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

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ "This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Agricultural and Food Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/jf2046202". P a g e 1 o f 36 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y 1 A COMPREHENSIVE COLORIMETRIC STUDY OF ANTHOCYANIC 2 COPIGMENTATION IN MODEL SOLUTIONS. EFECTS OF pH AND MOLAR 3 RATIO 4 5 Belén Go r dillo, F r ancisco J. Rod r ígu e z-Pulido, M. Lui sa E sc ud er o-Gil e t e , 6 M. Loudes González-Mi r et, F ra n c i sc o J . H ere di a* 7 Food Colour & Quality Lab., D ep t. N u triti on & F ood Sci en c e . F a c u lt ad de 8 Farmacia. Universidad de Sevill a . 41012 -S e vill a , S pa i n . 9 10 11 Abbreviated r unning title: 12 Color and copigmentation of ant ho cy an i n s 13 14 15 * Corresponding autho r : 16 Francisco J. Heredia 17 Food Colour & Quality Lab., D ep t. N u triti on & F ood Sci en c e . F a c u lt ad de 18 Farmacia. Universidad de Sevill a . 41012 -S e vill a , S pa i n 19 Tel.: +34 954556495 Fax: + 34 954557017 20 e-mail: [email protected] 21 22 23 24 25 ACS P a r a gon Plu s En v ironm e n t 1 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y P a g e 2 of 36 26 Abstract 27 New colorimetric variabl e s ha v e been de fi ned i n t he un if o rm CIE L AB 28 color space to assess the quantit a tiv e and qua lit a tiv e c o l o r c hange s i ndu c ed b y 29 copigmentation, and their incid en c e on vis ua l pe rc ep ti on . T he c op i g m en t a ti on 30 process was assayed in mod e l s o l u ti on s be tw een M a lvi d i n 3 - g l u c o si de and 31 three phenolic compounds (cat e c h i n , ep ic a t e c h i n and c a ff e ic a ci d ) a s a f un cti on 32 of the pH and the pigment/copi g m en t m o l a r r a ti o . Al ong t he p H v a ri a ti on , t he 33 greatest magnitude of copigm en t a ti on w a s ob t a i ned a t p H 3 . 0 , be i ng 34 significantly higher with epicatec h i n and c a ff e ic a ci d . At h i gh a ci d ic p H, t he m a i n 35 contribution of copigmentation t o t he t o t a l c o l o r w a s qua lit a tiv e w h il e be tw een 36 pH 2.0 and 4.0, the main c o l o rim e tric c on tri bu ti on w a s quan tit a tiv e . T he 37 contribution of epicatechin and c a ff e ic a ci d t o t he c o l o r c hange s w a s m o r e 38 marked for the quantitative char a ct e ristics. O n c on tr a st, pa rtic u l a rly a t h i ghe r p H 39 values, the qualitative contributi on w a s m o r e im po rt an t i n c a t e c h i n c op i g m en t ed 40 solutions. Increasing copigm en t c on c en tr a ti on i ndu c ed pe rc ep ti b l e c o l o r 41 changes at molar ratios higher t han 1 : 2 , c on sisti ng i n a b l u is h and da rk en i ng 42 effect of the anthocyanin solutio n s. Am ong t he d iff e r en t CIE L AB a ttri bu t e s, hue 43 differences was the best correl a t ed pa r a m e t e r wit h t he i n cr ea s e o f c op i g m en t 44 concentration, proving the relev an c e o f t h is ph ysic o c he mic a l pheno m enon on 45 the qualitative changes of anthocy an i n c o l o r. 46 47 Keywo r ds: Anthocyanin; color; c op i g m en t a ti on ; Tristim u l u s C o l o rim e try ACS P a r a gon Plu s En v ironm e n t 2 P a g e 3 o f 36 48 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y INTRODUCTION 49 Anthocyanins are the pigm en ts a cc oun ti ng f o r t he b rilli an t r ed , pu r p l e , 50 and blue colors in many fruits, v ege t ab l e s, and de riv ed f ood p r odu cts s u c h a s 51 fruits juices, jams, and red wines ( 1 ). 52 It is well known that th e st ab ility o f an t ho cy an ic p i g m en ts is g r ea tly 53 limited because their resonatin g str u ct u r e c on f e rs t he m i n tri n sic i n st ab ility 54 against several physical-chemical c ond iti on s. Ev a l ua ti on o f t he f a ct o rs a ff e cti ng 55 the stability of anthocyanins in d ic a t e s t ha t p H is t he m o st im po rt an t e xtri n sic 56 factor of anthocyanin degradati on . D epend i ng on t he p H o f t he m ed i u m, t he r ed 57 colored flavylium cation coexists a s an equ ili b ri u m mixt u r e wit h o t he r f o rms o f 58 anthocyanins: the blue-purple q u i nono i da l ba s e s, t he c o l o rl e ss he mi a c e t a l B, 59 and the pale yellow chalcones. T he r e f o r e , t he s a m e an t ho cy an i n s o l u ti on m a y 60 show different colors (2). 61 However, the chemical an d c o l o rim e tric st ab ility o f an t ho cy an i n s c an be 62 improved by associations with ot he r s ub st an c e s. C op i g m en t a ti on pheno m enon , 63 among others, represents one of t he m o st c o m p l e x and e ffici en t m e c han isms o f 64 anthocyanin chromophore stabiliz a ti on i n na t u r e and f ood syst e ms ( 3 ). I n f ood 65 science, this phenomenon is c on si de r ed a r e l e v an t i n t e r a cti on be c au s e 66 obtaining stable and attractive c o l o rs is a m a j o r f o c u s f o r qua lity c on tr o l 67 purposes (4). Especially, in win e m a ki ng , is a ss u m ed it p l a ys a k e y r o l e on t he 68 color evolution and stability of th e y oung r ed wi ne s ( 5 ). 