Effect of genuine non-anthocyanin phenolics and chlorogenic acid on color and stability of black carrot (Daucus carota ssp. sativus var. atrorubens Alef.) anthocyanins

    Effect of genuine non-anthocyaninic¡!–¡query id=”q7”¿¡ce:para¿Would you consider changing ”non-anthocyaninic phenolics” to ”non-anthocyanin phenolics”? Please check here and in subsequent occurrences and amend if necessary.¡/ce:para¿¡/query¿–¿ phenolics and chlorogenic acid on color and stability of black carrot (Daucus carota ssp. sativus var. atrorubens Alef.) anthocyanins Claudia C. Gras, Hanna Bogner, Reinhold Carle, Ralf M. Schweiggert PII: DOI: Reference:

S0963-9969(16)30193-4 doi: 10.1016/j.foodres.2016.05.006 FRIN 6280

To appear in:

Food Research International

Received date: Revised date: Accepted date:

11 February 2016 28 April 2016 8 May 2016

Please cite this article as: Gras, C.C., Bogner, H., Carle, R. & Schweiggert, R.M., Effect of genuine non-anthocyaninic¡!–¡query id=”q7”¿¡ce:para¿Would you consider changing ”non-anthocyaninic phenolics” to ”non-anthocyanin phenolics”? Please check here and in subsequent occurrences and amend if necessary.¡/ce:para¿¡/query¿–¿ phenolics and chlorogenic acid on color and stability of black carrot (Daucus carota ssp. sativus var. atrorubens Alef.) anthocyanins, Food Research International (2016), doi: 10.1016/j.foodres.2016.05.006

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ACCEPTED MANUSCRIPT Effect of genuine non-anthocyaninic phenolics and chlorogenic acid on color and

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stability of black carrot (Daucus carota ssp. sativus var. atrorubens Alef.) anthocyanins

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Claudia C. Grasa, Hanna Bognera, Reinhold Carlea,b, Ralf M. Schweiggerta,*

University of Hohenheim, Institute of Food Science and Biotechnology, Chair of Plant

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b

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Foodstuff Technology and Analysis, Garbenstrasse 25, D-70599 Stuttgart, Germany

King Abdulaziz University, Faculty of Science, Biological Science Department,

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P.O. Box 80257, Jeddah 21589, Saudi Arabia

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*Corresponding author: R.M. Schweiggert Tel: +49 - (0) 711-459-22995; Fax: +49 - (0) 711-459-24110

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Email: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT This work aimed at studying the color intensity and stability of black carrot anthocyanins as

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influenced by intermolecular co-pigmentation. For this purpose, purified anthocyanin

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solutions were supplemented with purified genuine black carrot phenolics, chlorogenic acid,

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and an aqueous phenolic-rich green coffee bean extract at various anthocyanin:co-pigment ratios (1:0-1:162; pH 3.6). The hyperchromic co-pigmentation effect depended on the concentration of added co-pigments, resulting in an absorbance increase of up to 22% at the

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absorption maximum. Anthocyanin stability during heating (90°C, 5h) was barely improved

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unless the concentrations of co-pigments exceeded those of their natural source. When adding co-pigments at ratios above 1:9.4, anthocyanin heat stability was significantly improved. As acylated anthocyanins were most stable, breeders might aim at increasing their content in the

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future, while breeding for high levels of colorless polyphenols may be unreachable. Nevertheless, we provided proof-of-concept for the successful color enhancement by the

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applications.

being useful for food-grade

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addition of a phenolic-rich green coffee bean extract,

KEYWORDS Co-pigmentation; acylated anthocyanins; food-grade; chlorogenic acid; coffee extract; HPLCDAD-MSn

ABBREVIATIONS USED CE co-pigmentation effect as determined by the hyperchromic effect of intermolecular copigmentation. 2

ACCEPTED MANUSCRIPT 1. INTRODUCTION In recent decades, safety concerns over a number of synthetic food colorants and the insect-

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based aluminum-lake carmine have been raised. In contrast, red colored anthocyanins from

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fruit and vegetables have been related to several health benefits in humans (Stintzing & Carle,

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2004), thus, currently representing the preferred natural colorants in processed food. However, compared to synthetic dyes and carmine, anthocyanins are significantly less stable. Apart from their chemical structure and concentration, anthocyanin stability is strongly

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affected by temperature, light, and oxygen. Furthermore, high water activity, the presence of

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deteriorative enzymes, phenolic co-factors, proteins, metal ions, and pH play important roles for anthocyanin stability (Stintzing & Carle, 2004). The pH dependence of their color and their poor stability are major drawbacks when adding anthocyanins to foods. In acidic media,

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anthocyanins exist primarily in an equilibrium between the red colored flavylium cation and colorless hemiketal forms (Figueiredo, George, Tatsuzawa, Toki, Saito, & Brouillard, 1999;

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Stintzing, Stintzing, Carle, Frei, & Wrolstad, 2002). At higher pH values, the flavylium cation is directly converted into blue colored quinoidal bases due to an acid-base equilibrium. The

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stability of these quinoidal pigments is poor, and thus, food applications of anthocyanins in low acidic or even neutral media are intricate, and often impossible (Asen, Stewart, & Norris, 1972; Mazza & Brouillard, 1987). Although their stability is considerably better at acidic pH, anthocyanin discoloration in foods still represents a major challenge. Therefore, the use of copigments has been proposed to enhance anthocyanin stability, and, simultaneously, to improve their tinctorial strength and color shade (Stintzing & Carle, 2004). Several phenolic compounds have been previously reported to serve as co-pigments, being thoroughly investigated regarding the color of wine (Cavalcanti, Santos, & Meireles, 2011), and proposed for color and stability enhancement in several fruit- or berry-based foodstuffs (Maccarone, Maccarone, & Rapisarda, 1985; Rein & Heinonen, 2004; Shikov, Kammerer, Mihalev, Mollov, & Carle, 2008; Wilska-Jeszka & Korzuchowska, 1996). Generally, four types of co3

ACCEPTED MANUSCRIPT pigmentation may be distinguished, namely metal complexation, self-association, intramolecular and intermolecular co-pigmentation. Except for metal ion interactions,

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anthocyanin-co-pigment complex formation is believed to be mainly driven by hydrophobic

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π-π-interactions of the delocalized electrons of neighboring phenolic ring systems (Asen et al.,

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1972; Dangles, Saito, & Brouillard, 1993), being responsible for the complex formation of anthocyanins and colorless phenolic co-pigments (Malien-Aubert, Dangles, & Amiot, 2001). The latter may be flavonoids and hydroxycinnamic acid derivatives such as ferulic, coumaric,

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caffeic, rosmarinic, and chlorogenic acids (Asen et al., 1972; Cavalcanti et al., 2011; Eiro &

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Heinonen, 2002; Maccarone et al., 1985; Shikov et al., 2008; Wilska-Jeszka & Korzuchowska, 1996). Co-pigments may also be covalently bound to the anthocyanin skeleton. The resulting effect is known as intramolecular co-pigmentation (Dangles et al.,

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1993). The steric conformation of anthocyanin-co-pigment complexes, i.e. the sandwich-type stacking of the phenolic acid moiety superposing the planar anthocyanin molecule, widely

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prevents or hinders the deteriorative nucleophilic attack of water (Goto & Kondo, 1991). As a consequence, the observed hydration constants of acylated anthocyanins (pKH of an

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feruloylated cyanidin derivative = 4.4) are substantially higher than those of the non-acylated ones (pKH from 1.3 to 3.3) (Figueiredo et al., 1999; Stintzing et al., 2002), ultimately improving anthocyanin stability. Intramolecular co-pigmentation is also responsible for the high stability of black carrot (Daucus carota ssp. sativus var. atrorubens Alef.) anthocyanins, being widely used to impart red shades to soft drinks, preserves, jams, fruit preparations, and confectionary (Kirca, Özkan, & Cemeroglu, 2006a; Stintzing & Carle, 2004). The major proportion of black carrot anthocyanins is glycosylated with two or three sugars moieties and acylated with hydroxycinnamic acid derivatives, such as sinapic, ferulic and p-coumaric acids, respectively (Kammerer, Carle, & Schieber, 2004b). In addition, several uncolored phenolic compounds,

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ACCEPTED MANUSCRIPT for instance, 5-trans-caffeoyl-quinic acid (chlorogenic acid) were found at high concentrations in carrot roots (Kammerer, Carle, & Schieber, 2004a).

