Comprehensive Colorimetric Study of Anthocyanic Copigmentation in Model Solutions. Effects of pH and Molar Ratio

Depósi o de in es igación de la Uni e sidad de Se illa h ps://idus.us.es/ "This documen is he Accep ed Manusc ip e sion o a Published Wo k ha appea ed in inal o m in Jou nal o Ag icul u al and Food Chemis y, copy igh © Ame ican Chemical Socie y a e pee e iew and echnical edi ing by he publishe . To access he inal edi ed and published wo k see h ps://doi.o g/10.1021/j 2046202". P a g e 1 o 36 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y 1 A COMPREHENSIVE COLORIMETRIC STUDY OF ANTHOCYANIC 2 COPIGMENTATION IN MODEL SOLUTIONS. EFECTS OF pH AND MOLAR 3 RATIO 4 5 Belén Go dillo, F ancisco J. Rod ígu e z-Pulido, M. Lui sa E sc ud e o-Gil e e , 6 M. Loudes González-Mi e , F a n c i sc o J . H e e di a* 7 Food Colou & Quali y Lab., D ep . N u i i on & F ood Sci en c e . F a c u l ad de 8 Fa macia. Uni e sidad de Se ill a . 41012 -S e ill a , S pa i n . 9 10 11 Abb e ia ed unning i le: 12 Colo and copigmen a ion o an ho cy an i n s 13 14 15 * Co esponding au ho : 16 F ancisco J. He edia 17 Food Colou & Quali y Lab., D ep . N u i i on & F ood Sci en c e . F a c u l ad de 18 Fa macia. Uni e sidad de Se ill a . 41012 -S e ill a , S pa i n 19 Tel.: +34 954556495 Fax: + 34 954557017 20 e-mail: he [email protected] 21 22 23 24 25 ACS P a a gon Plu s En i onm e n 1 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y P a g e 2 o 36 26 Abs ac 27 New colo ime ic a iabl e s ha e been de i ned i n he un i o m CIE L AB 28 colo space o assess he quan i a i e and qua li a i e c o l o c hange s i ndu c ed b y 29 copigmen a ion, and hei incid en c e on is ua l pe c ep i on . T he c op i g m en a i on 30 p ocess was assayed in mod e l s o l u i on s be w een M a l i d i n 3 - g l u c o si de and 31 h ee phenolic compounds (ca e c h i n , ep ic a e c h i n and c a e ic a ci d ) a s a un c i on 32 o he pH and he pigmen /copi g m en m o l a a i o . Al ong he p H a i a i on , he 33 g ea es magni ude o copigm en a i on w a s ob a i ned a p H 3 . 0 , be i ng 34 signi ican ly highe wi h epica ec h i n and c a e ic a ci d . A h i gh a ci d ic p H, he m a i n 35 con ibu ion o copigmen a ion o he o a l c o l o w a s qua li a i e w h il e be w een 36 pH 2.0 and 4.0, he main c o l o im e ic c on i bu i on w a s quan i a i e . T he 37 con ibu ion o epica echin and c a e ic a ci d o he c o l o c hange s w a s m o e 38 ma ked o he quan i a i e cha a c e is ics. O n c on a s , pa ic u l a ly a h i ghe p H 39 alues, he quali a i e con ibu i on w a s m o e im po an i n c a e c h i n c op i g m en ed 40 solu ions. Inc easing copigm en c on c en a i on i ndu c ed pe c ep i b l e c o l o 41 changes a mola a ios highe han 1 : 2 , c on sis i ng i n a b l u is h and da k en i ng 42 e ec o he an hocyanin solu io n s. Am ong he d i e en CIE L AB a i bu e s, hue 43 di e ences was he bes co el a ed pa a m e e wi h he i n c ea s e o c op i g m en 44 concen a ion, p o ing he ele an c e o h is ph ysic o c he mic a l pheno m enon on 45 he quali a i e changes o an hocy an i n c o l o . 46 47 Keywo ds: An hocyanin; colo ; c op i g m en a i on ; T is im u l u s C o l o im e y ACS P a a gon Plu s En i onm e n 2 P a g e 3 o 36 48 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y INTRODUCTION 49 An hocyanins a e he pigm en s a cc oun i ng o he b illi an ed , pu p l e , 50 and blue colo s in many ui s, ege ab l e s, and de i ed ood p odu c s s u c h a s 51 ui s juices, jams, and ed wines ( 1 ). 52 I is well known ha h e s ab ili y o an ho cy an ic p i g m en s is g ea ly 53 limi ed because hei esona in g s u c u e c on e s he m i n i n sic i n s ab ili y 54 agains se e al physical-chemical c ond i i on s. E a l ua i on o he a c o s a e c i ng 55 he s abili y o an hocyanins in d ic a e s ha p H is he m o s im po an e x i n sic 56 ac o o an hocyanin deg ada i on . D epend i ng on he p H o he m ed i u m, he ed 57 colo ed la ylium ca ion coexis s a s an equ ili b i u m mix u e wi h o he o ms o 58 an hocyanins: he blue-pu ple q u i nono i da l ba s e s, he c o l o l e ss he mi a c e a l B, 59 and he pale yellow chalcones. T he e o e , he s a m e an ho cy an i n s o l u i on m a y 60 show di e en colo s (2). 61 Howe e , he chemical an d c o l o im e ic s ab ili y o an ho cy an i n s c an be 62 imp o ed by associa ions wi h o he s ub s an c e s. C op i g m en a i on pheno m enon , 63 among o he s, ep esen s one o he m o s c o m p l e x and e ici en m e c han isms o 64 an hocyanin ch omopho e s abiliz a i on i n na u e and ood sys e ms ( 3 ). I n ood 65 science, his phenomenon is c on si de ed a e l e an i n e a c i on be c au s e 66 ob aining s able and a ac i e c o l o s is a m a j o o c u s o qua li y c on o l 67 pu poses (4). Especially, in win e m a ki ng , is a ss u m ed i p l a ys a k e y o l e on he 68 colo e olu ion and s abili y o h e y oung ed wi ne s ( 5 ). 