69 Copigmentation reactions c on sist o f non -c o v a l en t i n t e r a cti on s be tw een 70 anthocyanins among themselves (s e lfa ss o ci a ti on ) o r wit h a wi de v a ri e ty o f 71 colorless organic compounds na m ed c op i g m en ts o r c op i g m en t a ti on c o f a ct o rs ACS P a r a gon Plu s En v ironm e n t 3 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y P a g e 4 of 36 72 (basically other phenolic comp ound s, bu t a ls o a mi no a ci d s, s uga rs, o r gan ic 73 acids, polysaccharides, etc.). 74 From a molecular point of vi e w, t he an t ho cy an i n -c op i g m en ts c o m p l e x e s 75 adopt a typical sandwich co n fi gu r a ti on (- st a cki ng ) vi a h y d r ophob ic 76 interaction. This structural conform a ti on p r o t e cts t he r ed c o l o r ed fl a vyli u m c a ti on 77 against the nucleophilic attack of w a t e r, pe r o xi de and s u l phu r d i o xi de b l ea c h i ng , 78 and pH changes reducing the f o rm a ti on o f t he o t he r c o l o rl e ss s pe ci e s i n t he 79 anthocyanin equilibrium (hemiac e t a l and c ha lc one ) ( 6 ). 80 In addition, copigmentati on no t on ly c on f e rs g r ea t e r st ab ility t o 81 anthocyanins, but also induc e s c o l o r v a ri a ti on s. T h is ki nd o f m o l e c u l a r 82 association is responsible for t he ty p ic a l c hange s i n t he s pe ctr a l p r ope rti e s o f 83 the chromophore group, that is, an i n cr ea s e o f t he ab s o r p tivity and fr equen tly a 84 shift of the visible λmax tow a r d g r ea t e r w a v e l eng t h s. C on s equen tly, 85 copigmentation produces both quan tit a tiv e and qua lit a tiv e c o l o r c hange s on t he 86 anthocyanins solutions. In this se n s e , t he m ea s u r e m en t and e v a l ua ti on o f t he s e 87 colorimetric changes is of great i n t e r e st i n f ood i ndu stry si n c e c o l o r is one o f t he 88 main sensory parameter for the qua lity o f f ood s i n fl uen ci ng c u st o m e r s e l e cti on 89 (7). 90 The contribution of the c op i g m en t a ti on pheno m enon on c o l o r ha s been 91 widely studied using spectrop ho t o m e tric m e t hod s, bo t h i n m ode l o r f ood 92 systems which contain individual an t ho cy an i n s and added c op i g m en ts; o r i n r ed 93 wines where anthocyanins nat u r a lly c o - o cc u rs wit h o t he r pheno lic c o m pound s 94 (8-15). The evaluation of the ch ange s i n t he VIS s pe ctr u m o f an t ho cy an i n s, o r 95 specifically in the λmax (520 nm), ha s pe rmitt ed t o k no w t he i n fl uen c e o f 96 numerous factors on the effectiv ene ss o f t he c op i g m en t a ti on i n cl ud i ng t he ACS P a r a gon Plu s En v ironm e n t 4 P a g e 5 o f 36 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y 97 concentrations of pigments and c o f a ct o rs, t he ir c he mic a l str u ct u r e s, t he 98 cofactor/pigment molar ratio, t he p H o f t he m ed i u m, e tc. I n t he s a m e w a y, 99 several studies in red wines hav e s ho w n t ha t t he m agn it ude o f c op i g m en t a ti on 100 and its evolution during winem a ki ng is e xtr e m e ly v a ri ab l e a cc o r d i ng t o 101 viticultural, agronomical or oen o l og ic a l p r a ctic e s a cc oun ti ng app r o xim a t e ly fr o m 102 25 to 50% to the total color of yo ung r ed wi ne s ( 5 , 16 - 20 ). 103 In any case, although sp e ctr a l m e t hod s ha v e de m on str a t ed t o be a v a li d , 104 simple and quick tool to quantit a tiv e e stim a ti on s, is gene r a lly a cc ep t ed t ha t t he y 105 provide limited precision and a cc u r a cy f o r c o l o r s pe cific a ti on s. T he l o w e r 106 precision and accuracy achiev ab l e c an be e x p l a i n be c au s e an adequa t e 107 description of the color variations r equ ir e s i) t ha t s pe ctr a l v a ri a ti on s c on si de r ed 108 should be those affecting the e n tir e s pe ctr a l c u rv e , no t on ly its visi b l e λmáx, and 109 ii) the use of at least three colorim e tric a ttri bu t e s: hue , s a t u r a ti on and li gh t ne ss 110 (21). Moreover, the specific c hange s a t t he λmáx a r e a ls o o ft en i n t e r p r e t ed 111 incorrectly in colorimetric terms. It ha s been de scri bed t ha t t he h y pe rc h r o mic 112 and bathocromic effects make an t ho cy an i n s s o l u ti on s appea r b l ue r and wit h 113 more intense color (3, 22-23). H o w e v e r, t he v a ri a ti on i n a si ng l e w a v e l eng t h 114 does not explain the complete b eha vi o r o f t he c o l o r due t o t h is pheno m enon . 