined flours. Further enhncement of the qulity of wholegrin bred could be needed for mking more ppeling products Effect of bking on lutein nd other crotenoids Lutein is essentil for humn helth nd it hs been linked with reduced risk of AMD nd ctrcts (Abdel-Al et l. 2013). It cn`t be synthetized by humns, nd its dily intke is low worldwide (Fullmer nd Sho 200; O Neill et l. 2001; Lyle et l. 1999; Wright et l. 2003). Thus lutein-enriched foods could be good strtegy to promote the consumption of this importnt crotenoid. Incorportion of lutein into the bking formul is chllenge 31

42 becuse of its lipophilicity nd difficulty in evenly distributing it in dough system (Red et l. 2015). The ddition of lutein in oil emulsion ws effective in cookies nd muffins but not in fltbred. The form of lutein (e.g. free vs ester or powder vs oil-bsed) could ffect distribution nd concentrtion of lutein in the end product. In the current study, free lutein ws dded in 3 vrious formuls, i.e. powder form, in oil emulsion nd in ethnol suspension. Additionlly whey protein ws dded to the formul to enhnce dispersion of lutein in the colloidl system. As expected enrichment of flours with lutein produced enriched breds hving totl crotenoid content tht re significntly higher thn tht of the control bred mde from non-enriched flours (Tble 3.2). The enriched breds hd totl crotenoid content bout 5 to 8 times higher thn the control bred. The bking recipe or ingredients (bsic versus enriched) nd how lutein ws incorported in the formul slightly ffected the product totl crotenoid content. Since totl crotenoid content is not specific mesurement nd other components in the extrct could contribute to the mesurement, the comprison between products should be tken with cution. The method would provide rough ide regrding crotenoid content in the bred products, but it is essentil to mesure individul crotenoid compounds nd their concentrtion in the end products. Previous work on totl crotenoid content in comprison with tht mesured by HPLC method showed tht the colorimetric method overestimtes the content by 20% (Abdel-Al et l. 2007). The totl crotenoid content of the lof prts (crust, top crumb nd center crumb) in the enriched formul ws slightly lower compred with their corresponding prts of the bsic formul (Figure 3.1A). For crumb prts, however, no significnt differences were observed mong the 3 lof prts in ll enrichment tretments in both formuls (Figure 3.1B, C). These findings indicte tht bking ingredients re more pronounced on crotenoids in bred crust during bking process. Vritions in totl crotenoid content could be due to the ddition of lutein nd/or chnges in nturl pigments during bking process which could influence 32

43 spectrophotometer redings. Hidlogo et l. (2010) found tht bred crumbs lose 21% of the totl crotenoid content nd 47% in bred crusts. A less reduction occurred during kneding where crotenoids reduced by 9% in bred nd 6% in wter biscuit. Abdel-Al et l. (2010) reported tht bred levening hs smll effect on crotenoids, wheres significnt crotenoid losses occurred during bking. The long kneding time involved in pst production significntly decresed the mount of crotenoids, but the degrdtion ws not significnt during the drying phse (Hidlgo et l. 2010). Lipoxygense nd peroxidse could led to crotenoids oxidtion in the presence of oxygen nd wter in the dough. The concentrtion of lutein in the bred prts (crust, top crumb nd center crumb) vried by the bking formul nd form of lutein dded to the bking formul (Figure 3.2 nd Tble 3.3). In generl, the enriched formul resulted in significnt reductions in lutein concentrtion in ll prts of the bred, with crust showing much greter level of lutein degrdtion thn both crumbs (top nd center) in both bred formuls. Within ech formul, significnt differences were observed mong the 3 lof prts with the center crumb receiving the lowest lutein reduction, followed by the top crumb nd finlly the crust. This cn be nticipted since the center crumb hs the lowest temperture during oven bking. The stbility of lutein ws significntly influenced by bking formul nd lutein form, i.e. how lutein ws dded (powder form, oil emulsion or ethnol suspension). When lutein ws dded in powder form, the concentrtion of lutein in ll bred prts ws significntly higher thn those enriched with other lutein forms (oil emulsion or ethnol suspension) in both bking formuls. The incorportion of lutein in n oil emulsion produced breds with the lowest concentrtion of lutein (Figure 3.2). This finding ws very obvious in the enriched bking formul. The ddition of lutein in n oil emulsion to the enriched bking formul tht contins oil to mkes lutein (lipophilic compound) more ccessible nd vulnerble to bking conditions resulting in more degrdtion through oxidtion nd isomeriztion. Similr results 33

44 were obtined in cookies nd muffins (high ft products) compred with bred products (low ft products) (Abdel-Al et l. 2010). Red et l. (2015) indicted tht forms of lutein such s free vs ester or powder vs oil-bsed could hve n effect on the distribution of lutein in end products. Their study hs lso showed tht using whey protein in the bking formul improves crotenoids distribution in cookies nd muffin but not in flt bred. The current study is the first to evlute the content of lutein in wholegrin breds. The crumb seems to be good preserve of lutein through bking s compred to crust, nd the bsic formul enriched with lutein powder in dry mix ppers to be more effective wy for mking lutein-enriched breds. Since biovilbility of lutein is essentil for the delivery of nticipted helth benefits of lutein, its content in food long with biovilbility re needed to determine the product efficcy s functionl food. The concentrtion of zexnthin in the lutein-enriched nd non-enriched or control bred is presented in Tble 3.3. Interestingly, the lutein-enriched breds hd higher zexnthin concentrtions tht the control ones. Additionlly, the lutein-enriched breds bked form the bsic formul contined more zexnthin compred with tht mde from the enriched formul. Similr to lutein the presence of ft in the enriched formul nd lutein formul (oil emulsion) cused more zexnthin degrdtion during the bking process becuse of its solubility in ft s lipophilic compound. Among ll tretments, using the bsic formul bred enriched with lutein powder hd the highest lutein nd zexnthin concentrtions in ll lof prts which underscore the question of lutein forms nd bking ingredients. All-trns-lutein is the dominnt configurtion in whet flour with trce mount of cis-isomers (Abdel-Al et l. 2007). When the flours undergo therml processing or long term storge, potion of ll-trns-lutein could be converted into its cis-isomers. In this study, it ws noticeble tht trce mounts of cis-isomers such s 13-cis-lutein, 13`-cis-lutein, 9-cislutein, nd 9`-cis-lutein re found in breds enriched with lutein (Figure 3.3). Abdel-Al et l. 34