69 Copigmen a ion eac ions c on sis o non -c o a l en i n e a c i on s be w een 70 an hocyanins among hemsel es (s e l - a ss o ci a i on ) o wi h a wi de a i e y o 71 colo less o ganic compounds na m ed c op i g m en s o c op i g m en a i on c o a c o s ACS P a a gon Plu s En i onm e n 3 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y P a g e 4 o 36 72 (basically o he phenolic comp ound s, bu a ls o a mi no a ci d s, s uga s, o gan ic 73 acids, polysaccha ides, e c.). 74 F om a molecula poin o i e w, he an ho cy an i n -c op i g m en s c o m p l e x e s 75 adop a ypical sandwich co n i gu a i on (- s a cki ng ) i a h y d ophob ic 76 in e ac ion. This s uc u al con o m a i on p o e c s he ed c o l o ed l a yli u m c a i on 77 agains he nucleophilic a ack o w a e , pe o xi de and s u l phu d i o xi de b l ea c h i ng , 78 and pH changes educing he o m a i on o he o he c o l o l e ss s pe ci e s i n he 79 an hocyanin equilib ium (hemiac e a l and c ha lc one ) ( 6 ). 80 In addi ion, copigmen a i on no on ly c on e s g ea e s ab ili y o 81 an hocyanins, bu also induc e s c o l o a i a i on s. T h is ki nd o m o l e c u l a 82 associa ion is esponsible o he y p ic a l c hange s i n he s pe c a l p ope i e s o 83 he ch omopho e g oup, ha is, an i n c ea s e o he ab s o p i i y and equen ly a 84 shi o he isible λmax ow a d g ea e w a e l eng h s. C on s equen ly, 85 copigmen a ion p oduces bo h quan i a i e and qua li a i e c o l o c hange s on he 86 an hocyanins solu ions. In his se n s e , he m ea s u e m en and e a l ua i on o he s e 87 colo ime ic changes is o g ea i n e e s i n ood i ndu s y si n c e c o l o is one o he 88 main senso y pa ame e o he qua li y o ood s i n l uen ci ng c u s o m e s e l e c i on 89 (7). 90 The con ibu ion o he c op i g m en a i on pheno m enon on c o l o ha s been 91 widely s udied using spec op ho o m e ic m e hod s, bo h i n m ode l o ood 92 sys ems which con ain indi idual an ho cy an i n s and added c op i g m en s; o i n ed 93 wines whe e an hocyanins na u a lly c o - o cc u s wi h o he pheno lic c o m pound s 94 (8-15). The e alua ion o he ch ange s i n he VIS s pe c u m o an ho cy an i n s, o 95 speci ically in he λmax (520 nm), ha s pe mi ed o k no w he i n l uen c e o 96 nume ous ac o s on he e ec i ene ss o he c op i g m en a i on i n cl ud i ng he ACS P a a gon Plu s En i onm e n 4 P a g e 5 o 36 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y 97 concen a ions o pigmen s and c o a c o s, he i c he mic a l s u c u e s, he 98 co ac o /pigmen mola a io, he p H o he m ed i u m, e c. I n he s a m e w a y, 99 se e al s udies in ed wines ha e s ho w n ha he m agn i ude o c op i g m en a i on 100 and i s e olu ion du ing winem a ki ng is e x e m e ly a i ab l e a cc o d i ng o 101 i icul u al, ag onomical o oen o l og ic a l p a c ic e s a cc oun i ng app o xim a e ly o m 102 25 o 50% o he o al colo o yo ung ed wi ne s ( 5 , 16 - 20 ). 103 In any case, al hough sp e c a l m e hod s ha e de m on s a ed o be a a li d , 104 simple and quick ool o quan i a i e e s im a i on s, is gene a lly a cc ep ed ha he y 105 p o ide limi ed p ecision and a cc u a cy o c o l o s pe ci ic a i on s. T he l o w e 106 p ecision and accu acy achie ab l e c an be e x p l a i n be c au s e an adequa e 107 desc ip ion o he colo a ia ions equ i e s i) ha s pe c a l a i a i on s c on si de ed 108 should be hose a ec ing he e n i e s pe c a l c u e , no on ly i s isi b l e λmáx, and 109 ii) he use o a leas h ee colo im e ic a i bu e s: hue , s a u a i on and li gh ne ss 110 (21). Mo eo e , he speci ic c hange s a he λmáx a e a ls o o en i n e p e ed 111 inco ec ly in colo ime ic e ms. I ha s been de sc i bed ha he h y pe c h o mic 112 and ba hoc omic e ec s make an ho cy an i n s s o l u i on s appea b l ue and wi h 113 mo e in ense colo (3, 22-23). H o w e e , he a i a i on i n a si ng l e w a e l eng h 114 does no explain he comple e b eha i o o he c o l o due o h is pheno m enon . 115 Fo hese easons, o ad an c e i n he k no wl edge o he g l oba l c o l o im e ic 116 ole o copigmen a ion is necess a y o c on si de bo h quan i a i e and qua li a i e 117 colo changes. In his con ex , T is im u l u s C o l o im e y, w h ic h is ba s ed on 118 ansmi ance alues o he whol e s pe c a , ep e s en s a u s e u l m e hodo ly ha 119 widely imp o es he objec i e an a lysis o c o l o . 