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However, whether these non-colored phenolic compounds contribute to anthocyanin stability

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by intermolecular co-pigmentation remains unknown to date. Therefore, the main goal of our

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study was to investigate the effect of genuine black carrot phenolics on thermal stability and color expression of black carrot anthocyanins. The genuine anthocyanins and phenolics were isolated from a black carrot concentrate, and subsequently purified by column

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chromatography and liquid-liquid extraction. After identification and verification of their

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purity by HPLC-DAD-MSn, they were recombined at specific ratios to investigate the effect of co-pigmentation on color expression and heat stability. In addition, experiments with added chlorogenic acid and an aqueous green coffee bean extract were conducted to demonstrate the

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beneficial effect of co-pigment supplementation on potential applications in coloring foodstuffs.

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2. MATERIAL AND METHODS

2.1 RAW MATERIAL AND CHEMICALS

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A black carrot concentrate with 66.6 °Brix was obtained from Diana Naturals (Antrain, France). Green coffee beans (Coffea arabica L.) were provided by Colcafé (Medellin, Colombia), and stored at -20 °C until use. The authentic standard cyanidin 3-O-(2″xylosylgalactoside) chloride (cya-xyl-gal; 97%) was from Extrasynthèse (Genay, France), and 5-trans-caffeoylquinic acid (chlorogenic acid; 95%) from Sigma-Aldrich (Steinheim, Germany). Methanol was purchased from VWR International (Leuven, Belgium), while ethyl acetate, formic acid, acetic acid, hydrochloric acid (HCl, 37%), trifluoroacetic acid (TFA), sodium acetate and sodium hydroxide (NaOH) were from Merck (Darmstadt, Germany). The adsorber resin Lewatit® VP OC 1064 MD PH was obtained from Lanxess (Leverkusen, Germany), and purified water was prepared using a Sartorius arium 611 Ultrapure Water System (Göttingen, Germany). 5

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2.2. ISOLATION OF ANTHOCYANINS AND NON-ANTHOCYANINIC POLYPHENOLS

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Figure 1 provides an overview of the procedure applied for the separation and purification of

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anthocyanins and non-anthocyaninic polyphenols from black carrot, including their

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recombination for co-pigmentation and heat stability tests. Briefly, black carrot concentrate was diluted with purified water to obtain a black carrot juice (approximately 8.3 °Brix). To remove sugars, salts and amino acids, the black carrot juice was applied to a column (500 mm

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x 30 mm i.d.) filled with a macro-porous adsorber resin without functional groups (Lewatit

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VP OC 1064 MD PH). Semi-purification was performed according to a slightly modified procedure described by Fischer, Carle, & Kammerer (2013). In brief, the resin was

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equilibrated with two bed volumes (BV) of methanol, and four BV of water (pH 2, acidified

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with TFA). Subsequently, an aliquot of 50 mL of the black carrot juice was applied to the column. After washing the loaded column with four BV acidified water (pH 2), the pigment

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fraction, also containing non-anthocyaninic phenolics, was eluted with methanol/water (90/10, v/v, acidified to pH 2 with TFA). The organic portion of the solvent mixture was

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evaporated in vacuo at 30 °C, yielding the semi-purified extract. In order to separate anthocyanins from non-anthocyaninic polyphenols, the semi-purified extract was adjusted to pH 1.5 with aqueous HCl (0.1 M), and subsequently extracted four times with ethyl acetate. The separated organic phases, containing non-anthocyaninic polyphenols (Figure 1), were combined and evaporated to dryness in vacuo at 30 °C. The obtained solid residue was re-dissolved in sodium acetate buffer (0.1 M, pH 3.6) to obtain the phenolic extract. The aqueous fraction, containing anthocyanins, was concentrated under vacuum, and then diluted with sodium acetate buffer (pH 3.6), yielding the anthocyanin extract. If necessary, the solutions were adjusted to pH 3.6 using 0.1 M aqueous HCl and NaOH. Both extracts were membrane-filtered (0.45 µm, Macherey-Nagel, Düren, Germany), and stored at -20 °C until further use. Anthocyanins and non-anthocyaninic polyphenols were 6

ACCEPTED MANUSCRIPT identified and quantitated using HPLC-DAD-MSn as described in section 2.4. Noteworthy, the compound patterns of both fractions remained qualitatively unaltered during purification

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as shown by comparison of their HPLC chromatograms with that of the initial black carrot

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juice. Moreover, the extracts were checked for mutual impurities, displaying HPLC-DAD-

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based purities of 95.5%, and 100% for the anthocyanin and the non-anthocyaninic extract monitored at 320 nm, and at 520 nm, respectively. Purity of the commercial chlorogenic acid

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used for the preparation of co-pigmentation model solutions amounted to 95%.

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2.3 PREPARATION OF THE GREEN COFFEE BEAN EXTRACT Green coffee beans were ground with a laboratory mill ZM1 (Retsch, Haan, Germany)

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equipped with a 1 mm pore size sieve. Subsequently, an aliquot of 500 ± 1 mg of ground

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green coffee beans was extracted with 5 mL of purified water, applying a probe sonicator with a micro tip MS72 at 75% amplitude for 20 s (Sonopuls UW 3100, Bandelin electronic, Berlin,

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Germany). After centrifugation (1729 x g, 5 min), the supernatant was collected and the solid residue was re-extracted twice. The obtained aqueous extract was filtrated through a paper

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filter (615 1/4, Macherey-Nagel, Düren, Germany), concentrated under vacuum to approximately 2 mL, adjusted to pH 3.6 with 0.1 M HCl, made up to 5 mL using sodium acetate buffer (pH 3.6), and consecutively passed through membrane filters (1 µm glass fiber pre-filter, 0.45 µm polyester filter, and 0.2 µm cellulose filter). The content of hydroxycinnamic acids in the resulting extract was quantitated by HPLC-DAD as described in section 2.4.

2.4 HPLC-DAD-MSN ANALYSES OF ANTHOCYANINS AND NON-ANTHOCYANINIC PHENOLICS Analyses of anthocyanin and non-anthocyaninic polyphenol fractions from black carrot as well as of the phenolics of the green coffee bean extract were performed using an 1100 series HPLC-system (Hewlett Packard, Waldbronn, Germany), equipped with a G1315 diode array 7

ACCEPTED MANUSCRIPT detector (DAD). For HPLC-MSn analyses including collision induced dissociation (CID) experiments, the HPLC system was connected in series with a Bruker model Esquire 3000+

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ion trap mass spectrometer (Bremen, Germany) fitted with an ESI-source. Separation of all

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compounds of interest was performed on a Phenomenex Kinetex C18 core-shell column (250

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mm x 4.6 mm i.d., 5 µm particle size, Phenomenex, Torrance, CA, USA), equipped with a C18 ODS guard column (4.0 mm x 2.0 mm i.d., Phenomenex), as well as with water- and methanol-based solvents acidified with formic acid. Solvent composition, gradient program,

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and all further HPLC-PDA-MSn parameters were set as described in a previous study (Appel

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et al., 2015).

Individual compound identification was based on retention times, UV/Vis absorption spectra and mass spectrometric data. Individual anthocyanins were quantitated at 520 nm using an

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external linear calibration generated with the authentic standard cya-xyl-gal. Due to the lack of authentic reference materials, all other anthocyanins were quantitated by the cya-xyl-gal

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calibration applying molecular weight correction factors (Gras, Carle, & Schweiggert, 2015). All non-anthocyaninic phenolics from black carrot were quantitated at 320 nm, and phenolics

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in green coffee bean extract at 280 nm by an external linear calibration of chlorogenic acid.