115 For these reasons, to adv an c e i n t he k no wl edge o f t he g l oba l c o l o rim e tric 116 role of copigmentation is necess a ry t o c on si de r bo t h quan tit a tiv e and qua lit a tiv e 117 color changes. In this context, Tristim u l u s C o l o rim e try, w h ic h is ba s ed on 118 transmittance values of the whol e s pe ctr a , r ep r e s en ts a u s e f u l m e t hodo ly t ha t 119 widely improves the objective an a lysis o f c o l o r. 120 Thus, through Tristimul u s C o l o rim e try, i n t h is st ud y is pe rf o rm ed a 121 precise colorimetric interpretati on o f t he c op i g m en t a ti on pheno m enon u si ng t he ACS P a r a gon Plu s En v ironm e n t 5 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y P a g e 6 of 36 122 uniform 1976-(L * a * b * ) color spac e (CIE L AB), w h ic h ha s been r e c o mm ended b y 123 the CIE (Commission Internatio na l e de l ’ Écl a ir age ) a s a m o r e app r op ri a t e t oo l 124 for color specification in most i ndu stri a l app lic a ti on s. F o r t h is pu r po s e , d iv e rs e 125 colorimetric variables based both on t he sc a l a r ( L* , a* , b* ) and angu l a r ( L* , C * ab, 126 hab) color coordinates has b een de fi ned t o a ss e ss t he quan tit a tiv e and 127 qualitative color implications ass o ci a t ed t o c op i g m en t a ti on , and t he ir i n ci den c e 128 on visual perception. These c o l o rim e tric v a ri ab l e s ha v e been app li ed and 129 compared with the most used sim p lifi ed m e t hod i n t he e v a l ua ti on o f t he 130 copigmentation effects in model s o l u ti on s. 131 132 MATERIALS AND METHODS 133 134 Standards and copigmented m od e l s s olution s 135 The pigment malvidin 3-gl u c o si de (Mv 3 - g l) w a s is o l a t ed i n t he l abo r a t o ry 136 from skins of Vitis vinifera red g r ape s o f T e m p r an ill o v a ri e ty. Extr a cti on w a s 137 made with acidic methanol (met hano l: HCl 1 N; 95 : 5 v/v) and t he e xtr a cts w e r e 138 purificated by semipressure li qu i d c h r o m a t og r aph y u si ng a r e v e rs ed - pha s e 139 column, as described by Her ed i a , Fr an ci a -Aric ha , Riv a s-G on z a l o , Vic a ri o & 140 Santos-Buelga, 1998. The copigm en ts (+)-c a t e c h i n (CAT) and (-) - ep ic a t e c h i n 141 (EPI) and caffeic acid (CAF) w e r e pu rc ha s ed fr o m Si g m a C he mic a l C o . (St. 142 Louis, MO). 143 All the model solutions wer e p r epa r ed i n a wi ne -lik e m ed i u m c on t a i n i ng 5 144 g/L tartaric acid in 12% ethanol and i on ic str eng t h ad j u st ed t o 0 . 2 M b y add iti ng 145 sodium chloride. 146 In order to evaluate th e e ff e ct o f t he p H on t he c op i g m en t a ti on 147 phenomenon, three copigmente d s o l u ti on s o f m a lvi d i n 3 - g l:(+)-c a t e c h i n (MC), ACS P a r a gon Plu s En v ironm e n t 6 P a g e 7 o f 36 148 149 150 151 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y malvidin 3-gl:(-)-epicatechin (ME), and m a lvi d i n 3g l: c a ff e ic a ci d (MF), a s w e ll a s a reference solution (Mv 3-gl) w e r e p r epa r ed i n wi ne -lik e m ed i u m a t d iff e r en t p H values: 1.0, 2.0, 3.0, 4.0, and 5 . 0 . T he r e f e r en c e s o l u ti on c on t a i ned 200 m g / L (0.41 mM) of Mv 3-gl. Copigment ed s o l u ti on s c on t a i ned t he s a m e an t ho cy an i n 152 concentration and the corresp ond i ng c op i g m en t u si ng a p i g m en /c op i g m en t 153 molar ratio of 1:5. 154 The effect of the copigm en t c on c en tr a ti on w a s a ls o a ss e ss ed . Tw o 155 copigmented solutions of malvi d i n 3 - g l:(+)-c a t e c h i n (MC) and m a lvi d i n 3 - g l:(-)- 156 epicatechin (ME), and a referenc e s o l u ti on w e r e p r epa r ed i n t he s a m e wi ne -lik e 157 medium adjusted to pH 3.60. C op i g m en t ed s o l u ti on s c on t a i ned t he s a m e 158 anthocyanin concentration (0.41mM) and t he c o rr e s pond i ng c op i g m en ts t o g iv e 159 the required pigment/copigment m o l a r r a ti o : 1 : 1 , 1 : 2 , 1 : 5 , and 1 : 7 . 160 All of the solutions were p r epa r ed i n tri p lic a t e and equ ili b r a t ed t o r ea c h 161 the equilibrium for 2 hours, stor ed cl o s ed i n da rk ne ss a t 25 °C, a ft e r w h ic h t he ir 162 absorption spectra were recorde d . 163 Color analysis 164 The absorption spectra ( 380 - 770 n m) o f a ll t he m ode l s o l u ti on s w e r e 165 recorded at constant intervals (Δλ= 2 n m) wit h a H e wl e ttP a ck a r d UV-vis 166 HP8452 spectrophotometer (Pal o Alt o , CA), u si ng 2 mm pa t h l eng t h g l a ss c e lls 167 and distilled water as a referenc e . T he CIE L AB pa r a m e t e rs ( L* , a* , b* , C * ab, and 168 hab) were determined by usin g t he Cr o m aLab s o ftw a r e ( 24 ), f o ll o wi ng t he 169 Commission International de L’ Ecl a ri age’ s r e c o mm enda ti on s ( 25 ): t he 10 º 170 Standard Observer and the Stan da r d Ill u mi nan t D 65 . 171 The L * value is the vertical a xis and de fi ne s t he li gh t ne ss, t he p r ope rty 172 according to which each color ca n be c on si de r ed a s equ iv a l en t t o a m e m be r o f ACS P a r a gon Plu s En v ironm e n t 11 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y P a g e 12 of 36 272 E0) / E0 ] * 100. The results showe d t ha t c op i g m en t a ti on o cc u rr ed fr o m p H v a l ue s 273 close to 1 up to neutrality for all t he pheno lics u s ed a s c op i g m en ts. H o w e v e r, 274 the magnitude of the copigmentati on and its c o l o rim e tric e ff e ct w a s str ong ly p H275 dependent and influenced by th e na t u r e o f t he c op i g m en t u s ed . T he g r ea t e st 276 magnitude of copigmentation was ob t a i ned a t p H 3 . 