120 Thus, h ough T is imul u s C o l o im e y, i n h is s ud y is pe o m ed a 121 p ecise colo ime ic in e p e a i on o he c op i g m en a i on pheno m enon u si ng he ACS P a a gon Plu s En i onm e n 5 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y P a g e 6 o 36 122 uni o m 1976-(L * a * b * ) colo spac e (CIE L AB), w h ic h ha s been e c o mm ended b y 123 he CIE (Commission In e na io na l e de l ’ Écl a i age ) a s a m o e app op i a e oo l 124 o colo speci ica ion in mos i ndu s i a l app lic a i on s. F o h is pu po s e , d i e s e 125 colo ime ic a iables based bo h on he sc a l a ( L* , a* , b* ) and angu l a ( L* , C * ab, 126 hab) colo coo dina es has b een de i ned o a ss e ss he quan i a i e and 127 quali a i e colo implica ions ass o ci a ed o c op i g m en a i on , and he i i n ci den c e 128 on isual pe cep ion. These c o l o im e ic a i ab l e s ha e been app li ed and 129 compa ed wi h he mos used sim p li i ed m e hod i n he e a l ua i on o he 130 copigmen a ion e ec s in model s o l u i on s. 131 132 MATERIALS AND METHODS 133 134 S anda ds and copigmen ed m od e l s s olu ion s 135 The pigmen mal idin 3-gl u c o si de (M 3 - g l) w a s is o l a ed i n he l abo a o y 136 om skins o Vi is ini e a ed g ape s o T e m p an ill o a i e y. Ex a c i on w a s 137 made wi h acidic me hanol (me hano l: HCl 1 N; 95 : 5 / ) and he e x a c s w e e 138 pu i ica ed by semip essu e li qu i d c h o m a og aph y u si ng a e e s ed - pha s e 139 column, as desc ibed by He ed i a , F an ci a -A ic ha , Ri a s-G on z a l o , Vic a i o & 140 San os-Buelga, 1998. The copigm en s (+)-c a e c h i n (CAT) and (-) - ep ic a e c h i n 141 (EPI) and ca eic acid (CAF) w e e pu c ha s ed o m Si g m a C he mic a l C o . (S . 142 Louis, MO). 143 All he model solu ions we e p epa ed i n a wi ne -lik e m ed i u m c on a i n i ng 5 144 g/L a a ic acid in 12% e hanol and i on ic s eng h ad j u s ed o 0 . 2 M b y add i i ng 145 sodium chlo ide. 146 In o de o e alua e h e e e c o he p H on he c op i g m en a i on 147 phenomenon, h ee copigmen e d s o l u i on s o m a l i d i n 3 - g l:(+)-c a e c h i n (MC), ACS P a a gon Plu s En i onm e n 6 P a g e 7 o 36 148 149 150 151 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y mal idin 3-gl:(-)-epica echin (ME), and m a l i d i n 3g l: c a e ic a ci d (MF), a s w e ll a s a e e ence solu ion (M 3-gl) w e e p epa ed i n wi ne -lik e m ed i u m a d i e en p H alues: 1.0, 2.0, 3.0, 4.0, and 5 . 0 . T he e e en c e s o l u i on c on a i ned 200 m g / L (0.41 mM) o M 3-gl. Copigmen ed s o l u i on s c on a i ned he s a m e an ho cy an i n 152 concen a ion and he co esp ond i ng c op i g m en u si ng a p i g m en /c op i g m en 153 mola a io o 1:5. 154 The e ec o he copigm en c on c en a i on w a s a ls o a ss e ss ed . Tw o 155 copigmen ed solu ions o mal i d i n 3 - g l:(+)-c a e c h i n (MC) and m a l i d i n 3 - g l:(-)- 156 epica echin (ME), and a e e enc e s o l u i on w e e p epa ed i n he s a m e wi ne -lik e 157 medium adjus ed o pH 3.60. C op i g m en ed s o l u i on s c on a i ned he s a m e 158 an hocyanin concen a ion (0.41mM) and he c o e s pond i ng c op i g m en s o g i e 159 he equi ed pigmen /copigmen m o l a a i o : 1 : 1 , 1 : 2 , 1 : 5 , and 1 : 7 . 160 All o he solu ions we e p epa ed i n i p lic a e and equ ili b a ed o ea c h 161 he equilib ium o 2 hou s, s o ed cl o s ed i n da k ne ss a 25 °C, a e w h ic h he i 162 abso p ion spec a we e eco de d . 163 Colo analysis 164 The abso p ion spec a ( 380 - 770 n m) o a ll he m ode l s o l u i on s w e e 165 eco ded a cons an in e als (Δλ= 2 n m) wi h a H e wl e - P a ck a d UV- is 166 HP8452 spec opho ome e (Pal o Al o , CA), u si ng 2 mm pa h l eng h g l a ss c e lls 167 and dis illed wa e as a e e enc e . T he CIE L AB pa a m e e s ( L* , a* , b* , C * ab, and 168 hab) we e de e mined by usin g he C o m aLab s o w a e ( 24 ), o ll o wi ng he 169 Commission In e na ional de L’ Ecl a i age’ s e c o mm enda i on s ( 25 ): he 10 º 170 S anda d Obse e and he S an da d Ill u mi nan D 65 . 171 The L * alue is he e ical a xis and de i ne s he li gh ne ss, he p ope y 172 acco ding o which each colo ca n be c on si de ed a s equ i a l en o a m e m be o ACS P a a gon Plu s En i onm e n 11 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y P a g e 12 o 36 272 E0) / E0 ] * 100. The esul s showe d ha c op i g m en a i on o cc u ed o m p H a l ue s 273 close o 1 up o neu ali y o all he pheno lics u s ed a s c op i g m en s. H o w e e , 274 he magni ude o he copigmen a i on and i s c o l o im e ic e e c w a s s ong ly p H- 275 dependen and in luenced by h e na u e o he c op i g m en u s ed . T he g ea e s 276 magni ude o copigmen a ion was ob a i ned a p H 3 . 0 , be i ng si gn i ic an ly h i ghe 277 wi h (-)-epica echin and ca eic a ci d . T he y bo h i n c ea s ed e s pe c i e ly he o a l 278 colo o M 3-gl by 13.70% and 13 . 80% , w h il e (+)-c a e c h i n , w h ic h w a s he l e ss 279 e ec i e copigmen , only each ed an i n c ea s e o 9 . 30% . T h is ag ee wi h o he 280 s udies ha ha e shown ha am ong he d i e en l a an - 3 - o ls c op i g m en s, he 281 mo e plana molecules as (-)-epic a e c h i n , o wi h e l e c on - dono s ub s i uen s a s 282 cinnamic acids can be e s a ck wi h an ho cy an i n s e s u l i ng i n h i ghe 283 copigmen a ion e ec (11, 28 - 30 ). T he sli gh ly l o w e a l ue s o he 284 copigmen a ion magni ude ob ai ned i n h is s ud y e s pe c i ng o ho s e epo ed i n 285 he li e a u e could be due bo h o he l o w e p i g m en /c op i g m en m o l a a i o u s ed 286 and he chemical na u e o he e s ed c op i g m en s. I ha s been e s ab lis hed ha 287 he di e ences in he numbe , h e siz e o he s pa i a l l o c a i on o he s ub s i uen s 288 make monome ic la an-3-ols as c a e c h i n , ep ic a e c h i n o sim p l e pheno lic a ci d s, 289 exhibi weake copigmen a ion han m o e p l ana pheno lic c o m pound s a s 290 la onols (31). 