2.5 PREPARATION OF ANTHOCYANIN-CO-PIGMENT MODEL SOLUTIONS Since co-pigmentation was reported to be most pronounced at pH values close to 3.6 (Brouillard, Mazza, Saad, Albrecht-Gary, & Cheminat, 1989), and to ensure comparability to previous studies (Iliopoulou, Thaeron, Baker, Jones, & Robertson, 2015; Sadilova, Carle, & Stintzing, 2007), the pH of the model solutions was set to 3.6. Consequently, the purified anthocyanin and phenolic extracts were diluted with sodium acetate buffer (pH 3.6) to yield stock solutions of 108 ± 3 mg/L for the anthocyanin extracts, and 45.1 ± 0.2, 102.5 ± 9.6, 187.8 ± 8.0, and 379 ± 2.1 mg/L for the phenolic extracts (Figure 1). Subsequently, the model solutions were obtained by combining two equal volumes of the above mentioned stock 8

ACCEPTED MANUSCRIPT solutions resulting in a final anthocyanin concentration of 54 ± 1.4 mg/L, while concentrations of the co-pigments were 22.6 ± 0.1, 51.2 ± 4.8, 93.9 ± 4.0, and 189.7 ± 1.0

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mg/L. In addition, a control model solution without added co-pigments was prepared by

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combining the anthocyanin stock solution with an equal amount of sodium acetate buffer (pH

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3.6). Thereby, the anthocyanin:co-pigment ratios were 1:0, 1:0.43, 1:0.9, 1:1.7, and 1:3.6. Noteworthy, according to our HPLC analyses, anthocyanin and non-anthocyaninic polyphenol contents in the black carrot concentrate amounted to 1,315.3 and 423.5 mg/100 g,

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respectively. Hence, the genuine ratio of pigment to co-pigment in the carrot concentrate was

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approximately 1:0.32. In addition, purified anthocyanin stock solutions (116 ± 9 mg/L) were combined with equal amounts of aqueous chlorogenic acid (1,000, 10,000 and 20,000 mg/L), the major phenolic compound in black carrot, in order to obtain model solutions with

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increased anthocyanin:co-pigment ratios of 1:9.4, 1:85 and 1:162 (m/m; Figure 1). Finally, equal volumes of the above mentioned green coffee bean extract and an anthocyanin solution

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were combined to yield an anthocyanin:co-pigment ratio of 1:52 (m/m). The aqueous green coffee bean extract contained a total hydroxycinnamic acid content of 4,636.5 mg/L as

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determined by HPLC-DAD (expressed as chlorogenic acid).

2.6 HEAT STABILITY TESTS Thermal stability tests were performed by heating the aforementioned model solutions at 90 ± 1 °C in Pyrex glass tubes (VWR International, Darmstadt, Germany) with PTFE-coated silicon seal screw caps, using a heating block (Liebisch Labortechnik, Bielefeld, Germany). After 1, 2, 3, and 5 h, the tubes were removed from the block and immediately cooled in an ice bath. As indicated above, the heating temperature (90 °C) and time of 5 h were selected to demonstrate the effect of thermal processing and to enable comparison of our obtained data with other studies (Fischer et al., 2013; Iliopoulou et al., 2015; Sadilova et al., 2007). An aliquot of each sample was membrane-filtered (regenerated cellulose, Macherey-Nagel, 9

ACCEPTED MANUSCRIPT Düren, Germany) and analyzed by HPLC-DAD to quantitate individual anthocyanins. Furthermore, the remaining solutions were used for spectrophotometric measurements.

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Unheated samples (controls) were monitored for 5 h at room temperature, and analyzed in the

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same way. Each model solution was prepared in duplicate. According to Kirca et al. (2006b) ,

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thermal degradation of black carrot anthocyanins follows first-order kinetics (equation 1).

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(Eq. 1)

where C0 is the initial anthocyanin concentration at t= 0 h, Ct the anthocyanin content after t

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hours at a given temperature (T= 363 K), and k the kinetic constant. The natural logarithm of the ratio Ct/C0 was plotted against the heating time and the slope of the regression line was

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follows (equation 2):

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equated with -k, from which the corresponding half-life values (t1/2) were calculated as

(Eq. 2)

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t

2.7 SPECTROPHOTOMETRIC MEASUREMENTS

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Absorption spectra of all model solutions were recorded in the range of 400-800 nm with a UV/Vis-spectrophotometer (PowerwaveXS Microplate Reader, Biotek, Bad Friedrichshall, Germany) in order to determine the maximum absorbance (Amax) at its corresponding wave e gth λmax) between 510 and 540 nm. Based on the obtained data, the hyperchromic effect of co-pigmentation (CE) (Mistry, Cai, Lilley, & Haslam, 1991) was calculated according to equation 3: -

(Eq. 3)

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ACCEPTED MANUSCRIPT where A0 is the absorptio at λmax of the anthocyanin solution without added co-pigments (ratio 1:0) and ACP is the absorptio at λmax of an anthocyanin solution with added co-

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pigments.

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Additionally, the spectral shift of the absorption maximum Δλmax) was expressed as the

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difference of the wavelengths at the absorption maximum of a sample with λmaxCP) and without λmax0) added co-pigments. Positive values indicated a bathochromic shift.

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2.8 COLOR MEASUREMENTS

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CIE-L*a*b* color measurements (Gonnet, 1998) were performed using a HunterLab colorimeter (UltraScan Vis, HunterLab, Reston, VA, USA) equipped with EasyMatch ® QC

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software (HunterLab). After the heating experiments, a remainder of each sample was filled in

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1 cm path length cuvettes (Brand, Wertheim, Germany) and analyzed in the total transmission mode at 10° observer angle and illuminant D65 in duplicate. Chroma (C*), hue angle (h°), ar e, &

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Stintzing (2007).

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a d Δ *ab were ca cu ated from L*, a* a d b* coordi ates accordi g to Sadi ova,

2.9 STATISTICAL ANALYSIS Statistically significant differences of means were identified by analyses of variance (ANOVA) and Tu ey’s Test, usi g S S studio 9.4 (SAS Institute, Cary, NY, USA). Differences were considered significant at p < 0.05. All reported data represent means ± standard deviation. The standard deviations of the relative retention of anthocyanin contents, those of the hyperchromic effect of co-pigmentation, and those of the absorption difference Δλmax) were calculated by the Gaussian law of error propagation from the standard deviations of the respective mean values of individual anthocyanin contents, absorptions (ACP and A0), and wavelengths (λmax0 a d λmaxCP), respectively.

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ACCEPTED MANUSCRIPT 3. RESULTS AND DISCUSSION 3.1 ANTHOCYANIN AND NON-ANTHOCYANINIC POLYPHENOL PROFILES

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The genuine anthocyanin profile of the black carrot juice as well as the purified extracts

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obtained therefrom comprised five major anthocyanins, (compounds 1-5), exhibiting

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molecular ions [M]+ at m/z 743, 581, 949, 919, and 889, respectively (Figure 2A, Table 1). All major compounds were based on a cyanidin-aglycon, as revealed by their predominant CID fragment ion at m/z 287 in the MS2-experiment. By comparison to previously reported mass

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spectra (Algarra, Fernandes, Mateus, de Freitas, Esteves da Silva, & Casado, 2014; Kammerer et al., 2004b) compounds 1 and 2 were identified as cyanidin 3-O- ″-xylosyl-6″-

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glucosylgalactoside) (cya-xyl-glc-gal) and cyanidin 3-O- ″-xylosylgalactoside) (cya-xylgal), respectively. In addition, compound 3 was assigned to cyanidin 3-O- ″-xylosyl-6″-(6‴-

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sinapoylglucosyl)-galactoside) (cya-xyl-glc-gal sinapoyl), compound 4 to cyanidin 3-O- ″xylosyl-6″- 6‴-feruloylglucosyl)-galactoside) (cya-xyl-glc-gal feruloyl), and compound 5 to 3-O- ″-xylosyl-6″- 6‴-p-coumaroylglucosyl)-galactoside)