0 , be i ng si gn ific an tly h i ghe r 277 with (-)-epicatechin and caffeic a ci d . T he y bo t h i n cr ea s ed r e s pe ctiv e ly t he t o t a l 278 color of Mv 3-gl by 13.70% and 13 . 80% , w h il e (+)-c a t e c h i n , w h ic h w a s t he l e ss 279 effective copigment, only reach ed an i n cr ea s e o f 9 . 30% . T h is ag r ee wit h o t he r 280 studies that have shown that am ong t he d iff e r en t fl a v an - 3 - o ls c op i g m en ts, t he 281 more planar molecules as (-)-epic a t e c h i n , o r wit h e l e ctr on - dono r s ub stit uen ts a s 282 cinnamic acids can better st a ck wit h an t ho cy an i n s r e s u lti ng i n h i ghe r 283 copigmentation effect (11, 28 - 30 ). T he sli gh tly l o w e r v a l ue s o f t he 284 copigmentation magnitude obtai ned i n t h is st ud y r e s pe cti ng t o t ho s e r epo rt ed i n 285 the literature could be due both t o t he l o w e r p i g m en t/c op i g m en t m o l a r r a ti o u s ed 286 and the chemical nature of the t e st ed c op i g m en ts. It ha s been e st ab lis hed t ha t 287 the differences in the number, th e siz e o r t he s pa ti a l l o c a ti on o f t he s ub stit uen ts 288 make monomeric flavan-3-ols as c a t e c h i n , ep ic a t e c h i n o r sim p l e pheno lic a ci d s, 289 exhibit weaker copigmentation t han m o r e p l ana r pheno lic c o m pound s a s 290 flavonols (31). 291 As shown in Table 1, th e CIE L AB d iff e r en c e s (E * ab,  L* , C * ab,  h ab) 292 between Mv 3-gl solutions with and wit hou t c op i g m en ts w e r e a ls o c a lc u l a t ed . 293 Along the pH variation, the highest c o l o r d if e r en c e s (E * ab) w e r e p r odu c ed fr o m 294 pH 1.0 to pH 3.0, confirming th e c o l o rim e tric st ab iliz a ti on o f t he fl a vyli u m i on a t 295 lower than a higher pH values (Y an e t a l., 2011 ). S pe cific a lly, t he c o l o r 296 differences increased from 1.73 t o 3 . 06 , fr o m 2 . 88 t o 3 . 69 and fr o m 3 . 39 t o 3 . 87 ACS P a r a gon Plu s En v ironm e n t 12 P a g e 13 of 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 r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y CIELAB units, in Mv 3-gl solution c op i g m en t ed wit h (+)-c a t e c h i n , (-)- ep ic a t e c h i n and caffeic acid, respectively. Th e r e f o r e , a lt hough fl a v an - 3 - o ls ha s been u s ua lly considered poorer anthocyanin c op i g m en ts, a t m o st a ci d ic p H v a l ue s, t he c o l o r changes induced were always p e rc ep ti b l e b y t he hu m an e y e ( 32 ). H o w e v e r, a t pH 4.0 and 5.0, there was no si gn ific an t d iff e r en c e be tw een t he c o l o r e ff e ct induced by the flavanols tested, and a ll t he c o l o r d iff e r en c e s c a lc u l a t ed w e r e smaller than 2 CIELAB units, that is, no t vis ua lly d isti ngu is hed . In general, caffeic acid p r odu c ed t he l a r ge st c o l o r e ff e cts, w h ic h w a s coherent with the results obtain ed abou t t he m agn it ude o f c op i g m en t a ti on w hen it was assessed by Tristimulus C o l o rim e try. O n t he c on tr a ry, a s c an be c he ck ed in Table 1, at some pH values, w h il e c o l o r c hange s w e r e de t e ct ed i n t he CIELAB color space, no chan ge s a t A520 n m w a s ob s e rv ed . T h is d iv e r gen c e between both analytical methods ha s been p r e vi ou sly r epo rt ed b y G on z á l e zManzano (14) and confirms that t he m o st sim p lifi ed m e t hod s no t a lw a ys c an achieve a reliable evaluation of th e c op i g m en t a ti on p r o c e ss. With respect to the individ ua l c o l o r a ttri bu t e s, r ega r d l e ss o f p H v a l ue and the copigment used, copigment a ti on i ndu c ed si gn ific an t c o l o rim e tric c hange s consisting in a decrease of light ne ss ( L* ) and hue ( h ab), a s w e ll a s an i n cr ea s e of chroma (C * ab). The lightness and c h r o m a d iff e r en c e s ( L* and C * ab) w e r e more pronounced between pH 2 . 0 and 3 . 0 , w h il e t he l a r ge st c hange s on t he hue (hab) were produced at hi ghe r p H v a l ue s. T h is fi nd i ng m ean s t ha t i n quantitatively terms, across the d iff e r en t p H v a l ue s st ud i ed , t he o ri g i na l Mv 3 - g l control solution change toward d a rk e r and m o r e i n t en s e c o l o r due s pe cific a lly t o the copigmentation phenomen on . H o w e v e r, i n qua lit a tiv e ly t e rms, due t o t he notable influence of the pH on t he an t ho cy an i n c o l o r, t he tr end o f t he hue ACS P a r a gon Plu s En v ironm e n t 13 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y P a g e 14 of 36 322 changes were more variable. Fr o m p H 1 . 0 t o 4 . 0 , Mv 3 - g l c on tr o l s o l u ti on 323 decreased towards more bluish hue s w h il e de cr ea s ed t o w a r d s m o r e r edd is h a t 324 pH 5.0, as can be observed i n Fi gu r e 2 . T he f a ct t ha t hue s d iff e r en c e s a r e 325 almost indiscernible in the (a * b*) d i ag r a m a t p H 4 . 