45 (2010) found tht 9, 9`-, 13-, 13`-cis-isomers re, generlly, detected in bkery products. Also, they found tht the concentrtion of 13-, 13`-cis-lutein in enriched flt bred is lower thn in the respective flours. Besides lutein nd zenthin, other crotenoids including α- crotene, β-crotene nd β-cryptoxnthin re found in whet products (Abdel-Al et l. 2007; 2010). In this study, the verge mg of totl crotenoid, lutein nd zexnthin per serving (30g) in lutein-enriched nd control breds is presented in Figure 3.4. The enriched breds hd totl crotenoid of mg/serving, lutein mg/serving nd zexnthin bout mg/serving compred with pproximtely 0.1, <0.06 nd <0.01 mg/serving for control or non-enriched breds, respectively. In previous study on flt bred using einkorn flour (high crotenoid whet), lutein-enriched flt bred contined pproximtely mg lutein/serving, wheres the non-enriched einkorn hd < 0.2 mg/serving (Abdel-Al et l. 2010). In ddition, the lutein einkorn/corn pn bred contined zexnthin t pproximtely 0.1 mg/serving. These lutein enriched bked products would boost the dily intke of lutein nd other crotenoids on the bsis of the recommended 3-8 servings per dy Effect of bred storge on lutein nd other crotenoids Crotenoids re vulnerble molecules prticulrly when subjected to unfvorble conditions such s high temperture, oxygen, light, etc. In the present study, storge of luteinenriched breds t room temperture up to 7 dys hd no significnt effects on totl crotenoid content (Figure 3.5). Smples stored for 3 nd 7 dys showed no significnt differences in totl crotenoid content in the crust nd both crumbs of bsic nd enriched formuls compred with their corresponding fresh breds. We observed slight reduction in the concentrtion of totl crotenoid content in the crust nd top crumb of the breds mde from the enriched formul when lutein incorporting in suspension form. In generl, storge for 7 dys did not ffect totl crotenoid content for lutein-enriched breds. Storing 35

46 flt bred t mbient temperture for more thn 5 dys hd little effects on the ll-trns-lutein in non-enriched products, but liner losses foolowed first-order kinetics occurred in luteinenriched products (Abdel-Al et l. 2010). It is known tht crotenoid compounds cn be destroyed t higher storge tempertures. But storge time nd temperture re the fctor tht could ffect crotenoids stbility of bred whet (Hidlgo nd Brndolini 2008). Lutein nd zexnthin content of white nd golden corn preserved in sugr/slt brine solution in cns for 12 min t C did not chnge (Scott nd Eldridge 2005). Similr to crotenoids, storge for 7 dys t mbient temperture hd little effect on lutein content in bred curst, top crumb nd center crumb except for bred mde from enriched bking formul with lutein dded in suspension form (Figure 3.6). In generl the results showed tht different forms of lutein in wholegrin breds preserve the stbility of lutein nd totl crotenoid content for 7 dys storge t mbient temperture Conclusion The current study is the first to evlute the content of lutein in wholegrin bred mde from different bking recipes nd enriched with lutein in powder or liquid form. The crumb seems to be good preserve of lutein through bking s compred to crust, nd the bsic formul enriched with lutein powder in dry mix ppers to be more effective wy for mking lutein-enriched breds. The enriched formul resulted in significnt reductions in lutein concentrtion in ll prts of the bred, with crust showing much greter level of lutein degrdtion thn both crumbs (top nd center) for both bred formuls (bsic nd enriched). The reltively higher temperture of crust compred to tht of crumb during bking resulting in more lutein degrdtion. In generl, bking formul nd lutein form (powder versus liquid form) ffect the distribution nd concentrtion of lutein in lutein enriched breds. Since biovilbility of lutein is essentil for the delivery of nticipted helth benefits of lutein, its content in food long with biovilbility re needed to determine the product efficcy s 36

47 functionl food. Thus more reserch is needed to determine biovilbility of lutein. The storge of breds t room temperture for 7 dys did not ffect the concentrtion of lutein in most lutein-enriched nd non-enriched breds. A low ft-product (bsic formul) enriched with lutein in powder form could hold promise for the development of high lutein functionl bred nd boost the dily intke of lutein. 37

48 Tble 3.1: Lof volume, weight nd specific volume of non-enriched nd lutein-enriched wholegrin breds bked from bsic nd enriched formuls (men ±SD) Bked type Lof Volume Lof Weight Lof specific volume (cm 3 ) (g) (cm 3 /g) Bsic formul Control (non- enriched) 1760± ± ±0.03 Enriched- lutein powder 1740± ± ±0.01 Enriched lutein in oil emulsion 1720± ± ±0.02 Enriched -lutein in ethnol suspension 1760± ± ±0.01 Enriched formul Control (non- enriched) 1780± ± ±0.01 Enriched -powder lutein 1760± ± ±0.02 Enriched lutein in oil emulsion 1780± ± ±0.02 Enriched -lutein in ethnol suspension 1720± ± ±