(cya-xyl-glc-gal

p-

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cyanidin

coumaroyl) (Gläßgen, Wray, Strack, Metzger, & Seitz, 1992). In addition, several minor

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compounds were detected. As shown in Table 1 and Figure 2A, feruloylated triglycosides of perlargonidin (compound 6) and peonidin (compound 7) were also identified by comparison of their UV/Vis absorption and mass spectra with literature data (Algarra et al., 2014). Noteworthy, the mass spectral differentiation of acylated anthocyanins from non-acylated ones was complemented by measurements of their UV/Vis absorption spectra, providing further highly characteristic information about their chemical structure. Acylated anthocyanins revealed additional absorption bands in the range of 310-360 nm (Table 1), which are due to covalently bound hydroxycinnamic acids moieties of the anthocyanin (Giusti & Wrolstad, 2003). Acylated anthocyanins (compounds 3-5, Figure 2, Table 1) accounted for the major proportion (64.7%) of the total anthocyanin content in the purified anthocyanin stock solution 12

ACCEPTED MANUSCRIPT (see section 2.2). These findings are in accordance with a previous report of Kammerer et al. (2004b) They reported the abundance of acylated anthocyanins in diverse black carrot

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varieties to range from 55 to 99%. When calculating anthocyanin contents with molecular

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weight correction factors (see section 2.4), the most abundant individual anthocyanin was

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found to be the acylated cya-xyl-glc-gal feruloyl, contributing 34.5% to the total anthocyanin content.

Regarding the non-anthocyaninic phenolic extract, 12 phenolic compounds were tentatively

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identified by comparing their UV/Vis absorption and mass spectra with literature (Kammerer

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et al., 2004a; Kramer, Maksylewicz-Kaul, Baranski, Nothnagel, Carle, & Kammerer, 2013; Suzme, Boyacioglu, Toydemir, & Capanoglu, 2014) (Figure 2B, Table 1). The qualitative profile consisted of hydroxycinnamic acids and their derivatives, mostly derived from caffeic,

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ferulic and quinic acids. As verified by an authentic reference, 5-trans-caffeoyl quinic acid (chlorogenic acid, compound 11) was found to be the main compound, which is in accordance

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with previously published data (Kammerer et al., 2004a; Kammerer et al., 2004b). Moreover, according to Clifford, Johnston, Knight, & Kuhnert (2003), chlorogenic acid was

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differentiated from other caffeoyl quinic acids (compounds 9, 10 and 13) by its fragmentation pattern. In the polyphenol stock solution, chlorogenic acid represented approximately 67% of the total non-anthocyaninic polyphenols. Furthermore, quercetin-3-O-galactoside (compound 17) was identified as the sole flavonoid in black carrot, being also in agreement with earlier reports (Kammerer et al., 2004a; Kramer et al., 2013).

3.2 ANTHOCYANIN

DEGRADATION AND EFFECT OF INTERMOLECULAR CO-PIGMENTATION

UPON THERMAL TREATMENT

Stability of total anthocyanins In order to study the effect of phenolic co-pigments on thermal stability of black carrot anthocyanins, the purified anthocyanins were re-combined with different amounts of non13

ACCEPTED MANUSCRIPT anthocyaninic black carrot phenolics and chlorogenic acid, respectively, yielding mass ratios between 1:0 (without co-pigment) and 1:162. Irrespective of the added co-pigment amount,

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heat exposure to 90 °C resulted in a rapid degradation of total anthocyanins, following first-

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order kinetics (R2 > 0.9871; Table 2, Figure 3) as previously described (Kirca et al., 2006a). In

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contrast, the decline of total anthocyanins was negligible (max. 3%) in our control experiment (5 h at room temperature; Figure 3).

As illustrated by Figure 3, heating anthocyanins for 5 h at 90 °C without added co-pigments

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resulted in a decrease to 37.6% of their initial concentration (Figure 3A). When anthocyanin-

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co-pigment ratios were in the range of 1:0.43 to 1:3.6, the residual anthocyanin contents in all model solutions were only insignificantly higher (38.1-39.2%) than without the addition of co-pigments. These findings were also reflected by their corresponding t1/2 values (Table 2),

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being only slightly prolonged from 3.63 h (ratio 1:0) to a maximum of 3.79 h (ratio 1:3.6). Consequently, the genuine concentration of non-anthocyaninic phenolics did not suffice to

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evoke a substantial heat-stabilizing intermolecular co-pigmentation effect, therefore being considered widely irrelevant for anthocyanin stability in carrot.

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However, when adding higher amounts of chlorogenic acid (i.e., at ratios of 1:9.4 to 1:162), anthocyanin stability was significantly improved (Figure 3B). For instance, co-pigment addition at a ratio of 1:9.4 led to a 5.3% increase in anthocyanin retention after 5 h at 90 °C as compared to the control without added co-pigments. Interestingly, the addition of higher copigment concentrations (1:85 and 1:162) only resulted in an increase of 5.6% and 8.7%, which was less than expected. Nevertheless, the thus obtained half-life for the anthocyanins in the model solution at a pigment:co-pigment ratio of 1:162 was 4.72 h, being significantly greater than that without co-pigments (3.63 hours). These findings are in agreement with previous reports (Maccarone et al., 1985; Mazza & Brouillard, 1987; Wilska-Jeszka & Korzuchowska, 1996).

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ACCEPTED MANUSCRIPT In brief, our observations strongly suggest that the effect of naturally occurring levels of nonanthocyaninic phenolics on the stability of black carrot anthocyanins is negligible.

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Nevertheless, intermolecular co-pigmentation may be used to enhance stability of black carrot

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anthocyanins during thermal treatment when co-pigments are added at higher levels.

Stability of individual anthocyanins

In all our experiments, acylated anthocyanins exerted significantly better stability than the

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non-acylated ones when heating at 90 °C, irrespective of the added co-pigment levels (see

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examples for ratios 1:0 and 1:162 in Figure 4). Superior stability of acylated black carrot anthocyanins was previously reported by Iliopoulou, Thaeron, Baker, Jones, & Robertson (2015) and Sadilova, Carle, & Stintzing (2007) at pH values of 3.6 and 3.5, respectively.

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Regarding the individual pigments, the non-acylated diglycosylated anthocyanin cya-xyl-gal (compound 2) exhibited by far the poorest heat stability, presenting losses of up to 81.7%

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after 5 h at 90 °C without added co-pigments. At the same time, only 65.2% of the trisaccharide cya-xyl-glc-gal (compound 1) degraded. For comparison, among the acylated

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anthocyanins, only 56.2% of cya-xyl-glc-gal sinapoyl (compound 3), 53.8% of cya-xyl-glcgal p-coumaroyl (compound 5), and 51.1 % of the feruloyl derivative (compound 4) were degraded in the sample without added co-pigments. Previously, Stintzing et al. (2002) determined the lowest hydration constant (pKH = 3.1) for the disaccharide cya-xyl-gal, a slightly higher value (pKH = 3.3) for the trisaccharide cya-xylglc-gal, while cya-xyl-glc-gal feruloyl was the pigment exhibiting the highest value (pKH = 4.4). Thus, since our experiments were conducted at pH 3.6, cya-xyl-gal should have mainly existed in its hemiketal/chalcone form, followed by the cya-xyl-glc-gal also predominantly occurring as hemiketal/chalcone. In contrast, the flavylium form of the acylated anthocyanins should have prevailed at pH 3.6. The red flavylium cation has been previously described to be

15

ACCEPTED MANUSCRIPT more stable than the respective hemiketal/chalcones (Goto & Kondo, 1991), thus explaining the greater stability of acylated anthocyanins as observed in our experiments.