0 and 5 . 0 c an be e x p l a i ned 326 based on the important degra da ti on o f t he c o l o r ed f o rms i n t he an t ho cy an i n 327 equilibrium at higher pH valu e s, w h ic h m ade Mv 3 - g l s o l u ti on p r a ctic a lly 328 achromatic. 329 For a more comprehensiv e ana lysis o f t he c o l o rim e tric im p lic a ti on o f 330 copigmentation process as a f un cti on o f t he p H, t he r e l a tiv e c on tri bu ti on o f 331 lightness (%L), chroma (%C), and hue ( % H) t o t he t o t a l c o l o r d iff e r en c e f o r 332 each pigment/copigment soluti on w e r e a ls o c a lc u l a t ed . As c an be s een i n 333 Figure 3, the main contribution t o t he m ea s u r ed c o l o r d iff e r en c e s ΔE * ab a t p H 334 1.0 was qualitative, which was e vi den c ed b y t he si gn ific an t h i ghe r c on tri bu ti on 335 of hue %ΔH (80-88%) with res pe ct t o t he li gh t ne ss %  L o r c h r o m a % C ( 7 - 336 10% and 6-10%, respectively). T he s e r e s u lts a r e c ohe r en t si n c e a t p H  2 , t he 337 anthocyanin exists primarily in t he f o rm o f t he r ed c a ti on fl a vyli u m and t he 338 amount of the other colorless s pe ci e s is a lm o st i ne xist en t. T he r e f o r e , a t t h is p H 339 value, the copigmented complex e s c an i ndu c e ba sic a lly c o l o r v a ri a ti on bu t c an 340 not provide important displaceme n t o f t he an t ho cy n i n equ ili b ri u m t o w a r d t he r ed 341 colored flavylium cation, which is a ls o r e fl e ct ed b y t he l o w e r v a ri a ti on s ob t a i ned 342 for E and E * ab. On the other hand , be tw een p H 1 . 0 and p H 4 . 0 , t he m a i n 343 colorimetric contribution was quan tit a tiv e ; be i ng pa rtic u l a rly t he c h r o m a 344 modifications %C more mark ed t han li gh t ne ss %  L ( 35 - 65% and 22 - 38% , 345 respectively). In this pH range, t he p r opo rti on o f t he o t he r c o l o rl e ss s pe ci e s 346 increase coexisting with the r ed c o l o r ed fl a vyli u m c a ti on i n s o l u ti on , s o ACS P a r a gon Plu s En v ironm e n t 14 P a g e 15 of 36 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y copigmentation complexes can pr o vi de a t t he s a m e tim e c o l o r st ab iliz a ti on and variation, which coincides with t he g r ea t e st c hange s on t he E and E * ab v a l ue s. From pH 5.0 to neutrality, the m o st abundan t c o l o r ed s pe ci e s p r e s en t is t he quinoidal form and thus Mv 3-gl its e lf doe s no t c on f e r m u c h c o l o r t o a s o l u ti on , so again the contribution of the c op i g m en t a ti on on c o l o r is m a i n ly qua lit a tiv e , that is, significantly due to hue ch ange s. Moreover, the relative c on tri bu ti on o f li gh t ne ss ( %  L ), c h r o m a ( % C), and hue (%H) permitted us to c o m pa r e t he c o l o rim e tric e ff e ct i ndu c ed b y t he different flavanols tested. In g ene r a l t e rms, (-)- ep ic a t e c h i n and c a ff e ic a ci d produced similar effects on the t o t a l c o l o r o f t he Mv 3 - g l a cr o ss t o t he p H changes, contributing more mark ed wit h quan tit a tiv e c hange s t han qua lit a tiv e ones (%L + %C= 55-63%, an d % ΔH= 45 - 37% ). O n t he c on tr a ry, pa rtic u l a rly at the higher pH values, the qualit a tiv e c on tri bu ti on w a s m o r e im po rt an t i n (+)- catechin copigmented solutions. T hu s, it c an be s a i d t ha t t he c op i g m en t a ti on o f (-)-epicatechin and caffeic acid p r o vi de no t ab ly c o l o r st ab iliz a ti on and v a ri a ti on , while (+)-catechin provide more i n t en s e c o l o r v a ri a ti on t han st ab iliz a ti on . 363 364 Effect of the mola r r atio on copig me nt a tion 365 The influence of the pigm en t /c op i g m en t m o l a r r a ti o w a s a ls o a ss a y ed a t 366 pH 3.60. For this purpose, tw o c op i g m en t ed s o l u ti on s o f m a lvi d i n 3 - g l:(+)- 367 catechin (MC) and malvidin 3-gl:(-)- ep ic a t e c h i n (ME) w e r e p r epa r ed i n t he s a m e 368 wine-like medium at the molar r a ti o s 1 : 1 , 1 : 2 , 1 : 5 , and 1 : 7 . 369 The mean values of the tot a l c o l o r (E) o f t he o ri g i na l Mv 3 - g l s o l u ti on and 370 each copigmented solution as a f un cti on o f t he m o l a r r a ti o is p r e s en t ed i n T ab l e 371 2. For both assays, the positive eff e ct o f t he c op i g m en t a ti on on t he Mv 3 - g l t o t a l ACS P a r a gon Plu s En v ironm e n t 15 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y P a g e 16 of 36 372 color was increased with incr ea si ng c op i g m en t c on c en tr a ti on , w h ic h is i n 373 agreement with previous studi e s r epo rt ed i n b i b li og r aph y de s p it e be i ng l o w e r 374 the range of the pigment/copi g m en t m o l a r r a ti o u s ed ( 33 ). T he m a xim u m 375 displacement of hydration/dehy d r a ti on equ ili b ri u m t o w a r d s t he c o l o r ed s pe ci e s 376 was achieved using the highest m o l a r r a ti o ( 1 : 7 ) si n c e it c au s ed t he g r ea t e st 377 total color increases (from 49.26 t o 57 . 