291 As shown in Table 1, h e CIE L AB d i e en c e s (E * ab,  L* , C * ab,  h ab) 292 be ween M 3-gl solu ions wi h and wi hou c op i g m en s w e e a ls o c a lc u l a ed . 293 Along he pH a ia ion, he highes c o l o d i e en c e s (E * ab) w e e p odu c ed o m 294 pH 1.0 o pH 3.0, con i ming h e c o l o im e ic s ab iliz a i on o he l a yli u m i on a 295 lowe han a highe pH alues (Y an e a l., 2011 ). S pe ci ic a lly, he c o l o 296 di e ences inc eased om 1.73 o 3 . 06 , o m 2 . 88 o 3 . 69 and o m 3 . 39 o 3 . 87 ACS P a a gon Plu s En i onm e n 12 P a g e 13 o 36 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y CIELAB uni s, in M 3-gl solu ion c op i g m en ed wi h (+)-c a e c h i n , (-)- ep ic a e c h i n and ca eic acid, espec i ely. Th e e o e , a l hough l a an - 3 - o ls ha s been u s ua lly conside ed poo e an hocyanin c op i g m en s, a m o s a ci d ic p H a l ue s, he c o l o changes induced we e always p e c ep i b l e b y he hu m an e y e ( 32 ). H o w e e , a pH 4.0 and 5.0, he e was no si gn i ic an d i e en c e be w een he c o l o e e c induced by he la anols es ed, and a ll he c o l o d i e en c e s c a lc u l a ed w e e smalle han 2 CIELAB uni s, ha is, no is ua lly d is i ngu is hed . In gene al, ca eic acid p odu c ed he l a ge s c o l o e e c s, w h ic h w a s cohe en wi h he esul s ob ain ed abou he m agn i ude o c op i g m en a i on w hen i was assessed by T is imulus C o l o im e y. O n he c on a y, a s c an be c he ck ed in Table 1, a some pH alues, w h il e c o l o c hange s w e e de e c ed i n he CIELAB colo space, no chan ge s a A520 n m w a s ob s e ed . T h is d i e gen c e be ween bo h analy ical me hods ha s been p e i ou sly epo ed b y G on z á l e z- Manzano (14) and con i ms ha he m o s sim p li i ed m e hod s no a lw a ys c an achie e a eliable e alua ion o h e c op i g m en a i on p o c e ss. Wi h espec o he indi id ua l c o l o a i bu e s, ega d l e ss o p H a l ue and he copigmen used, copigmen a i on i ndu c ed si gn i ic an c o l o im e ic c hange s consis ing in a dec ease o ligh ne ss ( L* ) and hue ( h ab), a s w e ll a s an i n c ea s e o ch oma (C * ab). The ligh ness and c h o m a d i e en c e s ( L* and C * ab) w e e mo e p onounced be ween pH 2 . 0 and 3 . 0 , w h il e he l a ge s c hange s on he hue (hab) we e p oduced a hi ghe p H a l ue s. T h is i nd i ng m ean s ha i n quan i a i ely e ms, ac oss he d i e en p H a l ue s s ud i ed , he o i g i na l M 3 - g l con ol solu ion change owa d d a k e and m o e i n en s e c o l o due s pe ci ic a lly o he copigmen a ion phenomen on . H o w e e , i n qua li a i e ly e ms, due o he no able in luence o he pH on he an ho cy an i n c o l o , he end o he hue ACS P a a gon Plu s En i onm e n 13 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y P a g e 14 o 36 322 changes we e mo e a iable. F o m p H 1 . 0 o 4 . 0 , M 3 - g l c on o l s o l u i on 323 dec eased owa ds mo e bluish hue s w h il e de c ea s ed o w a d s m o e edd is h a 324 pH 5.0, as can be obse ed i n Fi gu e 2 . T he a c ha hue s d i e en c e s a e 325 almos indisce nible in he (a * b*) d i ag a m a p H 4 . 0 and 5 . 0 c an be e x p l a i ned 326 based on he impo an deg a da i on o he c o l o ed o ms i n he an ho cy an i n 327 equilib ium a highe pH alu e s, w h ic h m ade M 3 - g l s o l u i on p a c ic a lly 328 ach oma ic. 329 Fo a mo e comp ehensi e ana lysis o he c o l o im e ic im p lic a i on o 330 copigmen a ion p ocess as a un c i on o he p H, he e l a i e c on i bu i on o 331 ligh ness (%L), ch oma (%C), and hue ( % H) o he o a l c o l o d i e en c e o 332 each pigmen /copigmen solu i on w e e a ls o c a lc u l a ed . As c an be s een i n 333 Figu e 3, he main con ibu ion o he m ea s u ed c o l o d i e en c e s ΔE * ab a p H 334 1.0 was quali a i e, which was e i den c ed b y he si gn i ic an h i ghe c on i bu i on 335 o hue %ΔH (80-88%) wi h es pe c o he li gh ne ss %  L o c h o m a % C ( 7 - 336 10% and 6-10%, espec i ely). T he s e e s u l s a e c ohe en si n c e a p H  2 , he 337 an hocyanin exis s p ima ily in he o m o he ed c a i on l a yli u m and he 338 amoun o he o he colo less s pe ci e s is a lm o s i ne xis en . T he e o e , a h is p H 339 alue, he copigmen ed complex e s c an i ndu c e ba sic a lly c o l o a i a i on bu c an 340 no p o ide impo an displaceme n o he an ho cy n i n equ ili b i u m o w a d he ed 341 colo ed la ylium ca ion, which is a ls o e l e c ed b y he l o w e a i a i on s ob a i ned 342 o E and E * ab. On he o he hand , be w een p H 1 . 0 and p H 4 . 