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Comparing pigment retention of acylated anthocyanins with and without added co-pigments,

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the addition of co-pigments at a ratio of 1:162 resulted in a 10.7% enhanced retention after 5 h

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at 90 °C compared to the control without co-pigments. In contrast, the stability of nonacylated anthocyanins was only raised by 3.4% (Figure 4). The same observations were made for half-life values of acylated anthocyanins, which were increased from 4.7 h (without added

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co-pigments, ratio 1:0) to 6.7 h when co-pigment was added (1:162 anthocyanin:co-pigment

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ratio), while those of the non-acylated ones were merely increased from 2.3 to 2.6 h when raising pigment:co-pigment ratios from 1:0 to 1:162, respectively. Although being less pronounced and partly not reaching statistical significance, similar observations were made

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for all other anthocyanin:co-pigmentat ratios These findings were unexpected, since stabilization of non-acylated anthocyanins was supposed to be more effective by

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intermolecular co-pigmentation, because their perfectly planar structures would allow stacking on both sides of the molecules when co-pigments are added (Dangles et al., 1993;

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Eiro & Heinonen, 2002). However, enhanced co-pigmentation of non-acylated anthocyanins was not confirmed by our study. Instead, stability of acylated anthocyanins appeared to be more efficiently enhanced by the addition of co-pigments than that of the non-acylated ones. Noteworthy, intermolecular co-pigment-anthocyanin complexes were previously shown to be widely dissociated at high temperatures (Brouillard et al., 1989), while knowledge about the dissociation of intramolecular co-pigments from the anthocyanin core is lacking. Besides copigmentation, the added chlorogenic acid may also have acted as protective antioxidant. However, further studies with single isolated compounds may provide deeper insights, thus being indispensible.

16

ACCEPTED MANUSCRIPT 3.3 EFFECT OF CO-PIGMENTS ON VISIBLE ABSORPTION SPECTRA Since the anthocyanin-co-pigment complexes may have been partially or widely dissociated at

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the temperature of our heating experiment (90°C), the effect of co-pigmentation on the

at λmax (524 nm) was 1.21 for anthocyanin

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As shown in Figures 5A and B, absorptio

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absorption behavior of the anthocyanins was studied at room temperature (22 °C).

solutions without co-pigment (1:0). When adding non-anthocyaninic black carrot phenolics at the highest ratio (1:3.6; Figure 5A), the absorption was only slightly enhanced to 1.31.

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Therefore, the intermolecular co-pigmentation effect (CE; Table 3) resulted in a maximum of

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7.7%. The bathochromic shift at λmax in model solutions with genuine black carrot phenolics never exceeded 0.5 nm, regardless of the amount of added co-pigments from ratios of 1:0.43 to 1:3.6.

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However, when higher amounts of chlorogenic acid were added (ratio from 1:9.4 to 1:162; Figure 5B), absorptions markedly increased from 1.21 to 1.30-1.48, respectively.

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Consequently, a maximum hyperchromic co-pigmentation effect (CE) of 21.9% was detected at the highest anthocyanin:chlorogenic acid ratio of 1:162 (Table 3). Additionally, the

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supplementation of the anthocyanin solution with the above mentioned amounts of chlorogenic acid led to a bathochromic shift of the absorption maxima amounting to 1.0–10.5 nm. As described previously (Wilska-Jeszka & Korzuchowska, 1996), when adding hydroxycinnamic acids as copigments, the hyperchromic shift of intermolecular copigmentation was more pronounced than the bathochromic shift. Interestingly, the CE in the samples with added non-anthocyaninic black carrot phenolics at a ratio of 1:3.6 and chlorogenic acid at 1:9.4 were unexpectedly similar (CE of approximately 7.7% for both solutions), although a lower CE was anticipated at the ratio 1:3.6. Noteworthy, the relative abundance of chlorogenic acid in the genuine non-anthocyaninic black carrot phenolic extract amounted to approximately 67%, accompanied by a total of approximately 33% of other hydroxycinnamic acid derivatives and non-anthocyaninic phenolics (Table 1). The latter 17

ACCEPTED MANUSCRIPT hydroxycinnamic acid derivatives and phenolics might have additionally contributed to the unexpectedly high CE (7.7%; Table 3) observed in the model solution with genuine black

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carrot phenolics at the ratio of 1:3.6.

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3.4 COLOR CHANGES RESULTING FROM CO-PIGMENTATION AND HEATING The effect of co-pigment addition on color of the model solutions was monitored by measurements of CIE-L*, a*, b*, C* and h° values (Table 4). When compared to the sample

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being devoid of phenolic co-pigments (L* = 49.3), lightness (L*) was significantly decreased

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at the highest supplementation level of non-anthocyaninic phenolics (L* = 47.8 at the 1:3.6 ratio). L* steadily decreased further when the chlorogenic acid level was increased from the anthocyanin:co-pigment ratio of 1:9.4 (L* = 48.5), to 1:85 (L* = 45.4), and 1:162 (L* = 43.5).

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These findings are in agreement with literature data (Rein & Heinonen, 2004). Higher a* va ues “red ess” were observed at higher eve s of added co-pigments, although this

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relationship was not strictly concentration-dependent (Table 4). Moreover, b* values “ye ow ess” augme ted whe addi g co-pigments; however, possibly being related to the

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naturally pale yellowish color of phenolic co-pigments. Chroma C* of anthocyanin solutions has been previously observed to increase when adding co-pigments (Fischer et al., 2013; Gonnet, 1999; Rein & Heinonen, 2004; Shikov et al., 2008). Accordingly, this trend became obvious in our study when adding higher co-pigment levels (at ratios 1:3.6 to 1:162), where the color of the co-pigmented samples appeared to be more vivid than that of the non-copigmented control. When adding genuine co-pigments at higher levels (ratios of 1:1.7 to 1:3.6), the hue angle shifted significantly towards higher values, i.e. from red hues (18.620.2°) to slightly more brownish tonalities (20.6-21.1°). Moreover, total color difference Δ *ab of a samp es ge era y was visib e for the huma eye at Δ *ab >

as reported

previously (Gonnet, 1998), except for the anthocyanin:co-pigment ratios of 1:0.9 and 1:1.7.

18

ACCEPTED MANUSCRIPT In addition to the effect of co-pigment addition, color changes evoked by heating at 90 °C for 5 h were monitored. Generally, lightness L* of the heated samples was increased by

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approximately 26.4-28.9%, presumably due to the thermal decay of anthocyanins during heat

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treatment. Similarly, a relative decline of a*, b* and C* was observed. For instance, b* values

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revealed decreases from -16.1% (at a ratio of 1:3.6) to up to -25.6% (at a ratio 1:9.4, Table 4). A similar loss of color intensity (C*) after 6 h at 95 °C at pH 3.5 was also reported for purified black carrot anthocyanins by Sadilova et al. (2007). The heated anthocyanin sample,

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having an anthocyanin:co-pigment ratio of 1:162, revealed an exceptionally low relative

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decrease of the b* value (-9.2%), which may be attributed to the putative oxidation of chlorogenic acid during thermal treatment, resulting in brownish pigments. Fischer et al. (2013) determined similar tendencies of CIE-L*a*b* color values after thermal treatment of

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model solutions containing anthocyanins and genuine co-pigments from pomegranate where

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anthocyanins are non-acylated.

3.5 OUTLOOK ON INTERMOLECULAR CO-PIGMENTATION USING FOOD GRADE EXTRACTS

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According to the above described results, a significant co-pigmentation effect can only be achieved at comparably high co-pigment dosages. Challenging the applicability of our findings, a food-grade chlorogenic acid-rich green coffee bean extract was examined regarding its co-pigmentation properties. As determined by HPLC-DAD (Figure 6) the aqueous extract contained mainly hydroxycinnamic acid derivatives with a major contribution of 65.8% chlorogenic acid to the total phenolic content (4,637 mg/L), which was in agreement with previously published data (Alonso-Salces, Serra, Reniero, & Héberger, 2009; Esquivel & Jiménez, 2012). The extract was obtained by aqueous extraction and subsequent concentration, allowing a ratio of black carrot anthocyanins:co-pigments of 1:52. As shown in Figure 7, the combination of equal volumes of black carrot anthocyanins and the aqueous green coffee bean extract resulted in a CE of 8.4 ± 0.7% and a bathochromic shift of 6 ± 0 nm. 19

ACCEPTED MANUSCRIPT Objective color measurement confirmed the trend described above for our model solutions with added chlorogenic acid. The L* value significantly decreased from 51.7 to 45.7 when

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adding the green coffee bean extract, while the a* value remained widely constant, i.e. a*

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changed from 64.4 (anthocyanin:co-pigment ratio 1:0) to 64.1 (ratio 1:52). The strongest

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effect when adding the pale yellowish green coffee bean extract to black carrot anthocyanins was observed for the b* values, which increased from 19.9 (1:0) up to 28.2 (1:52). A color shift towards yellow/orange was also observed for the hue angle, which moved from 17.1° up

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to 23.7°. As described above, adding co-pigments to black carrot anthocyanins increased

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color saturation of the samples at different pigment:co-pigment ratios. Accordingly, the addition of coffee bean extract to the anthocyanin solution increased chroma C* from 67.4 (1:0) to 70.0 (1:52). All spectral shifts as well as the color changes were in approximate

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agreement with those expected according to our experiments with the chlorogenic acid model

4. CONCLUSIONS

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solutions.