32 and 57 . 90 CIE L AB u . i n MC and ME 378 solutions, respectively). 379 Moreover, significant differ en c e s on t he L* , a* , and b* v a l ue s a m ong t he 380 pure Mv 3-gl solution and its corr e s pond i ng c op i g m en t ed w e r e f ound , e s pe ci a lly 381 at molar ratios higher than 1:2, r e s u lti ng i n a d iff e r en t l o c a ti on o f t he s a m p l e s i n 382 the (a*b * ) colorimetric diagram (Fi gu r e 4 ). I n ab s en c e o f an y c op i g m en ts, t he 383 original anthocyanin color took p o sitiv e a* and b* v a l ue s and appea r ed l o c a t ed 384 near 0º, that is, in a well defined r edd is h c o l o r a r ea ( L* = 69 . 71 , a* = 38 . 84 , and 385 b*= 0.39 CIELAB u.). However, wit h t he i n cr ea si ng add iti on o f t he fl a v ano l 386 copigments, the samples a ppea r ed l o c a t ed p r og r e ssiv e ly f a r e r t o t he 387 coordinates origin and experim en t ed a r e m a rk ab l e e v o l u ti on fr o m t he first 388 toward the fourth quadrant, that is, fr o m t he r edd is h t o t he pu r p l e o r r ed - b l u is h 389 colors region. The behavior of t he c o l o r pa r a m e t e rs a s a f un cti on o f t he 390 concentration was similar for b o t h t he c op i g m en t u s ed . At t he h i ghe st m o l a r 391 ratio (1:7), copigmented samples r ea c hed t he l o w e st L* and b* v a l ue s bu t t he 392 highest a * values, and thus, they e x h i b it ed t he da rk e st and m o st vivi d b l u is h 393 color (L* = 64.18 and 63.05, a* = 45 . 44 and 43 . 76 , and b* = - 2 . 23 and - 2 . 21 394 CIELAB u. for MC and ME soluti on s, r e s pe ctiv e ly). 395 Regarding the changes o n t he c on tri bu ti on o f t he c op i g m en t a ti on t o t he 396 total anthocyanin color, the adde d c op i g m en ts c au s ed si gn ific an t c on c en tr a ti on - ACS P a r a gon Plu s En v ironm e n t 16 P a g e 17 of 36 397 398 399 400 401 402 403 404 405 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y dependent increases on the ma gn it ude o f t he e ff e ct, a s c an be s een i n Fi gu r e 2 . From 1:1 to 1:7 molar ratio, (+)-c a t e c h i n i ndu c ed an i n cr ea s e o f t he Mv 3 - g l t o t a l color from 1.2% to 16.0%, whil e wit h (-)- ep ic a t e c h i n , t he i n cr ea s e w a s fr o m 3.9% to 17.9%. Notwithstandin g , a lt hough t he pa tt e r n e v o l u ti on w a s simil a r between both flavanols copigm en ts, on c e aga i n , t he ab ility o f t he (-)- ep ic a t e c h i n to act as Mv-3gl copigments was str onge r t han f o r (+)-c a t e c h i n si n c e si gn ific an t highest values for copigmentati on w a s a c h i e v ed f o r m o st o f t he m o l a r r a ti o s tested. As was made in the pr e vi ou s s e cti on o f t h is st ud y, t he dependen c e 406 between the amplitude of the c o l o r e ff e ct i ndu c ed b y c op i g m en t a ti on w a s 407 evaluated by means of the CIE L AB c o l o r d iff e r en c e s (E * ab) (T ab l e 2 ). As 408 expected, the progressive form a ti on o f c op i g m en t a ti on c o m p l e x e s w a s 409 confirmed by a successive i n cr ea s e on t he c o l o r d iff e r en c e s be tw een 410 anthocyanins and flavanols s o l u ti on s w hen c op i g m en t c on c en tr a ti on s w e r e 411 increasingly added. For all the c on c en tr a ti on l e v e l t e st ed , (-)- ep ic a t e c h i n c au s ed 412 always significant increases (p  0 . 05 ) on t he c o l o r d iff e r en c e s. I n c on tr a st, 413 higher increases on the molar r a ti o w e r e ne c e ss a ry t o i ndu c e si gn ific a tiv e 414 changes with (+)-catechin copigm en t ed s o l u ti on s. 415 In any case, calculation o f t he sim p l e r eg r e ssi on c oe ffici en ts be tw een 416 total color differences and the c op i g m en t c on c en tr a ti on f o r ea c h c op i g m en t ed 417 solution confirmed that the relati on s h i p w a s si gn ific an tly h i gh r ega r d i ng t he 418 copigment used (r * values ran ged fr o m + 0 . 95 t o + 0 . 98 un its, p < 0 . 05 ). T he 419 lowest color differences were f ound be tw een pu r e Mv 3 - g l s o l u ti on and t he 420 copigmented ones at the lowest m o l a r r a ti o u s ed ( 1 : 1 ), t a ki ng v a l ue s o f 1 . 01 421 and 2.27 CIELAB u. with (+)-c a t e c h i n and (-)- ep ic a t e c h i n , r e s pe ctiv e ly. ACS P a r a gon Plu s En v ironm e n t 17 P a g e 23 of 36 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y 532 8. Brouillard, R.; Mazza, G.; S aad , Z.; Al b r e c h t-G a ry, A.M.; C he mi na t, A. T he 533 copigmentation reacti on o f an t ho cy an i n s: A micr op r obe f o r t he 534 structural study of a queou s s o l u ti on s. J ou r na l o f t he Am e ric an 535 Chemical Society. 1989, 111 , 2604 - 2610 . 536 9. Davies, A.J.; Mazza, G. C op i g m en t a ti on o f sim p l e and a cyl a t ed 537 anthocyanins with colorl e ss pheno lic c o m pound s. J. A g ric. F ood C he m. 538 1993, 41, 716-720. 