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HPLC-DAD-MS profiling of polyphenols responsible for yelloworange color in apple juices of different French cider apple varieties Erell LE DEUN, Remmelt van der Werf, Gildas Lebail, Jean-Michel Le Quéré, and Sylvain Guyot J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00988 • Publication Date (Web): 18 May 2015 Downloaded from http://pubs.acs.org on May 28, 2015

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Journal of Agricultural and Food Chemistry

HPLC-DAD-MS profiling of polyphenols responsible for yellow-orange color in apple juices of different French cider apple varieties Erell Le Deun, Remmelt Van der Werf, Gildas Le Bail, Jean-Michel Le Quéré and Sylvain Guyot*

*Corresponding author (Tel: 0033 2 23 48 52 09; Fax: 0033 2 23 48 52 10; E-mail: [email protected])

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ABSTRACT:

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The pigments responsible for the yellow-orange coloration of apple juices have remained

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largely unknown up to now. Four French cider apple juices were produced in conditions

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similar to those used in the cider-making industry. The oxidized juices, characterized using

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the CIE Lab parameters, displayed various colors depending on the apple variety and native

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phenolic composition. HPLC-DAD-MS revealed contrasting pigment profiles related to

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oxidized tanning and non-tanning molecules. The latter were divided into two groups

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according to their polarity and their visible spectra. Regarding phenolic classes, flavanol

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monomers and hydroxycinnamic acids played an essential role in the formation of oxidation

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products. Interestingly, dihydrochalcones appeared to include precursors of some yellow

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compounds. Indeed, the yellow pigment phloretin xyloglucoside oxidation product

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(PXGOPj), derived from phloretin xyloglucoside, was clearly identified in apple juices as a

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xyloglucose analogue of the yellow pigment phloridzin oxidation product (POPj), previously

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characterized in a model solution by Le Guernevé et al. (2004).

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KEYWORDS: Malus domestica, enzymatic browning, oxidation, dihydrochalcones, tannins

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INTRODUCTION

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The visual appearance of food and drinks is crucial because it affects the first impression felt

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by the consumer. As the main component of appearance, color is an indicator of palatability

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and quality of food and beverages1. It has to be within an expected range, which depends on

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the type and origin of the food product as well as the educational background, ethnic origin,

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age, and sex of the consumer. Consequently, color is likely to drive food preference and thus

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influence purchase2. In addition, color is known to influence other sensory characteristics

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such as identity and intensity of flavor or perception of sweetness3. Among the wide diversity

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of phenolic compounds, some are well-known natural pigments derived from raw plants or

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generated by further enzymatic or chemical reactions during processing4–6. They participate in

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oxidative browning7, which can be undesirable in the case of fresh fruits or vegetables, but

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can be associated with quality criteria for some food products, particularly some beverages.

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As an example, the yellow-orange coloration is beneficial to the image of apple juice and

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cider.

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Cider apple varieties are particularly rich in phenolic compounds displaying a wide variety of

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structures8. Concentrations can vary according to variety8, fruit maturity9,10, light exposure11

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or storage conditions12. In apples, several classes of polyphenols are found heterogeneously

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distributed in the various tissues of the fruits13. Flavan-3-ols are the most abundant class of

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apple phenolics, including monomers and procyanidins. Monomers consist mainly of (-)-

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epicatechin14, whereas (+)-catechin is only found in low concentrations. Procyanidins, also

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called condensed tannins, are catechin oligomers and polymers characterized by the nature of

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their constitutive units, their degree of polymerization, the nature and position of the

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interflavanic linkages and stereochemistry. Hydroxycinnamic acids - mainly quinic esters of

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caffeic and p-coumaric acids - are the second most abundant phenolics in fruits after

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flavanols. 5-O-caffeoylquinic acid, commonly known as chlorogenic acid, is clearly the most 3

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abundant14 and 4-O-para-coumaroylquinic acid is also present. Although the presence of

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dihydrochalcones has been reported in several plant species, mainly the Rosaceae and

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Ericaceae families (reviewed by Gosch et al15), this class of phenolic compound is more

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represented in apple tissues and their derived products14; it accounts for 1-5% of total

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polyphenols in French cider apple varieties8, in the form of phloretin glycosides. In cider

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apples, phloridzin and phloretin xyloglucoside are the most reported molecules. All these

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native compounds are colorless. Two other classes of native polyphenols are sources of

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colored molecules: anthocyanins, only found in the apple epidermis, except for red-fleshed

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apples16,17, and flavonols, responsible for the yellow color of apple skins. It should be noted

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that native flavonols do not diffuse significantly in juice during processing. Consequently,

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they are involved very little in the final coloration of apple-derived products.