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In brief conclusion, color and thermal stability of black carrot was only marginally improved by the presence of non-anthocyaninic polyphenols in their genuine concentration. However, strong intermolecular co-pigmentation, i.e. enhanced color expression and thermal stability, were achieved when adding the co-pigments at concentrations (m/m) being at least 9.4-fold higher than that of the anthocyanins. Therefore, breeding of black carrot roots with increased amounts of non-anthocyaninic phenolic acids for the obtainment of “ atura y co-pigme ted” carrots would require enormous gain of their co-pigment levels, representing the most challenging goal. Instead, color stability of black carrot preparations might be enhanced by selecting and breeding carrots with increased proportions of the highly-stable acylated anthocyanins, as has been previously suggested by Baranski, Goldman, Nothnagel, & Scott (2016). According to our findings, coloring foodstuff made from black carrot containing 20

ACCEPTED MANUSCRIPT increased amounts of acylated anthocyanins would also strongly benefit from added copigments. The supplementation of black carrot anthocyanins with non-anthocyaninic co-

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pigments, e.g., from plant extracts such as green coffee bean extract, may represent a

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promising option to stabilize and enhance red color shades from black carrot.

ACKNOWLEDGEMENTS

This work was financially supported by Diana Naturals SAS (Symrise Group, Rennes,

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France). We are grateful to Dr. Delphine Laroque (Diana Naturals SAS) for her helpful

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comments on the manuscript.

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ACCEPTED MANUSCRIPT References

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Algarra, M., Fernandes, A., Mateus, N., de Freitas, V., Esteves da Silva, J. C. G., & Casado, J. (2014). Anthocyanin profile and antioxidant capacity of black carrots (Daucus carota L. ssp. sativus var. atrorubens Alef.) from Cuevas Bajas, Spain. Journal of Food Composition and Analysis, 33(1), 71-76.

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Alonso-Salces, R. M., Serra, F., Reniero, F., & Héberger, K. (2009). Botanical and geographical characterization of green coffee (Coffea arabica and Coffea canephora): chemometric evaluation of phenolic and methylxanthine contents. Journal of Agricultural and Food Chemistry, 57(10), 4224-4235.

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Appel, K., Meiser, P., Millán, E., Collado, J. A., Rose, T., Gras, C. C., Carle, R., & Muñoz, E. (2015). Chokeberry (Aronia melanocarpa (Michx.) Elliot) concentrate inhibits NF-κB and synergizes with selenium to inhibit the release of pro-inflammatory mediators in macrophages. Fitoterapia, 105, 73-82. Asen, S., Stewart, R. N., & Norris, K. H. (1972). Co-pigmentation of anthocyanins in plant tissues and its effect on color. Phytochemistry, 11(3), 1139-1144.

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Baranski, R., Goldman, I., Nothnagel, T., & Scott, J. W. (2016). Improving color sources by plant breeding and cultivation. In R. Carle & R. M. Schweiggert, Handbook on Natural Pigments in Food and Beverages: Industrial Applications for Improving Color, (1st ed.) Philadelphia, PA, USA, in press.: Elsevier Inc.

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Brouillard, R., Mazza, G., Saad, Z., Albrecht-Gary, A., & Cheminat, A. (1989). The co-pigmentation reaction of anthocyanins: a microprobe for the structural study of aqueous solutions. Journal of the American Chemical Society, 111(7), 2604-2610.

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Cavalcanti, R. N., Santos, D. T., & Meireles, M. A. A. (2011). Non-thermal stabilization mechanisms of anthocyanins in model and food systems-An overview. Food Research International, 44(2), 499-509. Clifford, M. N., Johnston, K. L., Knight, S., & Kuhnert, N. (2003). Hierarchical scheme for LC-MSn identification of chlorogenic acids. Journal of Agricultural and Food Chemistry, 51(10), 2900-2911. Dangles, O., Saito, N., & Brouillard, R. (1993). Anthocyanin intramolecular copigment effect. Phytochemistry, 34(1), 119-124. Eiro, M. J., & Heinonen, M. (2002). Anthocyanin color behavior and stability during storage: Effect of intermolecular copigmentation. Journal of Agricultural and Food Chemistry, 50(25), 7461-7466. Esquivel, P., & Jiménez, V. M. (2012). Functional properties of coffee and coffee byproducts. Food Research International, 46(2), 488-495. Figueiredo, P., George, F., Tatsuzawa, F., Toki, K., Saito, N., & Brouillard, R. (1999). New features of intramolecular copigmentation by acylated anthocyanins. Phytochemistry, 51(1), 125-132. 22

ACCEPTED MANUSCRIPT Fischer, U. A., Carle, R., & Kammerer, D. R. (2013). Thermal stability of anthocyanins and colourless phenolics in pomegranate (Punica granatum L.) juices and model solutions. Food Chemistry, 138(2-3), 1800-1809.

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Giusti, M. M., & Wrolstad, R. E. (2003). Acylated anthocyanins from edible sources and their applications in food systems. Biochemical engineering journal, 14(3), 217225.

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Gläßgen, W. E., Wray, V., Strack, D., Metzger, J. W., & Seitz, H. U. (1992). Anthocyanins from cell suspension cultures of Daucus carota. Phytochemistry, 31(5), 1593-1601.

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Gonnet, J. (1999). Colour effects of co-pigmentation of anthocyanins revisited—2. A colorimetric look at the solutions of cyanin co-pigmented by rutin using the CIELAB scale. Food Chemistry, 66(3), 387-394.

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Gonnet, J. (1998). Colour effects of co-pigmentation of anthocyanins revisited—1. A colorimetric definition using the CIELAB scale. Food Chemistry, 63(3), 409-415.

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Goto, T., & Kondo, T. (1991). Structure and molecular stacking of anthocyanins: flower color variation. Angewandte Chemie International Edition in English, 30(1), 1733.

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Gras, C. C., Carle, R., & Schweiggert, R. M. (2015). Determination of anthocyanins from black carrots by UHPLC-PDA after ultrasound-assisted extraction. Journal of Food Composition and Analysis, 44, 170-177.

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Iliopoulou, I., Thaeron, D., Baker, A., Jones, A., & Robertson, N. (2015). Analysis of the thermal degradation of the individual anthocyanin compounds of black carrot (Daucus carota L.): A new approach using high-resolution proton nuclear magnetic resonance spectroscopy. Journal of Agricultural and Food Chemistry, 63(31), 70667073. Kammerer, D., Carle, R., & Schieber, A. (2004a). Characterization of phenolic acids in black carrots (Daucus carota ssp. sativus var. atrorubens Alef.) by highperformance liquid chromatography/ electrospray ionization mass spectrometry. Rapid Communications in Mass Spectrometry, 18(12), 1331-1340. Kammerer, D., Carle, R., & Schieber, A. (2004b). Quantification of anthocyanins in black carrot extracts (Daucus carota ssp. sativus var. atrorubens Alef.) and evaluation of their color properties. European Food Research and Technology, 219(5), 479-486. Kirca, A., Özkan, M., & Cemeroglu, B. (2006a). Effects of temperature, solid content and pH on the stability of black carrot anthocyanins. Food Chemistry, 101(1), 212218. Kirca, A., Özkan, M., & Cemeroglu, B. (2006b). Stability of black carrot anthocyanins in various fruit juices and nectars. Food Chemistry, 97(4), 598-605.