0 , he m a i n 343 colo ime ic con ibu ion was quan i a i e ; be i ng pa ic u l a ly he c h o m a 344 modi ica ions %C mo e ma k ed han li gh ne ss %  L ( 35 - 65% and 22 - 38% , 345 espec i ely). In his pH ange, he p opo i on o he o he c o l o l e ss s pe ci e s 346 inc ease coexis ing wi h he ed c o l o ed l a yli u m c a i on i n s o l u i on , s o ACS P a a gon Plu s En i onm e n 14 P a g e 15 o 36 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y copigmen a ion complexes can p o i de a he s a m e im e c o l o s ab iliz a i on and a ia ion, which coincides wi h he g ea e s c hange s on he E and E * ab a l ue s. F om pH 5.0 o neu ali y, he m o s abundan c o l o ed s pe ci e s p e s en is he quinoidal o m and hus M 3-gl i s e l doe s no c on e m u c h c o l o o a s o l u i on , so again he con ibu ion o he c op i g m en a i on on c o l o is m a i n ly qua li a i e , ha is, signi ican ly due o hue ch ange s. Mo eo e , he ela i e c on i bu i on o li gh ne ss ( %  L ), c h o m a ( % C), and hue (%H) pe mi ed us o c o m pa e he c o l o im e ic e e c i ndu c ed b y he di e en la anols es ed. In g ene a l e ms, (-)- ep ic a e c h i n and c a e ic a ci d p oduced simila e ec s on he o a l c o l o o he M 3 - g l a c o ss o he p H changes, con ibu ing mo e ma k ed wi h quan i a i e c hange s han qua li a i e ones (%L + %C= 55-63%, an d % ΔH= 45 - 37% ). O n he c on a y, pa ic u l a ly a he highe pH alues, he quali a i e c on i bu i on w a s m o e im po an i n (+)- ca echin copigmen ed solu ions. T hu s, i c an be s a i d ha he c op i g m en a i on o (-)-epica echin and ca eic acid p o i de no ab ly c o l o s ab iliz a i on and a i a i on , while (+)-ca echin p o ide mo e i n en s e c o l o a i a i on han s ab iliz a i on . 363 364 E ec o he mola a io on copig me n a ion 365 The in luence o he pigm en /c op i g m en m o l a a i o w a s a ls o a ss a y ed a 366 pH 3.60. Fo his pu pose, w o c op i g m en ed s o l u i on s o m a l i d i n 3 - g l:(+)- 367 ca echin (MC) and mal idin 3-gl:(-)- ep ic a e c h i n (ME) w e e p epa ed i n he s a m e 368 wine-like medium a he mola a i o s 1 : 1 , 1 : 2 , 1 : 5 , and 1 : 7 . 369 The mean alues o he o a l c o l o (E) o he o i g i na l M 3 - g l s o l u i on and 370 each copigmen ed solu ion as a un c i on o he m o l a a i o is p e s en ed i n T ab l e 371 2. Fo bo h assays, he posi i e e e c o he c op i g m en a i on on he M 3 - g l o a l ACS P a a gon Plu s En i onm e n 15 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y P a g e 16 o 36 372 colo was inc eased wi h inc ea si ng c op i g m en c on c en a i on , w h ic h is i n 373 ag eemen wi h p e ious s udi e s epo ed i n b i b li og aph y de s p i e be i ng l o w e 374 he ange o he pigmen /copi g m en m o l a a i o u s ed ( 33 ). T he m a xim u m 375 displacemen o hyd a ion/dehy d a i on equ ili b i u m o w a d s he c o l o ed s pe ci e s 376 was achie ed using he highes m o l a a i o ( 1 : 7 ) si n c e i c au s ed he g ea e s 377 o al colo inc eases ( om 49.26 o 57 . 32 and 57 . 90 CIE L AB u . i n MC and ME 378 solu ions, espec i ely). 379 Mo eo e , signi ican di e en c e s on he L* , a* , and b* a l ue s a m ong he 380 pu e M 3-gl solu ion and i s co e s pond i ng c op i g m en ed w e e ound , e s pe ci a lly 381 a mola a ios highe han 1:2, e s u l i ng i n a d i e en l o c a i on o he s a m p l e s i n 382 he (a*b * ) colo ime ic diag am (Fi gu e 4 ). I n ab s en c e o an y c op i g m en s, he 383 o iginal an hocyanin colo ook p o si i e a* and b* a l ue s and appea ed l o c a ed 384 nea 0º, ha is, in a well de ined edd is h c o l o a ea ( L* = 69 . 71 , a* = 38 . 84 , and 385 b*= 0.39 CIELAB u.). Howe e , wi h he i n c ea si ng add i i on o he l a ano l 386 copigmen s, he samples a ppea ed l o c a ed p og e ssi e ly a e o he 387 coo dina es o igin and expe im en ed a e m a k ab l e e o l u i on o m he i s 388 owa d he ou h quad an , ha is, o m he edd is h o he pu p l e o ed - b l u is h 389 colo s egion. The beha io o he c o l o pa a m e e s a s a un c i on o he 390 concen a ion was simila o b o h he c op i g m en u s ed . A he h i ghe s m o l a 391 a io (1:7), copigmen ed samples ea c hed he l o w e s L* and b* a l ue s bu he 392 highes a * alues, and hus, hey e x h i b i ed he da k e s and m o s i i d b l u is h 393 colo (L* = 64.18 and 63.05, a* = 45 . 44 and 43 . 76 , and b* = - 2 . 23 and - 2 . 21 394 CIELAB u. o MC and ME solu i on s, e s pe c i e ly). 