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Apple juice color develops early during the first stages of fruit processing. This phenomenon

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is often referred to as enzymatic browning and starts as soon as the apples are crushed, i.e.

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when cellular integrity is disrupted. Indeed, plastidial polyphenol oxidases (PPOs) catalyze

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oxidation of vacuolar phenolic compounds in the presence of oxygen generating colored o-

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quinones. These species are very unstable and are rapidly involved in non-enzymatic

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reactions18,19, thus forming secondary compounds of which most are colorless, but some new

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pigments are produced. Differences in hue and intensity of browning appear to be related to

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apple phenolic content, speed of enzymatic reaction and environmental factors such as pH,

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temperature, presence of reducing substances and metallic ions. Many studies have attempted

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to assess the correlation between browning intensity and phenolic content or PPO activity4,7,

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but there are some contradictory results in the literature14. The first difficulty was to find an

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adequate way of measuring the degree of browning. Amiot et al.20,

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susceptibility of apples to browning by measuring the absorbance at 400 nm and the lightness

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(L* parameter in CIE Lab color system) of soluble and insoluble pigments, respectively. It

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estimated the

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can be noted that most studies were carried out in model solutions. Overall, research has

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highlighted the crucial role of the relative balance between hydroxycinnamic derivatives and

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flavanol monomers in oxidative browning21–24. Multiple linear regression analysis has shown

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that both contribute to darkness (decrease in L*) and yellowness (increase in b*)22.

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Chlorogenic acid is the best natural PPO substrate in apples25. Its oxidation leads to slightly

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yellow quinones, but secondary products are lighter, suggesting that they are not the most

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significant compounds involved in color development. Through coupled oxidation reduction

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reactions, chlorogenic acid quinones accelerate the degradation of flavanol monomers of

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which the neo-formed products can be intensely colored21–24,26 with absorbance maxima at

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380 nm and 420 nm23. Some colored dimers are believed to be similar to dehydrodicatechin

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A27. In addition, procyanidins do not appear to be good PPO substrates; they have proved to

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result in PPO inhibition28. Nevertheless, Oleszek et al.29 showed that enzymatic oxidation of

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procyanidins B2 and C1 could generate the same color intensity as catechins, but with a

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slower browning rate. Consequently, their contribution to the final coloration of juice should

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be considered. Moreover, dihydrochalcones play a role in this coloration despite their low

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concentration. First, as PPO substrates, they can directly lead to the formation of yellow

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compounds29,30. For example, Le Guernevé et al.31 characterized, in a model solution, yellow

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Phloridzin Oxidation Product (POPj), a pigment displaying a bright yellow color up to pH 5

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and turning orange at higher pH32. This compound may contribute to the yellow color of apple

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juice. Secondly, phloretin glycosides may also have synergistic effects on color development

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when in the presence of chlorogenic acid or catechins29,30, and could play a significant role in

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the complex color of real apple juice .

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To our knowledge, no previous study has focused on identifying color compounds present in

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real apple juices. Our aim was to investigate HPLC-DAD-MS profiles of yellow-orange

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pigments found in juices resulting from processing of four French cider apple varieties. 5

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Phenolic content in these varieties differed clearly and juices covered a wide range of colors.

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We assessed varietal diversity of the chromophores and we discussed the first structural

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elements regarding these natural pigments.

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MATERIAL & METHODS

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Chemicals

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Methanol and acetonitrile were HPLC grade (Carlo Erba reagents, Val de Reuil, France).