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ACCEPTED MANUSCRIPT Kramer, M., Maksylewicz-Kaul, A., Baranski, R., Nothnagel, T., Carle, R., & Kammerer, D. R. (2013). Effects of cultivation year and growing location on the phenolic profile of differently coloured carrot cultivars. Journal of Applied Botany and Food Quality, 85(2), 235-247.

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Maccarone, E., Maccarone, A., & Rapisarda, P. (1985). Stabilization of anthocyanins of blood orange fruit juice. Journal of Food Science, 50(4), 901-904.

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Malien-Aubert, C., Dangles, O., & Amiot, M. J. (2001). Color stability of commercial anthocyanin-based extracts in relation to the phenolic composition. Protective effects by intra- and intermolecular copigmentation. Journal of Agricultural and Food Chemistry, 49(1), 170-176.

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Mazza, G., & Brouillard, R. (1987). Recent developments in the stabilization of anthocyanins in food products. Food Chemistry, 25(3), 207-225.

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Mistry, T. V., Cai, Y., Lilley, T. H., & Haslam, E. (1991). Polyphenol interactions. Part 5. Anthocyanin co-pigmentation. Journal of the Chemical Society, Perkin Transactions 2, (8), 1287-1296.

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Rein, M. J., & Heinonen, M. (2004). Stability and enhancement of berry juice color. Journal of Agricultural and Food Chemistry, 52(10), 3106-3114.

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Sadilova, E., Carle, R., & Stintzing, F. C. (2007). Thermal degradation of anthocyanins and its impact on color and in vitro antioxidant capacity. Molecular Nutrition and Food Research, 51(12), 1461-1471.

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Shikov, V., Kammerer, D. R., Mihalev, K., Mollov, P., & Carle, R. (2008). Heat stability of strawberry anthocyanins in model solutions containing natural copigments extracted from rose (Rosa damascena Mill.) petals. Journal of Agricultural and Food Chemistry, 56(18), 8521-8526. Stintzing, F. C., & Carle, R. (2004). Functional properties of anthocyanins and betalains in plants, food, and in human nutrition. Trends in Food Science and Technology, 15(1), 19-38. Stintzing, F. C., Stintzing, A. S., Carle, R., Frei, B., & Wrolstad, R. E. (2002). Color and antioxidant properties of cyanidin-based anthocyanin pigments. Journal of Agricultural and Food Chemistry, 50(21), 6172-6181. Suzme, S., Boyacioglu, D., Toydemir, G., & Capanoglu, E. (2014). Effect of industrial juice concentrate processing on phenolic profile and antioxidant capacity of black carrots. International Journal of Food Science and Technology, 49(3), 819-829. Wilska-Jeszka, J., & Korzuchowska, A. (1996). Anthocyanins and chlorogenic acid copigmentation - Influence on the colour of strawberry and chokeberry juices. Zeitschrift fur Lebensmittel -Untersuchung und -Forschung, 203(1), 38-42.

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ACCEPTED MANUSCRIPT FIGURE CAPTIONS Figure 1: Separation and purification of anthocyanins and non-anthocyaninic polyphenols

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from black carrot and preparation of model solutions for the evaluation of anthocyanin heat

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stability.

Figure 2: HPLC-DAD chromatograms of aqueous anthocyanin (A) and non-anthocyaninic polyphenol extracts (B) from black carrot detected at 520 and 320 nm, respectively, obtained

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after liquid-liquid extraction.

Figure 3: Degradation of total anthocyanins in black carrot model solutions (pH 3.6) with different anthocyanin:co-pigment-ratios (1:0 – 1:162) at room temperature and during heating

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at 90 °C for 5 hours. The co-pigments ratios were obtained by the addition of genuine nonanthocyaninic phenolics (A) and chlorogenic acid (B). Mean value data points marked with

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different symbols are significantly different (p<0.05) to those data points with a different or

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no symbol within one sampling point (0 h, 1 h, 2 h, 3 h, and 5 h).

Figure 4: Degradation of total acylated and non-acylated black carrot anthocyanins during heating at 90 °C without copigment (1:0) and with chlorogenic acid (1:162) at pH 3.6. Mean value data points marked with different symbols are significantly different (p<0.05) to those data points with a different or no symbol within one sampling point (0 h, 1 h, 2 h, 3 h, and 5 h).

Figure 5: Visible absorption spectra (400-700 nm) of black carrot anthocyanins co-pigmented with genuine non-anthocyaninic polyphenols (A) and chlorogenic acid (B) recorded at 22 °C.

25

ACCEPTED MANUSCRIPT Figure 6: HPLC-DAD chromatogram of phenolic compounds from an aqueous green coffee bean extract as monitored at 280 nm. Compound identification was accomplished using

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authentic standards (compounds c, d) and by comparing elution order and UV-spectra

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(compounds a,b,e-i) with literature (Alonso-Salces et al., 2009): a, b, e-i: hydroxycinnamic

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acid derivatives; c: 5-trans-caffeoylquinic acid (chlorogenic acid); d: caffeine. Photo of green coffee beans used with permission (R. Schweiggert).

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Figure 7: Visible absorption spectra (400-700 nm) of black carrot anthocyanins without (1:0)

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and with co-pigments (1:52) from an aqueous green coffee bean extract. Spectra were

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recorded at 22 °C.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Table 1: Retention times, UV and mass spectral characteristics of purified anthocyanin and non-anthocyaninic polyphenol-extracts from black carrot concentrate. UV-Vis

HPLC/ESI

HPLC/ESI

HPLC/ESI (+/-)-MSn m/z

time (min)

absorption

(+) [M]+

(-)

(relative intensity %)

maxima

m/z

[M-H]m/z

23.1

919

TE D

MS2 [919]: 287 (100)

34.5

889

MS2 [889]: 287 (100)

6.3

903

MS2 [903]: 271 (100)

0.3

933

MS2 [933]: 301 (100)

0.5

MS2 [365]: 179 (21), 185 (41), 203 (100)

1.2

cyanidin xyl-gal

17.1

279, 519

581

3

cyanidin xyl-glc-gal sinapoyl

17.6

285, 334, 533

949

4

cyanidin xyl-glc-gal feruloyl

19.1

284, 332, 531

5

cyanidin

p-

20.3

284, 317, 527

xyl-glc-gal

20.9

n.d.

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2

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MS2 [949]: 287 (100)

743

feruloyl 7

peonidin xyl-glc-gal feruloyl

21.2

280, 335, 524

8

caffeic acid derivative

11.2

240, 302sh, 326

365

the

extracts [%]

27.5

279, 518

perlargonidin

in

MS2 [581]: 287 (100)

15.3

6

proportionc

7.8

cyanidin xyl-glc-gal

coumaroyl

relative

MS2 [743]: 287 (100)

1

xyl-glc-gal

US

(nm)

T

retention

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compd name

CR

no.