49 Tble 3.2: Totl crotenoid content of non-enriched nd lutein-enriched wholegrin bred bked from bsic nd enriched formuls (µg/g, wb) Lutein form Crust Top Crumb Center Full lof Bsic Formul Control (non- enriched) Enriched -powder lutein Enriched lutein in oil emulsion Enriched -lutein in ethnol suspension 5.12± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.05 Enriched formul Control (non- enriched) Enriched -powder lutein 7.59± ± ± ± ± ± ± ±2.92 Enriched lutein in oil emulsion 37.61± ± ± ±1.50 Fortified-lutein in ethnol suspension 35.55± ± ± ±

50 Tble 3.3: Concentrtions of lutein nd zexnthin (µg/g, wb) of non-enriched nd luteinenriched wholegrin breds bked from bsic nd enriched formuls Bred prt Lutein Zexnthin Bsic formul Control LP LOE LES Control LP LOE LES Crust 1.64± ± ± ± ± ± ± ±0.1 Top crumb 1.73± ± ± ± ± ± ± ±0.07 Center crumb 1.86± ± ± ± ± ± ± ±0.02 Enriched formul Crust 0.70± ± ± ± ± ± ± ±0.01 Top crumb 1.10± ± ± ± ± ± ± ±0.04 Center crumb 1.12± ± ± ± ± ± ±0.07 LP, LOE nd LES stnd for lutein powder, lutein in oil emulsion, nd lutein in ethnol suspension, respectively. 40

51 A Totl crotenoids (µg/g ) b b b b b LP- B LOE- B LES- B LP- E LOE- E LES- E B Totl crotenoids (µg/g) LP- B LOE- B LES- B LP- E LOE- E LES- E C Totl crotenoids (ug/g) Figure 3.1: Effect of bking on totl crotenoid content in crust (A), top crumb (B) nd center crumb (C) of breds mde from bsic nd enriched formuls. LP-B, LOE-B, LES-B, LP-E, LOE-E nd LES-E stnd for lutein powder, lutein in oil emulsion, nd lutein in ethnol suspension with bsic bking formul; nd lutein powder, lutein in oil emulsion nd lutein in ethnol suspension with enriched bking formul, respectively Different letters indicte significnt differences between mens t p<0.05(stndrd devition vlues presented by error brs). 41 LP- B LOE- B LES- B LP- E LOE- E LES- E

52 A 50 Lutein (ug/g) bc b c d d 0 LP- B LOE- B LES- B LP- E LOE- E LES- E B 50 Lutein (ug/g) bc b cd e de 0 LP- B LOE- B LES- B LP- E LOE- E LES- E C 50 Lutein (ug/g) b b c d c 0 LP- B LOE- B LES- B LP- E LOE- E LES- E Figure 3.2: Effect of bking on lutein content in crust (A), top crumb (B) nd center crumb (C) of breds mde from bsic nd enriched formuls. LP-B, LOE-B, LES-B, LP-E, LOE-E nd LES-E stnd for lutein powder, lutein in oil emulsion, lutein in ethnol suspension with bsic bking formul; nd lutein powder, lutein in oil emulsion nd lutein in ethnol suspension with enriched bking formul, respectively. Different letters indicte significnt differences between mens t p<0.05(stndrd devition vlues presented by error brs). 42

53 Figure 3.3: High-performnce liquid chromtogrm of crotenoids extrcted from nonenriched bred (control) (A), enriched bred with lutein (bsic formul) (B) nd enriched bred with lutein (enriched formul) (C). Peks: 1, ll-trns-lutein; 2, ll-trns-zexnthin; 3, 13 -cis-lutein; 4, 13 cis lutein; 5, 9-cis-lutein; 6, 9 -cis-lutein. 43

54 A TCC (mg/serving 30g) b b b b b c c Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E Lutein (mg/serving 30g) B c b d e f g g Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E Zexnthin (mg/serving 30g) C b b c cd cd cd d Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E Figure 3.4: Amount per serving (mg/30g) of totl crotenoids (A), lutein (B) nd zexnthin (C) in full lof. LP-B, LOE-B, LES-B, LP-E, LOE-E nd LES-E stnd for lutein powder, lutein in oil emulsion, nd lutein in ethnol suspension with bsic bking formul; nd lutein powder, lutein in oil emulsion nd lutein in ethnol suspension with enriched bking formul, respectively. Different letters indicte significnt differences between mens t p<0.05 (stndrd devition vlues presented by error brs). 44

55 A Totl crotenoids (ug/g) b b LP- B LOE- B LES- B LP- E LOE- E LES- E Dy0 Dy3 Dy7 B Totl crotenoids (ug/g) b LP- B LOE- B LES- B LP- E LOE- E LES- E Dy0 Dy3 Dy7 Totl crotenoids (ug/g) C LP- B LOE- B LES- B LP- E LOE- E LES- E Dy0 Dy3 Dy7 Figure 3.5: Effect of storge t mbient temperture on totl crotenoids in crust (A), top crumb (B) nd center crumb (C) of breds mde from bsic nd enriched formuls. LP-B, LOE-B, LES-B, LP-E, LOE-E nd LES-E stnd for lutein powder, lutein in oil emulsion, nd lutein in ethnol suspension with bsic bking formul; nd lutein powder, lutein in oil emulsion nd lutein in ethnol suspension with enriched bking formul, respectively. Different letters indicte significnt differences between mens t p<0.05(stndrd devition vlues presented by error brs). 45