395 Rega ding he changes o n he c on i bu i on o he c op i g m en a i on o he 396 o al an hocyanin colo , he adde d c op i g m en s c au s ed si gn i ic an c on c en a i on - ACS P a a gon Plu s En i onm e n 16 P a g e 17 o 36 397 398 399 400 401 402 403 404 405 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y dependen inc eases on he ma gn i ude o he e e c , a s c an be s een i n Fi gu e 2 . F om 1:1 o 1:7 mola a io, (+)-c a e c h i n i ndu c ed an i n c ea s e o he M 3 - g l o a l colo om 1.2% o 16.0%, whil e wi h (-)- ep ic a e c h i n , he i n c ea s e w a s o m 3.9% o 17.9%. No wi hs andin g , a l hough he pa e n e o l u i on w a s simil a be ween bo h la anols copigm en s, on c e aga i n , he ab ili y o he (-)- ep ic a e c h i n o ac as M -3gl copigmen s was s onge han o (+)-c a e c h i n si n c e si gn i ic an highes alues o copigmen a i on w a s a c h i e ed o m o s o he m o l a a i o s es ed. As was made in he p e i ou s s e c i on o h is s ud y, he dependen c e 406 be ween he ampli ude o he c o l o e e c i ndu c ed b y c op i g m en a i on w a s 407 e alua ed by means o he CIE L AB c o l o d i e en c e s (E * ab) (T ab l e 2 ). As 408 expec ed, he p og essi e o m a i on o c op i g m en a i on c o m p l e x e s w a s 409 con i med by a successi e i n c ea s e on he c o l o d i e en c e s be w een 410 an hocyanins and la anols s o l u i on s w hen c op i g m en c on c en a i on s w e e 411 inc easingly added. Fo all he c on c en a i on l e e l e s ed , (-)- ep ic a e c h i n c au s ed 412 always signi ican inc eases (p  0 . 05 ) on he c o l o d i e en c e s. I n c on a s , 413 highe inc eases on he mola a i o w e e ne c e ss a y o i ndu c e si gn i ic a i e 414 changes wi h (+)-ca echin copigm en ed s o l u i on s. 415 In any case, calcula ion o he sim p l e eg e ssi on c oe ici en s be w een 416 o al colo di e ences and he c op i g m en c on c en a i on o ea c h c op i g m en ed 417 solu ion con i med ha he ela i on s h i p w a s si gn i ic an ly h i gh ega d i ng he 418 copigmen used ( * alues an ged o m + 0 . 95 o + 0 . 98 un i s, p < 0 . 05 ). T he 419 lowes colo di e ences we e ound be w een pu e M 3 - g l s o l u i on and he 420 copigmen ed ones a he lowes m o l a a i o u s ed ( 1 : 1 ), a ki ng a l ue s o 1 . 01 421 and 2.27 CIELAB u. wi h (+)-c a e c h i n and (-)- ep ic a e c h i n , e s pe c i e ly. ACS P a a gon Plu s En i onm e n 17 P a g e 23 o 36 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y 532 8. B ouilla d, R.; Mazza, G.; S aad , Z.; Al b e c h -G a y, A.M.; C he mi na , A. T he 533 copigmen a ion eac i on o an ho cy an i n s: A mic op obe o he 534 s uc u al s udy o a queou s s o l u i on s. J ou na l o he Am e ic an 535 Chemical Socie y. 1989, 111 , 2604 - 2610 . 536 9. Da ies, A.J.; Mazza, G. C op i g m en a i on o sim p l e and a cyl a ed 537 an hocyanins wi h colo l e ss pheno lic c o m pound s. J. A g ic. F ood C he m. 538 1993, 41, 716-720. 539 10. Ba anac, J.M.; Pe ano ic, N.A.; Dimi ic-M a k o ic, J.M. 540 Spec opho ome ic S ud y o A n ho cy an C op i g m en a i on R ea c i on s. 2 . 541 Mal in and he Nonglyc o si d iz ed Fl a one Q ue c e i n . J. A g ic. F ood 542 Chem. 1997, 45, 1694-1 697 . 543 11. Ei o, M.J.; Heinonen, M. A n ho cy an i n C o l o B eha i o and S ab ili y du i ng 544 S o age:Çë E ec o I n e m o l e c u l a C op i g m en a i on . J. A g ic. F ood 545 Chem. 2002, 50, 7461-7 466 . 546 12. Talco , S.T.; B enes, C.H.; Pi e s, D.M.; D e l P o z o -I n s an , D. 547 Phy ochemical S abili y and C o l o R e en i on o C op i g m en ed and 548 P ocessed Muscadine G ape J u ic e . J. A g ic. F ood C he m. 2003 , 51 , 549 957-963. 550 13. G is, E.; Fe ei a, E.; Falc ao , L .; B o d i gnon - Lu iz, M. I n l uen c e o e u lic 551 acid on s abili y o an hocy an i n s o m C abe ne S au i gnon g ape s i n a 552 model sys em and a y ogu sys e m. I n e na i ona l j ou na l o ood 553 science and echnology. 2007 , 42 , 992 - 998 . ACS P a a gon Plu s En i onm e n 23 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y P a g e 24 o 36 554 14. González-Manzano, S.; D ueña s, M.; Ri a s-G on z a l o , J.C.; Esc i bano - 555 Bailón, M.T.; San os-B ue l ga , C. S ud i e s on he c op i g m en a i on 556 be ween an hocyanins and l a an - 3 - o ls and he i i n l uen c e i n he c o l ou 557 exp ession o ed wine. F ood C he mis y. 2009 , 114 , 649 - 656 . 558 15. Lambe , S.G.; Asens o e , R.E.; Willi a ms on , N.M.; Il and , P.G.; J one s, 559 G.P. Copigmen a ion b e w een m a l i d i n - 3 - g l u c o si de and s o m e wi ne 560 cons i uen s and i s imp o an c e o c o l ou e x p e ssi on i n ed wi ne . F ood 561 Chemis y. 2011, 125, 106 - 115 . 562 16. He mosín-Gu ié ez, I. In l uen c e o E hano l C on en on he Ex en o 563 Copigmen a ion in a C en ci be l Y oung R ed Wi ne . J. A g ic. F ood C he m. 564 2003, 51, 4079-4083. 565 17. Gu ié ez, I.H.; Lo enzo, E.S.-P.; Es p i no s a , A.V. P heno lic c o m po si i on and 566 magni ude o copigme n a i on i n y oung and s ho ly aged ed wi ne s 567 made om he cul i a s, C abe ne S au i gnon , C en ci be l, and Sy ah . 568 Food Chemis y. 2005, 92 , 269 - 283 . 569 18. Da ias-Ma ín, J.; Ca illo-L ópe z, M.; Ec ha a i-G anado , J.F.; í a z-R o m e o , 570 C. The magni ude o co p i g m en a i on i n he c o l ou o aged ed wi ne s 571 made in he Cana y Isl and s. E u opean F ood R e s ea c h and 572 Technology. 2007, 224, 643 - 648 . 