MS3 [365203]: 69 (32), 97 (100), 127 (19), 141 (25), 185 (17) 9

caffeoylquinic acid

12.4

240, 303sh, 325

353

MS2 [353]: 135 (21), 179 (38), 191 (100)

1.8

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MS3 [353191]: 85 (93), 171 (62), 173 (100), acid

21.3

239, 304sh, 326

MS2 [353]: 135 (10), 173 (100), 179 (55), 191

353

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4-caffeoylquinic

4.5

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10

(57)

CR

(cryptochlorogenic acid)

MS3 [353173]: 93 (100)

5-trans-caffeoylquinic

acid

22.7

707b (353)

US

11

236, 303sh, 325

5-feruloylquinic acid

27.1

36.6

240, 302sh, 325

355

235, 315

239, 305sh, 325

15

caffeic acid derivative

38.2

239, 302sh, 327

66.9

MS3 [707353]: 191 (100) MS2 [355]: 134 (10), 175 (43), 191 (25), 193

10.0

(100), 217 (53) MS3 [355193]: 134 (100), 178 (20)

353

MS2 [353]: 191 (100)

3.1

MS3 [353191]: 127 (100) 367

AC

14

caffeoylquinic acid

24.7

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13

ferulic acid derivative

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12

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(chlorogenic acid)a

MS2 [707]: 353 (100)

MS2 [367]: 191 (100)

1.3

MS3 [367191]: 85 (100) 365

MS2 [365]: 179 (21), 185 (41), 203 (100)

7.6

MS3 [365203]: 69 (32), 97 (100), 127 (19), 141 (25), 185 (17) 16

caffeic acid derivative

40.7

242, 305sh, 326

367

MS2 [367]: 135 (65), 179 (100), 191 (39)

2.2

MS3 [367179]: 135 (100)

35

ACCEPTED MANUSCRIPT

17

quercetin-3-o-galactoside

54.9

257, 356

MS2 [463]: 301 (100)

463

0.7

di-caffeic acid derivative

56.7

239, 304sh, 327

MS2 [527]: 185 (17), 203 (36), 365 (100)

527

0.7

CR

18

IP

T

MS3 [463301]: 151 (100), 179 (67)

n.d.

531

MS2 [531]: 175 (18), 193 (29), 337 (100), 365

n.d.

(15) MS3 [531337]: 175 (100), 193 (80)

TE D

Identification was corroborated with authentic standard.

b

Dimeric 5-trans-caffeoylquinic acid ion [2M-H]-

Value refers to the proportion of a given anthocyanin or phenolic acid to the total anthocyanins or total phenolic acids, respectively.

CE P

c

73.5

AC

a

ferulic acid derivative

MA N

19

US

MS3 [527365]: 185 (55), 203 (100)

36

ACCEPTED MANUSCRIPT Table 2: Reaction rate constant k and half-life values (t1/2) of anthocyanins in model solutions supplemented with different levels (m/m) of co-pigments (genuine black carrot phenolics and chlorogenic acid). co-pigment

-k [h-1]

anthocyanin:co-pigment

t1/2 [h]

ratio (m/m) 0.191a (0.9937)*

1:0.43b#

0.188a (0.9971)

anthocyaninic

1:0.9

0.183a (0.9904)

black

1:1.7

0.188a (0.9963)

3.69b

1:3.6

0.186a (0.9916)

3.73b

1:9.4

0.170a,b (0.9952)

4.08a,b

1:85

0.166 a,b(0.9985)

4.18a,b

1:162

0.147b (0.9871)

4.72a

phenolics

chlorogenic acid

*a

IP

SC R

carrot

NU

non-

3.63b

MA

genuine

T

1:0 heated

3.69b 3.79b

Numbers in parentheses represent coefficients of determination (R2).

b#

D

Genuine anthocyanin:co-pigment ratio in the used black carrot concentrate: 1:0.32.

AC

CE P

TE

Different letters within a column indicate significantly different means (p<0.05).

37

ACCEPTED MANUSCRIPT Table 3: Visible hyperchromic effect of co-pigme tatio a d Δλmax at t h of mode so utio s usi g different anthocyanin:co-pigment ratios.Absorbance at absorption maximum (Amax), wavelength at absorption maximum λmax), hyperchromic effect of co-pigmentation (CE), and bathochromic effect of co-pigmentation Δλmax = λmax, with added co-pigments - λmax, without added co-pigments) in unheated model solutions using different anthocyanin:co-pigment ratios. anthocyanin:

at λmax [%]

Amax

1:0

1.21±0.02d

1:0.43*a

SC R

ratio (m/m) -

524±1c

-

1.21±0.00d

-0.01±1.3d

524±0c

0.5±0.7c

1:0.9

1.25±0.01d

2.8±1.5c,d

524±0c

0.5±0.7c

1:1.7

1.24±0.01d

2.1±1.7c,d

524±0c

0.5±0.7c

1:3.6

1.31±0.00c

7.7±1.4c

524±0c

0.5±0.7c

1:9.4

1.30±0.01c

7.6±1.8c

525±1c

1.0±1.0c

1:85

1.39±0.02b

14.3±2.3b

530±0b

6.5±0.7b

1:162

1.48±0.02a

21.9±2.3a

534±0a

10.5±0.7a

non-

carrot

MA

phenolics

chlorogenic

D

acid

Genuine anthocyanin:co-pigment ratio in the used black carrot concentrate: 1:0.32.

TE

a*

NU

anthocyaninic black

Δλmax [nm]

IP

co-pigment

genuine

λmaxΛmax [nm]

T

co-pigment

AC

CE P

Different letters within a column indicate significantly different means (p<0.05).

38

ACCEPTED MANUSCRIPT

T

Table 4: CIE-L*, a*, b*, C*, and h° color values of solutions at different anthocyanin:co-pigment ratios obtained with genuine phenolics from black carrot and chlorogenic acid at a d after 5 h at 9 ° . The tota co or differe ce Δ *ab expressed the geometric distance of the color coordinates of a solution with added co-pigments to those at t= 0 h of a solution without added co-pigments at t= 0 h. anthocyanin:co-pigment ratio

IP

heating time

C*

h°

Δ *ab

49.3±0.0a

48.9±0.0b,c

49.2±0.2a,b

5 h at 90 °C

62.6±0.2a

62.3±0.2a,b

62.2±0.3a,b

0h

65.3±0.0d

64.6±0.0e

5 h at 90 °C

50.8±0.4c

49.4±0.1e

0h

24.1±0.0c,d

21.7±0.1e

5 h at 90 °C

17.6±0.1e

17.7±0.1e

0h

69.6±0.0d,e

68.2±0.1f

5 h at 90 °C

53.8±0.4d

52.5±0.2e

0h

20.2±0.0c

5 h at 90 °C

48.9±0.1b,c

chlorogenic acid 1:3.6

1:9.4

1:85

1:162

47.8±0.0e

48.5±0.0d

45.4±0.0f

43.5±0.0g

62.2±0.1a,b

61.4±0.1b

61.4±0.2b

58.4±0.2c

55.3±0.5d

65.3±0.0d

65.5±0.0d

65.4±0.1d

66.1±0.0c

68.0±0.0a

67.3±0.0b

50.5±0.3c,d

50.1±0.3c,d

49.6±0.2d,e

52.5±0.1b

54.8±0.2a

55.6±0.3a

23.9±0.2d

24.6±0.1b

25.3±0.3a

24.0±0.0c,d

24.4±0.1b,c

25.5±0.0a

18.5±0.0d

19.6±0.1c

21.2±0.0b

17.8±0.1e

19.4±0.1c

23.1±0.2a

69.6±0.1e

69.9±0.1c,d

70.1±0.2b,c

70.3±0.0b

72.3±0.0a

72.0±0.0a

53.7±0.2d

53.8±0.3d

53.9±0.1d

55.5±0.1c

58.1±0.2b

60.3±0.2a

18.6±0.1e

20.1±0.1c,d

20.6±0.1b

21.1±0.2a

20.0±0.0c,d

19.8±0.1d

20.7±0.0b

19.1±0.0d

19.8±0.1c

20.1±0.1c

21.3±0.2b

23.2±0.1a

18.8±0.1e

19.5±0.0c,d

22.6±0.3a

0.0±0.0.

2.6±0.0

0.3±0.0

0.7±0.1

2.0±0.1

1.1±0.1

MA N

0h

TE D

b*

1:1.7

CE P

a*

1:0.9

AC

L*

1:0.43

US

1:0

CR

genuine black carrot co-pigments

4.8±0.0

6.4±0.0

Different letters within a line indicate significantly different means (p< 0.05).

39

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Graphical abstract

40

ACCEPTED MANUSCRIPT Highlights Color intensity and thermal stability of black carrot anthocyanins were studied.



Anthocyanins were combined with plant extracts and chlorogenic acid as co-

T



Chlorogenic acid addition particularly enhanced stability of acylated

SC R



IP

pigments.

anthocyanins.

CE P

TE

D

MA

NU

A green coffee bean extract enabled effective food-grade applications.

AC



41