56 A Lutein (ug/g) b b Dy0 Dy3 Dy7 0 LP- B LOE- B LES- B LP- E LOE- E LES- E B Lutein (ug/g) Dy0 Dy3 Dy7 0 LP- B LOE- B LES- B LP- E LOE- E LES- E C Lutein (ug/g) b b Dy0 Dy3 Dy7 0 LP- B LOE- B LES- B LP- E LOE- E LES- E Figure 3.6: Effect of storge t mbient temperture on lutein in crust (A), top crumb (B) nd center crumb (C) of breds mde from bsic nd enriched formuls. LP-B, LOE-B, LES-B, LP-E, LOE-E nd LES-E stnd for lutein powder, lutein in oil emulsion, nd lutein in ethnol suspension with bsic bking formul; nd lutein powder, lutein in oil emulsion nd lutein in ethnol suspension with enriched bking formul, respectively. Different letters indicte significnt differences between mens t p<0.05(stndrd devition vlues presented by error brs). 46

57 CHAPTER 4: Antioxidnt Properties of Wholegrin Bred Enriched with Lutein 4.1. Abstrct Dietry ntioxidnts such s crotenoids hve significnt role in disese prevention due to their bility to scvenge rective oxygen species nd other free rdicls. Thus, improving ntioxidnt properties of wholegrin products could hve significnt helth benefits in reducing the risk of chronic diseses. With this gol, we hve exmined the ntioxidnt properties of two different bred formuls (bsic nd enriched) nd three forms of lutein enrichment (lutein powder, lutein in oil emulsion, nd lutein in ethnol suspension) on ntioxidnt properties of wholegrin bred. Becuse of the presence of diverse free rdicls present in wholegrin bred, multiple types of oxidtive ssys were used. In the current study ntioxidnt cpcity of bred products ws mesured using the DPPH, ABTS nd ORAC ssys. Using the DPPH ssy, there ws generl trend for higher ntioxidnt cpcity in the enriched formul bred smples. In ddition, the bred crust hd higher DPPH scvenging cpcity thn tht of bred crumb. When the ntioxidnt cpcity mesured using the ABTS ssy, there were no significnt differences between bred formuls (bsic versus enriched), bred portions (crust versus crumb), or lutein enrichment method (powder versus liquid). The ORAC ssy reveled significntly higher ntioxidnt cpcity in bred enriched with lutein thn non-enriched bred. The results indicte tht enrichment of wholegrin products with lutein could provide n importnt source of dietry ntioxidnts. 47

58 4.2. Introduction Dietry ntioxidnts could prevent or mitigte oxidtive dmge to cellulr components through their bility to scvenge rective oxygen species nd other free rdicls (Abdel-Al nd Rblski 2013). Dietry ntioxidnts include vriety of components such s crotenoids, phenolic cids, flvonoids, tocols nd nthocynins. They re present in wholegrin foods t vrious extents subject to grin type, product type nd processing technology. At present number of ntioxidnt ssy re vilble which re either bsed on hydrogen tom trnsfer such s inhibition of induced low-density lipoprotein utoxidtion nd Oxygen rdicl bsorbnce cpcity (ORAC), or bsed on electron trnsfer such s 2, 2- diphenyl-1 picrylhydrzyl (DPPH) nd 2, 2 zino-bis (3-ethylbenzthizoline-6sulfonic cid) (ABTS) (Hung et l. 2005). Due to the vriety of ntioxidnts in food mtrix the use of single ntioxidnt ssy to mesure ntioxidnt cpcity is indequte nd more thn one ssy should be used to properly determine the ntioxidnt cpcity (Abdel-Al nd Rblski 2013). In the current study, the ntioxidnt properties were mesured bsed on three ssys, DPPH, ABTS nd ORAC using QUENCHER method which is QUick, Esy, New, CHEp nd Reproducible (Serpen et l. 2008). The method is bsed on the direct mesurement of ntioxidnt cpcity by mixing smples with the regents followed by subsequent spectrometric mesurement (Serpen et l. 2008). Wholegrin products re considered good source of dietry fiber nd phytonutrients known s bioctive compounds. The bioctive compounds found in grins nd seeds include crotenoids, phenolic cids, tocopherols, tocotrienols, nthocynins nd flvonoids (Rgee et l. 2012). They re ssocited with the promotion of humn helth nd reduced risk of chronic diseses such s crdiovsculr disese (Osgnin et l. 2003), cncer (Michud et l. 2000) nd dibetes (Slvin et l. 2001). Severl high-lutein functionl foods using nturlly high-lutein grin mterils or 48

59 lutein-enrichment grin flours s source of ntioxidnts nd dietry fiber were developed (Abdel-Al et l. 2010). Lutein is the primry yellow pigment crotenoid found in whet. Its concentrtion ws found to be between 77-83% of the totl crotenoids in high lutein whet species such s einkorn, Khorsn nd durum (Abdel-Al et l. 2007). It ws found t reltively lower concentrtions in white nd red whet s compred to einkorn nd durum whet. Lutein ws lso found t high concentrtion in vegetbles such s spinch nd kle (Perry et l. 2009), eggs yolk (Scheffer et l. 1988) nd pst (Humphries nd Khchik 2003). Lutein plys significnt roles in promoting the helth of eyes nd skin (Abdel-Al nd Akhtr 2006) nd in reducing the risk of ge-relted mculr degenertion (AMD) (Bone et l. 2001), ctrcts (Olmedill et l. 2001), cncer (Michud et l. 2000) nd crdiovsculr disese (CVD) (Osgnin et l. 2003). Abde-Al et l. (2010) hve studied the stbility of lutein in bkery products tht indicted significnt reductions of lutein (28-62% loss) subject to product type nd bking ingredients nd conditions. But when the products were enriched with lutein, resonble concentrtions of lutein were found in the finl bked products. The use of einkorn s high lutein whet with or without enrichment of flours with lutein showed the potentil to boost lutein content in bked products (Red 2011). Abdel-Al nd Rblski (2013) hve demonstrted the bility of high-lutein wholegrin bred, cookie nd muffin to scvenge peroxyl, ABTS nd DPPH rdicls. Since lutein is lipophilic compound, its incorportion into bred formul could be big chllenge. In previous study, lutein ws successfully dded to cookie nd muffin s compred with flt bred due to their higher ft content (Red et l. 2015). The current study is imed to investigte the effect of two different bred formuls (bsic nd enriched) nd three forms of lutein (lutein powder, lutein in oil emulsion nd lutein in ethnol suspension) on ntioxidnt cpcity of bred products using three ntioxidnt ssys nmely, DPPH, ABTS nd ORAC. Stbility of lutein nd its content in bred ws discussed in chpter 3 nd lso reported in previous studies 49