539 10. Baranac, J.M.; Petr ano vic, N.A.; Dimitric-M a rk o vic, J.M. 540 Spectrophotometric St ud y o f A n t ho cy an C op i g m en t a ti on R ea cti on s. 2 . 541 Malvin and the Nonglyc o si d iz ed Fl a v one Q ue rc e ti n . J. A g ric. F ood 542 Chem. 1997, 45, 1694-1 697 . 543 11. Eiro, M.J.; Heinonen, M. A n t ho cy an i n C o l o r B eha vi o r and St ab ility du ri ng 544 Storage:Çë Effect of I n t e rm o l e c u l a r C op i g m en t a ti on . J. A g ric. F ood 545 Chem. 2002, 50, 7461-7 466 . 546 12. Talcott, S.T.; Brenes, C.H.; Pir e s, D.M.; D e l P o z o -I n sfr an , D. 547 Phytochemical Stability and C o l o r R e t en ti on o f C op i g m en t ed and 548 Processed Muscadine Gr ape J u ic e . J. A g ric. F ood C he m. 2003 , 51 , 549 957-963. 550 13. Gris, E.; Ferreira, E.; Falc ao , L .; B o r d i gnon - Lu iz, M. I n fl uen c e o f f e r u lic 551 acid on stability of anthocy an i n s fr o m C abe r ne t S au vi gnon g r ape s i n a 552 model system and a y ogu rt syst e m. I n t e r na ti ona l j ou r na l o f f ood 553 science and technology. 2007 , 42 , 992 - 998 . ACS P a r a gon Plu s En v ironm e n t 23 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y P a g e 24 of 36 554 14. González-Manzano, S.; D ueña s, M.; Riv a s-G on z a l o , J.C.; Escri bano - 555 Bailón, M.T.; Santos-B ue l ga , C. St ud i e s on t he c op i g m en t a ti on 556 between anthocyanins and fl a v an - 3 - o ls and t he ir i n fl uen c e i n t he c o l ou r 557 expression of red wine. F ood C he mistry. 2009 , 114 , 649 - 656 . 558 15. Lambert, S.G.; Asenstorfer, R.E.; Willi a ms on , N.M.; Il and , P.G.; J one s, 559 G.P. Copigmentation b e tw een m a lvi d i n - 3 - g l u c o si de and s o m e wi ne 560 constituents and its imp o rt an c e t o c o l ou r e x p r e ssi on i n r ed wi ne . F ood 561 Chemistry. 2011, 125, 106 - 115 . 562 16. Hermosín-Gutiérrez, I. Infl uen c e o f Et hano l C on t en t on t he Ext en t o f 563 Copigmentation in a C en ci be l Y oung R ed Wi ne . J. A g ric. F ood C he m. 564 2003, 51, 4079-4083. 565 17. Gutiérrez, I.H.; Lorenzo, E.S.-P.; Es p i no s a , A.V. P heno lic c o m po siti on and 566 magnitude of copigme n t a ti on i n y oung and s ho rtly aged r ed wi ne s 567 made from the cultivars, C abe r ne t S au vi gnon , C en ci be l, and Syr ah . 568 Food Chemistry. 2005, 92 , 269 - 283 . 569 18. Darias-Martín, J.; Carrillo-L ópe z, M.; Ec ha v a rri-Gr anado , J.F.; í a z-R o m e r o , 570 C. The magnitude of co p i g m en t a ti on i n t he c o l ou r o f aged r ed wi ne s 571 made in the Canary Isl and s. E u r opean F ood R e s ea rc h and 572 Technology. 2007, 224, 643 - 648 . 573 19. Guadalupe, Z.; Ayestarán, B. Eff e ct o f C o mm e rci a l M annop r o t e i n A dd iti on 574 on Polysaccharide, Poly pheno lic, and C o l o r C o m po siti on i n R ed Wi ne s. 575 J. Agric. Food Chem. 20 08 , 56 , 9022 - 9029 . ACS P a r a gon Plu s En v ironm e n t 24 P a g e 25 of 36 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y 576 20. Gordillo, B.; López-Infante, M.I.; R a mír e z-P e ¦ ü r e z, P.; G on z á l e z-Mir e t, 577 M.L.; Heredia, F.J. Influe n c e o f Pr e f e rm en t a tiv e C o l d M a c e r a ti on on t he 578 Color and Anthocyanic C op i g m en t a ti on o f Or gan ic T e m p r an ill o Wi ne s 579 Elaborated in a Warm Clim a t e . J. A g ric. F ood C he m. 2010 , 58 , 6797 - 580 6803. 581 21. Gonnet, J.F. Colour effects o f c o - p i g m en t a ti on o f an t ho cy an i n s r e visit ed - 582 1. A colorimetric definiti on u si ng t he CIE L AB sc a l e . F ood C he mistry. 583 1998, 63, 409-415. 584 22. Asen, S.; Stewart, R.N.; N o rris, K.H. C o - p i g m en t a ti on o f an t ho cy an i n s i n 585 plant tissues and its eff e ct on c o l o r. P h yt o c he mistry. 1972 , 11 , 1139 - 586 1144. 587 23. Dangles, O.; Brouillard, R. A s pe ctr o sc op ic m e t hod ba s ed on t he 588 anthocyanin copigmentati on i n t e r a cti on and app li ed t o t he quan tit a tiv e 589 study of molecular compl e x e s. J ou r na l o f t he C he mic a l S o ci e ty, P e rki n 590 Transactions 2. 1992, 247 - 257 . 591 24. Heredia, F.J.; Álvarez, C.; G on z á l e z-Mir e t, M. L .; & R a mír e z, A. Cr o m aLab , 592 análisis de color. Registr o G ene r a l de l a Pr op i edad I n t e l e ct ua l. 2004 . 593 25. CIE Technical Report C o l o rim e try; C o mmissi on I n t e r na ti ona l e de 594 l'Eclairage Central Bure au : Vi enna , A u stri a , 2004 . 595 26. Melgosa, M.; Hita, E.; Poza, A.J.; Alm an , D.H.; B e r n s, R.S. S up r a t h r e s ho l d 596 color-difference ellipsoi d s f o r s u rf a c e c o l o rs. C o l. R e s. A p l. 1997 , 22 , 597 148-155. ACS P a r a gon Plu s En v ironm e n t 25 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y P a g e 26 of 36 598 27. Heredia, F.J.; Francia-Aric ha , E.M.; Riv a s-G on z a l o , J.C.; Vic a ri o , I.M.; 599 Santos-Buelga, C. Chr o m a tic c ha r a ct e riz a ti on o f an t ho cy an i n s fr o m r ed 600 grapes - I. pH effect. Fo od C he mistry. 1998 , 63 , 491 - 498 . 