573 19. Guadalupe, Z.; Ayes a án, B. E e c o C o mm e ci a l M annop o e i n A dd i i on 574 on Polysaccha ide, Poly pheno lic, and C o l o C o m po si i on i n R ed Wi ne s. 575 J. Ag ic. Food Chem. 20 08 , 56 , 9022 - 9029 . ACS P a a gon Plu s En i onm e n 24 P a g e 25 o 36 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y 576 20. Go dillo, B.; López-In an e, M.I.; R a mí e z-P e ¦ ü e z, P.; G on z á l e z-Mi e , 577 M.L.; He edia, F.J. In lue n c e o P e e m en a i e C o l d M a c e a i on on he 578 Colo and An hocyanic C op i g m en a i on o O gan ic T e m p an ill o Wi ne s 579 Elabo a ed in a Wa m Clim a e . J. A g ic. F ood C he m. 2010 , 58 , 6797 - 580 6803. 581 21. Gonne , J.F. Colou e ec s o c o - p i g m en a i on o an ho cy an i n s e isi ed - 582 1. A colo ime ic de ini i on u si ng he CIE L AB sc a l e . F ood C he mis y. 583 1998, 63, 409-415. 584 22. Asen, S.; S ewa , R.N.; N o is, K.H. C o - p i g m en a i on o an ho cy an i n s i n 585 plan issues and i s e e c on c o l o . P h y o c he mis y. 1972 , 11 , 1139 - 586 1144. 587 23. Dangles, O.; B ouilla d, R. A s pe c o sc op ic m e hod ba s ed on he 588 an hocyanin copigmen a i on i n e a c i on and app li ed o he quan i a i e 589 s udy o molecula compl e x e s. J ou na l o he C he mic a l S o ci e y, P e ki n 590 T ansac ions 2. 1992, 247 - 257 . 591 24. He edia, F.J.; Ál a ez, C.; G on z á l e z-Mi e , M. L .; & R a mí e z, A. C o m aLab , 592 análisis de colo . Regis o G ene a l de l a P op i edad I n e l e c ua l. 2004 . 593 25. CIE Technical Repo C o l o im e y; C o mmissi on I n e na i ona l e de 594 l'Eclai age Cen al Bu e au : Vi enna , A u s i a , 2004 . 595 26. Melgosa, M.; Hi a, E.; Poza, A.J.; Alm an , D.H.; B e n s, R.S. S up a h e s ho l d 596 colo -di e ence ellipsoi d s o s u a c e c o l o s. C o l. R e s. A p l. 1997 , 22 , 597 148-155. ACS P a a gon Plu s En i onm e n 25 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y P a g e 26 o 36 598 27. He edia, F.J.; F ancia-A ic ha , E.M.; Ri a s-G on z a l o , J.C.; Vic a i o , I.M.; 599 San os-Buelga, C. Ch o m a ic c ha a c e iz a i on o an ho cy an i n s o m ed 600 g apes - I. pH e ec . Fo od C he mis y. 1998 , 63 , 491 - 498 . 601 28. Da ias-Ma ín, J.; Ma ín-L u is, B.; C a ill o - Lópe z, M.; La m ue l a -R a en ó ¦s, 602 R.; Díaz-Rome o, C.; B ou l on , R. E e c o c a e ic a ci d on he c o l o o 603 ed wine. J. Ag ic. Food C he m. 2002 , 50 , 2062 - 2067 . 604 29. Gómez-Míguez, M.; Gonz a l e z-M an z ano , S.; G on z á l e z-Mi e , M. L .; T e e s a 605 Esc ibano-Bailón, M.; H e ed i a , F.J.; S an o s-B ue l ga , C. I n l uen c e o 606 di e en phenolic copi g m en s on he c o l o o m a l i d i n 3 - g l u c o si de . J. 607 Ag ic. Food Chem. 2006, 54 , 5422 - 5429 . 608 30. C uz, L.; B ás, N.F.; Teixei a , N.; M a eu s, N.; R a m o s, M.J.; D ang l e s, O.; 609 De F ei as, V. Vinylca e c h i n d im e s a e m u c h be e c op i g m en s o 610 an hocyanins han ca ec h i n d im e p o cy an i d i n B 3 . J. A g ic. F ood C he m. 611 2010, 58, 3159-3166. 612 31. Kunsági-Ma é, S.; Szabó, K.; Nik a d j a m, M.P.; K o ll a , L . D e e mi na i on o 613 he he modynamic pa a m e e s o he c o m p l e x o m a i on be w een 614 mal idin-3-O-glucoside and po ly pheno ls. C op i g m en a i on e e c i n ed 615 wines. Jou nal o Biochemic a l and Bi oph ysic a l M e hod s. 2006 , 69 , 113 - 616 119. 617 32. Ma ínez, J.A.; Melgosa, M.; P é e z, M.M.; Hi a , E.; N egue ue l a , A.I. N o e . 618 Visual and ins umen al c o l o e a l ua i on i n ed wi ne s. F ood Sci en c e 619 and Technology In e na i ona l. 2001 , 7 , 439 - 444 . ACS P a a gon Plu s En i onm e n 26 P a g e 27 o 36 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y 620 33. Be ké, B.; De F ei as, V. A c o l o im e ic s ud y o oen i n c op i g m en ed b y 621 p ocyanidins. Jou nal o he Sci en c e o F ood and A g ic u l u e . 2007 , 87 , 622 260-265. 623 624 ACS P a a gon Plu s En i onm e n 27 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y P a g e 28 o 36 625 Table 1. Mean alues o he A 520 and he o al colo (E) ob ained o he pu e 626 an hocyanic solu ion and i s espec i e copigmen ed a each pH alue; as well as, he 627 CIELAB di e ences (E* ab , L*, C* ab , h ab ) be ween M 3-gl solu ions wi h and 628 wi hou copigmen s. 629 Pigmen pH M 3-gl Pigmen /Copigmen MC ME MF 1 0 . 86 ± 0 . 001 a 2 0. 73 ± 0 . 002 a A 520 3 0. 32 ± 0 . 001 a 4 0. 10 ± 0 . 003 a 5 0 . 06 ± 0 . 001 a 1 59 . 51 ± 0 . 98 a 2 54 . 37 ± 1 . 67 a E 3 2 9 . 13 ± 0 . 98 a 4 9 . 47 ± 1 . 67 a 5 5 . 12 ± 0 . 60 a 1 - 2 -  E* ab 3 - 4 - 5 -1 - 2 -  L* 3 - 4 - 5 -1 - 2 -  C *ab 3 - 4 - 5 -1 - 2 -  h ab 3 - 4 - 5 - 0 . 84 ± 0 . 001 b 0 . 78 ± 0 . 03 a 0 . 34 ± 0 . 001 b 0 . 10 ± 0 . 003 a 0 . 05 ± 0 . 005 a 60 . 10 ± 0 . 23 b 57 . 51 ± 1 . 77 b 31 . 85 ± 0 . 17 b 10 . 22 ± 0 . 27 b 4 . 93 ± 0 . 38 a 1 . 73 ± 0 . 06 a 2 . 92 ± 0 . 48 a 3 . 06 ± 0 . 17 a 1 . 29 ± 0 . 08 a 1 . 01 ± 0 . 14 a - 0 . 46 ± 0 . 11 a - 2 . 64 ± 0 . 23 a - 0 . 89 ± 0 . 01 a - 0 . 03 ± 0 . 20 a + 0 . 23 ± 0 . 28 a + 0 . 41 ± 0 . 21 a + 0 . 47 ± 0 . 20 a + 2 . 59 ± 0 . 18 a + 0 . 