60 (Abdel-Al et l. 2010) Mterils nd Methods Mterils Wholegrin flour ws obtined from P&H milling group (Cmbridge, ON, Cnd). The wholegrin flour contins protein, sh nd totl dietry fiber t verge of 13.7, 1.4 nd 11.5%, respectively. Crisco shortening nd oil, commercil dry yest, sugr, slt nd whey protein were purchsed from the retil mrket in Guelph, ON, Cnd. Fluorescein, 2,2 - Azobis (2-methylpropion-midine) dihydrochloride (AAPH), 2, 2 zino-bis (3- ethylbenzthizoline-6sulfonic cid) (ABTS), 2, 2-diphenyl-1 picrylhydrzyl (DPPH), 6- hydroxy-2,5,7,8-tetrmethylchromn-2-crboxylic cid (Trolox) nd potssium persulfte were purchsed from Sigm (Sigm-Aldrich Cnd Ltd., Okville, ON, Cnd). Ethnol (95%) ws purchsed from Commercil Alcohols (Brmpton, ON, Cnd) Methods Preprtion of lutein formultions Lutein ws obtined in two forms, lutein powder (85%) nd lutein pste (20%) from (Lyc-O-Lutein, LycoRed Corp., Ornge, NJ, USA). The concentrtion of lutein in ech product ws confirmed using HPLC s outlined lter. Lutein powder ws used s is, nd ws dded to the wholegrin flour in dry mix. Lutein pste ws used to prepre lutein oil emulsion nd lutein ethnol suspension. Lutein oil emulsion ws prepred by solubilizing 160 mg of the lutein pste in 5 ml oil in the presence of 160 mg whey protein s stbilizer. Lutein in ethnol suspension ws prepred by solubilizing 160 mg of the lutein pste in 5 ml ethnol in the presence of 160 mg whey protein. Lutein enrichment ws performed to chieve level of bout 1.0 mg of free lutein/serving of bked product (30 g) Preprtion of wholegrin bred The pproved methods, optimized stright dough (Method ) nd bsic 50

61 stright dough (Method ) (AACCI 2011) were used to prepre one bound pn bred lof from wholegrin flour with nd without lutein enrichment. Wter bsorption of wholegrin flours ws previously determined using Frinogrph-E (C.W. Brbender Inc., Hckensck, NJ, USA). The verge wter bsorption of wholegrin flour ws pproximtely 70%. Wholegrin flour ws enriched with the three lutein forms (powder, oil emulsion, ethnol suspension) during mixing with other bking ingredients. The lutein powder were crefully nd evenly distributed with flour in dry mix, while lutein in oil emulsion nd lutein in ethnol suspension were dded into flours with other ingredients (yest suspension, sugr solution, wter) during mixing step Bred preprtion for nlysis Control (non-enriched) nd lutein-enrichment breds were tken from the oven nd left t room temperture for bout 2 hr to cool down. Bred loves were cut with n electric knife into crust 21.5% (crefully removed without crumb), top crumb 37.6% (under crust crumb tht close to crust tken bout 1 cm lyer) nd center crumb 40.8% (the inside center of crumb). Ech prt ws cut into slices nd dried in n oven t 35 o C. Dried bred smples were milled using UDY Cyclone mill equipped with 0. 5 mm screen nd kept t -20 C for further nlysis DPPH rdicl scvenging cpcity ssy A stock solution of 2, 2-diphenyl-1 picrylhydrzyl (DPPH) ws prepred dily by dissolving 40 mg of DPPH in 100 ml of ethnol, nd then diluted with 100 ml of deionized wter. A working DPPH solution ws prepred by diluting 200 ml of stock solution with pproximtely 800 ml of 50% ethnol to obtin solution hving n bsorbnce vlue of t 525 nm (Brnd-Willims et l. 1995; Serpen et l. 2012). Direct QUENCHER method ws used in this study (Serpen et l. 2012). Ten mg ±1.0 mg of ground bred smples ws weighed into centrifuge tube. The ntioxidnt rection ws initited by dding 10 ml 51