601 28. Darias-Martín, J.; Martín-L u is, B.; C a rrill o - Lópe z, M.; La m ue l a -R a v en t ó ¦s, 602 R.; Díaz-Romero, C.; B ou lt on , R. Eff e ct o f c a ff e ic a ci d on t he c o l o r o f 603 red wine. J. Agric. 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-Mir e t, M. L .; T e r e s a 605 Escribano-Bailón, M.; H e r ed i a , F.J.; S an t o s-B ue l ga , C. I n fl uen c e o f 606 different phenolic copi g m en ts on t he c o l o r o f m a lvi d i n 3 - g l u c o si de . J. 607 Agric. Food Chem. 2006, 54 , 5422 - 5429 . 608 30. Cruz, L.; Brás, N.F.; Teixeir a , N.; M a t eu s, N.; R a m o s, M.J.; D ang l e s, O.; 609 De Freitas, V. Vinylcat e c h i n d im e rs a r e m u c h be tt e r c op i g m en ts f o r 610 anthocyanins than catec h i n d im e r p r o cy an i d i n B 3 . J. A g ric. F ood C he m. 611 2010, 58, 3159-3166. 612 31. Kunsági-Maté, S.; Szabó, K.; Nikf a r d j a m, M.P.; K o ll a r, L . D e t e rmi na ti on o f 613 the thermodynamic par a m e t e rs o f t he c o m p l e x f o rm a ti on be tw een 614 malvidin-3-O-glucoside and po ly pheno ls. C op i g m en t a ti on e ff e ct i n r ed 615 wines. Journal of Biochemic a l and Bi oph ysic a l M e t hod s. 2006 , 69 , 113 - 616 119. 617 32. Martínez, J.A.; Melgosa, M.; P é r e z, M.M.; Hit a , E.; N egue r ue l a , A.I. N o t e . 618 Visual and instrumental c o l o r e v a l ua ti on i n r ed wi ne s. F ood Sci en c e 619 and Technology Internati ona l. 2001 , 7 , 439 - 444 . ACS P a r a gon Plu s En v ironm e n t 26 P a g e 27 of 36 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y 620 33. Berké, B.; De Freitas, V. A c o l o rim e tric st ud y o f oen i n c op i g m en t ed b y 621 procyanidins. Journal of t he Sci en c e o f F ood and A g ric u lt u r e . 2007 , 87 , 622 260-265. 623 624 ACS P a r a gon Plu s En v ironm e n t 27 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y P a g e 28 of 36 625 Table 1. Mean values of the A 520 and the total color (E) obtained for the pure 626 anthocyanic solution and its respective copigmented at each pH value; as well as, the 627 CIELAB differences (E* ab , L*, C* ab , h ab ) between Mv 3-gl solutions with and 628 without copigments. 629 Pigment pH Mv 3-gl Pigment/Copigment 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 r a gon Plu s En v ironm e n t 28 P a g e 29 of 36 630 631 632 633 634 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y Table 2. Mean values of the A 520 and the total color (E) obtained for the pure anthocyanic solution and its respective copigmented at each molar ratio; as well as, the CIELAB differences (E* ab , L*, C* ab , h ab ) between Mv 3-gl solutions with and without copigments. Colorimetric parameters 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 Molar Ratio 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 r a gon Plu s En v ironm e n t 29 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y P a g e 30 of 36 635 FIGURE CAPTIONS 636 Figure 1. Change in the locatio n o f t he pu r e Mv 3 - g l and c op i g m en t ed s o l u ti on s 637 (MC, ME, and MF) within (a) the ( a*b* ) d i ag r a m and ( b ) li gh t ne ss v a l ue s ( L* ) a s 638 a function of the pH value. 639 640 Figure 2. (a) Changes on the m agn it ude o f c op i g m en t a ti on f o r t he Mv 3 - g l 641 solutions copigmented with (+)-c a t e c h i n (MC), (-)- ep ic a t e c h i n (ME), and c a ff e ic 642 acid (MF) as a function of the pH, and ( b ) wit h (+)-c a t e c h i n (MC), (-)- ep ic a t e c h i n 643 (ME) as a function of the molar r a ti o , e v a l ua t ed b y Tristim u l u s C o l o rim e try a s 644 the ratio [(ECE0) / E0 ] * 100. 645 646 Figure 3. Relative contribution o f li gh t ne ss ( %  L ), c h r o m a ( % C), and hue 647 (%H) to the total color differenc e f o r ea c h p i g m en t/c op i g m en t a s a f un cti on o f 648 the pH value. 649 650 Figure 4. Change in the locatio n o f t he pu r e Mv 3 - g l and c op i g m en t ed s o l u ti on s 651 (MC, ME) within (a) the (a * b * ) d i ag r a m and ( b ) li gh t ne ss v a l ue s ( L* ) a s a 652 function of the molar ratio. 653 654 Figure 5. CIELAB hue differ en c e s ( h ab) a s a f un cti on o f m o l a r r a ti o f o r 655 corresponding to Mv 3-gl: CAT and Mv 3 - g l: EPI s o l u ti on s. 656 657 Figure 6. Relative contribution o f li gh t ne ss ( %  L ), c h r o m a ( % C), and hue 658 (%H) to the total color differenc e f o r ea c h p i g m en t/c op i g m en t a s a f un cti on o f 659 the molar ratio. ACS P a r a gon Plu s En v ironm e n t 30 ACS P a r a gon Plu s En v ironm e n t 34  P a g e 35 of 36 J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y Figure 5. 2 1 0 -1 Mv 3-gl : CAT Mv 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 Molar Ratio (MR) ACS P a r a gon Plu s En v ironm e n t 35 Ea J ou r n a l of Agri c ul t ur a l a nd Food Ch e mi st r y P a g e 36 of 36 Figure 6. a ) MC % %C %L 100 75 50 25 0 1:1 1:2 1:5 1:7 Molar ratio pigment/copigment b ) ME %  %  C %  L 100 75 50 25 0 1 : 1 1 : 2 1 : 5 1 : 7 Molar Ra t io pigmen t/ copigmen t ACS P a r a gon Plu s En v ironm e n t 36