91 ± 0 . 19 a + 0 . 02 ± 0 . 06 a - 1 . 74 ± 0 . 07 a - 1 . 30 ± 0 . 75 a - 2 . 91 ± 0 . 08 a - 6 . 65 ± 0 . 65 a - 17 . 28 ± 2 . 30 a 0 . 84 ± 0 . 008 b 0 . 73 ± 0 . 002 a 0 . 36 ± 0 . 004 c 0 . 11 ± 0 . 001 b 0 . 06 ± 0 . 001 a 60 . 77 ± 0 . 26 c 55 . 83 ± 0 . 17 b 32 . 68 ± 0 . 34 c 10 . 82 ± 0 . 01 c 5 . 40 ± 0 . 12 a 2 . 88 ± 0 . 04 b 2 . 29 ± 0 . 12 a 3 . 69 ± 0 . 37 b 1 . 38 ± 0 . 02 a 0 . 54 ± 0 . 09 a - 0 . 91 ± 0 . 29 ab - 1 . 16 ± 0 . 02 a - 1 . 97 ± 0 . 20 b - 0 . 77 ± 0 . 21 b - 0 . 29 ± 0 . 10 a + 0 . 95 ± 0 . 15 b + 1 . 03 ± 0 . 19 a + 2 . 95 ± 0 . 27 a + 1 . 09 ± 0 . 01 a + 0 . 05 ± 0 . 01 a - 2 . 74 ± 0 . 20 b - 1 . 93 ± 0 . 07 a - 2 . 12 ± 0 . 29 b - 2 . 75 ± 0 . 84 b - 5 . 56 ± 0 . 92 b 0 . 83 ± 0 . 004 b 0 . 77 ± 0 . 03 a 0 . 37 ± 0 . 003 c 0 . 12 ± 0 . 00 5 b 0 . 05 ± 0 . 002 a 60 . 79 ± 0 . 16 c 57 . 88 ± 1 . 4 2 b 32 . 79 ± 0 . 2 0 c 11 . 06 ± 0 . 38 c 5 . 52 ± 0 . 47 a 3 . 39 ± 0 . 13 c 3 . 78 ± 1 . 06 a 3 . 87 ± 0 . 24 b 1 . 59 ± 0 . 27 a 0 . 71 ± 0 . 14 a - 0 . 95 ± 0 . 10 b - 2 . 27 ± 0 . 8 2 a - 2 . 14 ± 0 . 1 3 b - 1 . 03 ± 0 . 4 0 b - 0 . 20 ± 0 . 4 0 a + 0 . 94 ± 0 . 1 4 b + 2 . 77 ± 1 . 17 b + 2 . 98 ± 0 . 1 5 a + 1 . 17 ± 0 . 1 6 a + 0 . 44 ± 0 . 24 b - 3 . 35 ± 0 . 15 c - 1 . 99 ± 0 . 5 3 a - 2 . 61 ± 0 . 35 ab - 2 . 20 ± 0 . 1 7 b - 6 . 41 ± 3 . 07 b ACS P a a gon Plu s En i onm e n 28 P a g e 29 o 36 630 631 632 633 634 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y Table 2. Mean alues o he A 520 and he o al colo (E) ob ained o he pu e an hocyanic solu ion and i s espec i e copigmen ed a each mola a io; as well as, he CIELAB di e ences (E* ab , L*, C* ab , h ab ) be ween M 3-gl solu ions wi h and wi hou copigmen s. Colo ime ic pa ame e s A 520 E 0 0 . 66 ± 0 . 00 1 a 49 . 26 ± 0 . 45 a 1 0 . 68 ± 0 . 001 a 49 . 84 ± 0 . 12 a Mola Ra io 2 0 . 77 ± 0 . 008 b 52 . 64 ± 0 . 58 b 5 0 . 77 ± 0 . 008 b 54 . 40 ± 0 . 50 b 7 0 . 84 ± 0.004 c 57 . 3 2 ± 0.90 c MC ME E* ab - L* - C* ab - h ab - A 520 0 . 67 ± 0 . 004 a E 49 . 26 ± 0 . 001 a E* ab - L* -C* ab - h ab 1 . 01 ± 0 . 33 a - 0 . 16 ± 0 . 54 a + 0 . 60 ± 0 . 26 a - 1 . 21 ± 0 . 60 a 0 . 72 ± 0 . 01 b 51 . 21 ± 0 . 10 b 2 . 27 ± 0 . 20 a - 1 . 77 ± 0 . 23 a + 1 . 08 ± 0 . 30 a - 1 . 02 ± 0 . 54 a 4 . 89 ± 0 . 37 b - 4 . 51 ± 0 . 33 b + 0 . 65 ± 1 . 06 a - 2 . 56 ± 0 . 99 a 0 . 76 ± 0 . 004 c 52 . 51 ± 0 . 28 c 4 . 53 ± 0 . 91 b - 3 . 99 ± 1 . 12 b + 0 . 90 ± 0 . 95 a - 2 . 39 ± 0 . 83 ab 6 . 36 ± 0 . 19 b - 3 . 95 ± 0 . 66 b + 3 . 40 ± 1 . 17 a - 5 . 05 ± 0 . 13 b 0 . 78 ± 0 . 008 d 55 . 50 ± 0 . 38 d 6 . 65 ± 0 . 33 c - 4 . 16 ± 0 . 37 b + 4 . 67 ± 0 . 34 b - 3 . 11 ± 0 . 47 b 9 . 7 6 ± 1.27 c - 8 . 56 ± 0.99 c + 2 . 5 9 ± 3.60 a - 5 . 1 2 ± 0.13 b 0 . 82 ± 0.002 e 57 . 90 ± 0.36 e 10 . 69 ± 0.44 d - 5 . 5 3 ± 0.24 b + 6 . 64 ± 0.63 c - 3 . 3 8 ± 0.50 b ACS P a a gon Plu s En i onm e n 29 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y P a g e 30 o 36 635 FIGURE CAPTIONS 636 Figu e 1. Change in he loca io n o he pu e M 3 - g l and c op i g m en ed s o l u i on s 637 (MC, ME, and MF) wi hin (a) he ( a*b* ) d i ag a m and ( b ) li gh ne ss a l ue s ( L* ) a s 638 a unc ion o he pH alue. 639 640 Figu e 2. (a) Changes on he m agn i ude o c op i g m en a i on o he M 3 - g l 641 solu ions copigmen ed wi h (+)-c a e c h i n (MC), (-)- ep ic a e c h i n (ME), and c a e ic 642 acid (MF) as a unc ion o he pH, and ( b ) wi h (+)-c a e c h i n (MC), (-)- ep ic a e c h i n 643 (ME) as a unc ion o he mola a i o , e a l ua ed b y T is im u l u s C o l o im e y a s 644 he a io [(EC- E0) / E0 ] * 100. 645 646 Figu e 3. Rela i e con ibu ion o li gh ne ss ( %  L ), c h o m a ( % C), and hue 647 (%H) o he o al colo di e enc e o ea c h p i g m en /c op i g m en a s a un c i on o 648 he pH alue. 649 650 Figu e 4. Change in he loca io n o he pu e M 3 - g l and c op i g m en ed s o l u i on s 651 (MC, ME) wi hin (a) he (a * b * ) d i ag a m and ( b ) li gh ne ss a l ue s ( L* ) a s a 652 unc ion o he mola a io. 653 654 Figu e 5. CIELAB hue di e en c e s ( h ab) a s a un c i on o m o l a a i o o 655 co esponding o M 3-gl: CAT and M 3 - g l: EPI s o l u i on s. 656 657 Figu e 6. Rela i e con ibu ion o li gh ne ss ( %  L ), c h o m a ( % C), and hue 658 (%H) o he o al colo di e enc e o ea c h p i g m en /c op i g m en a s a un c i on o 659 he mola a io. ACS P a a gon Plu s En i onm e n 30 ACS P a a gon Plu s En i onm e n 34  P a g e 35 o 36 J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y Figu e 5. 2 1 0 -1 M 3-gl : CAT M 3-gl : EP I  h ab = 0.1158 -1.8512*MR + 0.1595*MR 2 (CAT)  h ab = 0.0229 - 1.1267*MR + 0.0956*MR 2 (EPI) -2 -3 -4 -5 -6 1 : 0 1 : 1 1 : 2 1 : 5 1 : 7 Mola Ra io (MR) ACS P a a gon Plu s En i onm e n 35 Ea J ou n a l o Ag i c ul u a l a nd Food Ch e mi s y P a g e 36 o 36 Figu e 6. a ) MC % %C %L 100 75 50 25 0 1:1 1:2 1:5 1:7 Mola a io pigmen /copigmen b ) ME %  %  C %  L 100 75 50 25 0 1 : 1 1 : 2 1 : 5 1 : 7 Mola Ra io pigmen / copigmen ACS P a a gon Plu s En i onm e n 36