62 of the working DPPH solution. The tube ws shken rigorously for 1 min nd plced on n orbitl shker in the drk. The mixture ws shken t rpm t room temperture to fcilitte the surfce rection between the solid bred prticles nd the regent. Centrifugtion ws performed t 9,200g for 2 min. The cler superntnt (2 ml) ws trnsferred into cuvette nd the bsorbnce ws mesured t 525 nm t room temperture. The inflection point (where the curve chnges from convex to concve or vice vers) of DPPH ws clculted by using regression nlysis nd pproximtion Polynomils to determine reding times (Tble 4.1). Four different times (5, 31, 48 nd 60 min) were chosen for the DPPH depletion mesurement. The DPPH scvenge cpcity of non-enriched nd enriched wholegrin bred extrct ws mesured nd clculted s µmole trolox equivlents/g smple. A stndrd trolox solution ws prepred in ethnol t concentrtion rnge between 0 nd 600 µg/ml. Exctly 0.1 ml of ech trolox concentrtion ws dded to 9.9 ml of DPPH rdicl solution. After 30 min of incubtion t room temperture, 2 ml of the rection solution ws trnsferred into cuvette nd the bsorbnce ws mesured t 525 nm. A regent blnk ws prepred using deionized wter insted of trolox solution. The determintion coefficient (R 2 ) of the method is nd regression eqution is y=0.0096x ABTS ction rdicl scvenging cpcity ssy A solution of 7 mmol/l ABTS ws prepred by dding 5 ml of deionized wter to mg of ABTS nd 2.45 mmol/l potssium persulfte solution ws prepred by dding 5 ml of deionized wter to mg potssium persulfte. Five ml from ech solution were mixed to stock solution of ABTS which is kept in the drk t room temperture for hr before use (Re et l. 1999). A working ABTS solution ws prepred dily by diluting the 10 ml of stock solution with pproximtely 800 ml of 50% ethnol to obtin solution hving n bsorbnce vlue of t 734 nm (Serpen et l. 2012). Direct QUENCHER 52

63 method ws prepred s described bove in DPPH ssy. The inflection point of ATS ws lso clculted to determine reding times (Tble 4.1). Four different times (5, 26, 45 nd 60 min) were chosen for the ABTS depletion mesurement. The ABTS scvenge cpcity of non-enriched nd lutein-enriched wholegrin bred smples ws mesured nd clculted s µmole trolox equivlents/g smple. A stndrd trolox solution ws prepred s explined bove in DPPH ssy. The determintion coefficient of the method is nd regression eqution is y=0.0112x ORAC ssy The ORAC method is bsed on the method of Ou et l. (2001). Ten mg of ground smples ws weighed into eppendorf tube with 1mL of 75 mm phosphte buffer ph 7.5. The mixture ws vortexed for 1min nd centrifuged t 9,000g for 45 sec. The smple extrcts were diluted severl times until n pproprite curve ws obtined over the test period. Twenty-five µl smple extrct, trolox stndrd solution or nno pure wter (blnk) were mixed with 150 µl of fluorescein in ech of the 96 micro-plte well. The mixture ws conditioned t 37 C for 30 min, then 25 µl of 2,2 Azobis (2-methylpropion-midine) dihydrochloride (AAPH) s peroxyl rdicl genertor ws dded to strt the decying of fluorescein. The degrdtion of fluorescein progressed for 60 min in the heted chmber of BioTech FLX800TBI with the following settings: the fluorescence excittion 485 nm, the emission wvelength 528 nm, nd reding ws tken every min for 1 hr. The micro-plte fluorescent reder ws operted by Gen 5 softwre version (BioTek). In this ssy, the scvenge cpcity of wholegrin bred extrcts ws lso clculted s µmole trolox equivlents/g smple. The determintion coefficient of the method is nd regression eqution is y=0.2552x Sttisticl Anlysis All nlyses were performed t lest in duplicte for (crust, top crumb nd center 53

64 crumb) nd the men vlues re reported. Anlysis of vrince ws performed using IBM SPSS Sttistics 21 softwre (Armonk, New York, USA). Significnt differences (P <0.05) mong mens were detected using Tukey s multiple rnge test t fixed level of α= Results nd Discussion Antioxidnt properties mesured by DPPH ssy Dietry ntioxidnts hve the bility to scvenge rective oxygen species nd free rdicls tht could dmge cellulr components. Wholegrin foods re good source of ntioxidnts nd phytonutrients which could promote humn helth nd/or reduce the risk of chronic diseses (Abdel-Al nd Rblski 2013). Lutein-enriched wholegrin bred could ply significnt roles in humn diet s being good source of lutein nd other ntioxidnt components such s polyphenols. The current study ssessed ntioxidnt properties of luteinenriched wholegrin bred products mde from bsic nd enriched bking formuls in comprison with non-enriched bred. QUENCHER method ws used to evlute the ntioxidnt cpcity of wholegrin bred products to void the problems ssocited with extrction of ntioxidnts due to their diverse chemicl structures s being hydrophilic nd lipophilic compounds. In this method food smples directly interct with rdicl regents. The QUENCHER method ws pplied for DPPH nd ABTS ssys. No doubt, it would be preferble to use single test to evlute ntioxidnt or rdicl scvenging cpcity, but currently there is no single test tht could chrcterize ntioxidnt properties of foods due to the presence of multiple free rdicls nd oxidnts in foods or biologicl systems. Such complex system would require more thn one test for ntioxidnt mesurement nd chrcteriztion (Abdel-Al nd Rblski 2013). In the DPPH ssy, the bility of food or food extrct to scvenge the DPPH free rdicls is mesured through the reduction of the color intensity of the rdicl reduced in the presence of n ntioxidnt. The color intensity of DPPH rdicls with no ntioxidnts or 54

65 grin extrcts remins stble over the test time (Rgee et l. 2012). Scvenging of DPPH rdicl llows evlution of the electron-donting potency of food components (Brnd- Willims et l. 1995). The DPPH rdicl scvenging cpcity of lutein-enriched nd nonenriched breds expressed s micromole trolox equivlents per grm is presented in Figure 4.1. The DPPH scvenging cpcity ws mesured in the bred crust (Figure 4.1A), top crumb (Figure 4.1B) nd center crumb (Figure 4.1C) to understnd their contribution to ntioxidnt properties nd chnges in ntioxidnt components during bking process. The bred crust showed the highest bility to scvenge DPPH rdicls despite its lowest content of lutein s it received the highest degrdtion in the bred crust (chpter 3). This indictes tht other ntioxidnt components contribute to the DPPH scvenging cpcity. Millrd rection products could contribute to the overll scvenging cpcity (Yilmz nd Toledo 2005). Lutein-enriched bred crusts hd higher DPPH scvenging cpcity thn non-enriched bred crust. Previous studies hve shown DPPH scvenging cpcity of lutein-enriched flt bred, cookie nd muffin products ws significntly higher thn non-enriched ones (Abdel- Al nd Rblski 2013). DPPH scvenging cpcity of top crumb of breds from lutein-enriched nd enriched formul showed higher DPPH scvenging cpcity s compred to tht mde from bsic formuls (Figure 4.1B). In ddition, top crumbs from lutein-enriched breds hd higher bility to scvenge DPPH thn those from non-enriched or control breds. Once gin this indictes the contribution of lutein to the DPPH scvenging ttribute s previously reported (Abdel-Al nd Rblski 2013). Figure 4.1C presents the DPPH scvenging cpcity of the center crumb of lutein-enriched nd non-enriched breds. In generl, the center crumb hd slightly lower DPPH scvenging cpcity thn tht of the top crumb (Figure 4.1B). In other words the DPPH scvenging cpcity progressively incresed from the bred center to the top. This could reflect the extent of non-browning rections occurred in the crumb nd crust 55

66 during bking. Yu nd Nnguet (2013) hve reported tht the verge scvenging bility decresed by 32% for whole whet bred during mking process. This loss might be becuse of the loss of phytochemicls due to high temperture during bking. The extrction of ntioxidnts from vrious food mtrices (e.g. whet flour versus bred) my lso ffect the level of ntioxidnt cpcity (Yu nd Nnguet 2013). The kinetics of scvenging cpcity of DPPH in the presence of non-enriched nd lutein-enriched wholegrin bred crust, top crumb nd center crumb is shown in Figure 4.2A, B nd C. The DPPH rdicl rection exhibited non-liner rection pttern with rpid increse during the first 30 min nd fter tht the rection rte stedily incresed up to 60 min. The pttern hd more thn one inflection point indicting vrious rection rtes over the test period (Tble 4.1). It ws reported tht the effectiveness of crotenoids s DPPH rdicl scvengers incresed by the length of the effective conjugted double-bond system nd by the ddition of hydroxyl groups on the terminl rings (Jiménez Escrig et l. 2000). In ddition to the conjugted double-bond system, lutein contins 2 hydroxyl groups tht could contribute to the DPPH scvenging property of bred products Antioxidnt properties mesured by ABTS ssy The ABTS ssy mesures the bility of ntioxidnt compounds to scvenge ABTS free rdicl ction bsed on electron-trnsfer donted by ntioxidnt compounds (Brnd- Willims et l. 1995). The ABTS rdicl ction hs reltively stble blue-green color which is in the presence of n ntioxidnt such s 6-hydroxy-2,5,7,8-tetrmethylchromn- 2- crboxylic cid (trolox) or potentil ntioxidnts in mteril extrcts, the color production will be suppressed to certin extent in proportionl to the concentrtion of ntioxidnts (Rgee et l. 2012). The ABTS scvenging cpcities of bred crust, top crumb nd center crumb re presented in Figure 4.3A, B nd C, respectively. There were no significnt differences mong the bred crusts of lutein-enriched nd control breds mde from both 56

67 formuls, bsic nd enriched, except for the crust from bred enriched with lutein in n oil emulsion in the enriched formul. This tretment ws the lowest compred to other tretments. The ABTS scvenging cpcity of top crumb showed insignificnt differences between non-enriched (control) nd enriched bred mong ll tretments except for the top crumb from bred enriched with lutein in n oil emulsion in the enriched formul. A similr trend ws lso found for the ABTS scvenging cpcity of center crumb. Once gin, only the center crumb from bred enriched with lutein in n oil emulsion in enriched formul significntly hd lower ABTS scvenging cpcity compred to the other tretments. The ddition of lutein in n oil emulsion system my result in lowering the ntioxidnt rection rte leding to lower bility to scvenge ABTS rdicl ction. The kinetics of scvenging cpcity of ABTS in the presence of non-enriched nd enriched wholegrin crust, top crumb nd center crumb re shown in Figure 4.4A, B nd C. The rection is not liner nd rpidly incresed during the first 26 min nd then the rte of rection stedily incresed up to 60 min. The ABTS rection showed more thn one inflection points indicting vrious rection rtes (Tble 4.1). Abdel-Al nd Rblski (2008) reported tht scvenge cpcity ginst ABST rdicl ction by whet ntioxidnts would vry depending upon concentrtion of individul bioctive compounds in whet extrcts nd their synergic effects Antioxidnt properties mesured by ORAC ssy ORAC ssy mesures the bility of compounds to rect with physiologiclly relevnt peroxyl rdicls (Moore et l. 2009). It mesures scvenging cpcity of peroxyl rdicl (ROO. ) bsed on clculting the net protection re under the time recorded for the fluorescein decy curve in the presence of ntioxidnts. The ORAC vlues, similr to DPPH nd ABTS, re expressed s micromole trolox per grm. The ORAC vlues of the crust of breds mde from enriched formul were significntly higher thn tht mde with the bsic formul (Figure 4.5A). Similr trends were found for top crumb nd center crumb smples 57

68 (Figure 4.5B nd C). For the enriched formul, the lutein-enriched bred crust, top crumb nd center crumb hd significntly higher ORAC vlues. This ws lso true for the top crumb of breds from the bsic formul (Figure 4.5B). It seems tht the ddition of lutein to the enriched formul improve exposure of lipophilic ntioxidnts (e.g. lutein) to the peroxyl rdicls. Yu nd Nnguet (2013) reported tht the incresing of ntioxidnt cpcity fter bred mking process using ORAC ssy is becuse tht phytochemicls re more likely to be detected by ORAC method. Another reson for the increse in ntioxidnt cpcity, mesured by ORAC ssy, fter bking could be due to Millrd rections (Yilmz nd Toledo 2005). The study lso reported tht the ntioxidnts properties could be ffected by the temperture, ph, wter ctivity, rectnts type nd vilbility of oxygen Conclusion This study investigted the ntioxidnt cpcity of the breds (crust, top crumb nd center crumb) enriched with three different forms of lutein. The bred crusts showed the highest DPPH scvenging cpcity compred with top nd center crumbs. The crust nd both crumbs of enriched formul breds, in generl, provide higher ntioxidnt cpcity thn the prts of bsic formul breds due to Millrd rection. The ABTS scvenging cpcity, in generl, did not show significnt differences mong severl enrichment breds prts nd formuls. The ORAC vlues in bred crust nd both crumbs in enriched formul presented better scvenging cpcity of peroxyl rdicl compred with bsic formul. The lutein enrichment of bred by using enriched formul could improve the consumption of dietry ntioxidnts nd thus promote humn helth 58

69 Tble 4.1: Regression equtions, determintion coefficients nd inflection points of DPPH nd ABTS rections with wholegrin bred smples Regression Eqution nd determintion coefficients Inflection points (min) Assy 2 nd order 3 rd order 4 th order 3 rd order 4 th order DPPH y= 7E -05 x x y= 3E -06 x x x y= 6E -0 8x x x & 48.2 R 2 = R 2 = R 2 = ABTS y= x x R 2 = y= 8E -06 x x x R 2 = y= 3E -07 x 4 5E -05 x x x R 2 = &

70 DPPH scvenging cpcity (µmol TE/g) A b bc bc bc c c Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E DPPH scvenging cpcity (µmol TE/g) B b c c c d Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E C DPPH scvenging cpcity (µmol TE/g) c cd bc Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E Figure 4.1: DPPH scvenging cpcity of crust (A), top crumb (B) nd center crumb (C) of breds mde from bsic nd enriched formuls. LP-B, LOE-B, LES-B, LP-E, LOE-E nd LES-E stnd for lutein powder, lutein in oil emulsion, nd lutein in ethnol suspension with bsic bking formul; nd lutein powder, lutein in oil emulsion nd lutein in ethnol suspension with enriched bking formul, respectively. Different letters indicte significnt differences between mens t p<0.05(stndrd devition vlues presented by error brs). 60 cd b b

71 A DPPH scvenging cpcity (µmol TE/g) Time (min) Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E B DPPH scvenging cpcity (µmol TE/g) Time (min) Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E C DPPH scvenging cpcity (µmol TE/g) Time (min) Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E Figure 4.2: Kinetics of DPPH with non-enriched nd enriched wholegrin bred crust (A), top crumb (B) nd center crumb (C) in breds mde from bsic nd enriched formuls. LP-B, LOE-B, LES-B, LP-E, LOE-E nd LES-E stnd for lutein powder, lutein in oil emulsion, nd lutein in ethnol suspension with bsic bking formul; nd lutein powder, lutein in oil emulsion nd lutein in ethnol suspension with enriched bking formul, respectively 61

72 A ABTS scvenging cpcity (µmol TE/g) b Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E B ABTS scvenging cpcity (µmol TE/g) b b b b Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E ABTS scvenging cpcity (µmol TE/g) C b b b Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E Figure 4.3: ABTS scvenging cpcity of crust (A), top crumb (B) nd center crumb (C) of breds mde from bsic nd enriched formuls. LP-B, LOE-B, LES-B, LP-E, LOE-E nd LES-E stnd for lutein powder, lutein in oil emulsion, nd lutein in ethnol suspension with bsic bking formul; nd lutein powder, lutein in oil emulsion nd lutein in ethnol suspension with enriched bking formul, respectively. Different letters indicte significnt differences between mens t p<0.05(stndrd devition vlues presented by error brs). b b b 62

73 A ABTS scvenging cpcity (µmol TE/g) Time (min) Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E ABTS scvenging cpcity (µmol TE/g) B Time (min) Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E C ABTS scvenging cpcity (µmol TE/g) Time (min) Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E Figure 4.4: Kinetics of ABTS with non-enriched nd enriched wholegrin bred crust (A), top crumb (B) nd center crumb (C) in breds mde from bsic nd enriched formuls. LP-B, LOE-B, LES-B, LP-E, LOE-E nd LES-E stnd for lutein powder, lutein in oil emulsion, nd lutein in ethnol suspension with bsic bking formul; nd lutein powder, lutein in oil emulsion nd lutein in ethnol suspension with enriched bking formul, respectively 63

74 ORAC scvenging cpcity (µmol TE/g) A b b b b b Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E B ORAC scvenging cpcity (µmol TE/g) b c c c c d Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E C ORAC scvenging cpcity (µmol TE/g) b b b b b Control- B LP- B LOE- B LES- B Control- E LP- E LOS- E LES- E Figure 4.5: ORAC scvenging cpcity of crust (A), top crumb (B) nd center crumb (C) of breds mde from bsic nd enriched formuls. LP-B, LOE-B, LES-B, LP-E, LOE-E nd LES-E stnd for lutein powder, lutein in oil emulsion, nd lutein in ethnol suspension with bsic bking formul; nd lutein powder, lutein in oil emulsion nd lutein in ethnol suspension with enriched bking formul, respectively. Different letters indicte significnt differences between mens t p<0.05(stndrd devition vlues presented by error brs). 64