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Acetic acid was analytical grade (Carlo Erba reagents, Val de Reuil, France). Formic acid and

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sodium fluoride were purchased from NORMAPUR (VWR Prolabo, Fontenay sous Bois,

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France). Hydrochloric acid and sodium acetate were purchased from Emprove® exp, Merck

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(Darmstadt, Germany). Ultra-pure water was purified with a water purification system

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(PURELAB Classic model, Elga Véolia, Antony, France). Gelatin was obtained from

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RousselotTM (Courbevoie, France). Phloroglucinol, (+)-catechin, (−)-epicatechin, 5-O-

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caffeoylquinic acid, p-coumaric acid, phloridzin, and avicularin (quercetin-3-O-arabinoside)

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phenolic standards were provided by Sigma-Aldrich (Lyon, France). Hyperoside (quercetin-3-

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O-galactoside), ideain chloride (cyanidin-3-O-galactoside chloride), procyanidin B1, and

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procyanidin B2 standards were obtained from Extrasynthèse (Genay, France).

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Plant material

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French cider apples have various “taste types” that are described according to the acidity and

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total polyphenol content of the variety considered33. Here, four of the most grown French

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cider apple varieties were used to produce apple juices: ‘Dous Moen’ (bittersweet), ‘Marie

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Ménard’, (bitter), ‘Petit Jaune’ (sour) and ‘Guillevic’ (sharp). Fruits were picked in a local

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orchard (Iffendic, Brittany, France) in autumn 2012, except for ‘Guillevic’ fruits, which were

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picked in the Surzur orchard (Brittany, France). When technological ripeness was reached 6

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(50% fruit fallen to the ground), fruits were picked off the tree by hand to avoid bruising that

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causes flesh oxidation and deterioration. Fruits were kept at 10 °C until 100% starch

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regression (i.e. complete physiological ripeness) was confirmed using the visual iodine test34.

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Oxidized and O2-deprived juice preparation

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Juices were prepared according to two distinct procedures. In the first case, oxidation was

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promoted by presence of air to generate colored juices, just like when cider-makers process

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their apples. In the second case, the presence of oxygen was avoided throughout all

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processing steps in order to prevent oxidation and thus limit juice browning. Regarding the

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oxidation processing mode, two 20 kg batches of ripe apples were pressed separately for each

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variety. After crushing, the grated apple flesh was vatted for 20 min in air and pressed at 2.5

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bars with a hydraulic press (Hydropresse IT80, Fourage CTI, La Haye Fouassière, France)

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modified by Demoisy (Baune, France). Pectic substances were degraded enzymatically at 10

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°C by adding 2.5 mL of pectinolytic enzyme preparation per hL of crude juice (Endozym

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Pectofruit, Spindal, Gretz-Armainvilliers, France). The treated juice was stored overnight at 2

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°C to let most of crude juice particles settle. Must was then racked, micro-filtered using 0.14

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µm tangential filtration (Inside Céram membrane, 39 channels, hydraulic diameter of 2.5,

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Siva Diam, Nyons, France) and kept at -25 °C until use. For the O2-deprived processing

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mode, ripe fruits were first placed under vacuum for 15 minutes to remove any air inside the

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fruits. The vacuum was then broken by incorporating pure CO2. This operation was repeated

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once. All the other processing steps were similar to the previous mode, but were achieved in

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an inert atmosphere with CO2 or N2 instead of O2.

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Solid phase polyphenol extraction

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Polyphenols were extracted from the juices using C18-solid phase extraction (5 g Sep-Pak®

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Vac 20cc C18 cartridge, Waters, Milford, USA). Silica sorbent was first pre-conditioned with 7

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30 mL of methanol and 40 mL of acetic acid 1%, V/V. Potential residual polyphenol oxidase

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activity was inhibited in micro-filtered juices by addition of sodium fluoride (0.2 M). Juices

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were diluted twice with acidified water (acetic acid 1%) and then transferred to the solid

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phase. The sample volumes were adapted so as not to exceed the retention capacity of the

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solid phase. Forty milliliters of acetic acid 1%, V/V, were used to wash the cartridge and

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eliminate sugars. Polyphenols retained on the solid phase were then eluted with 40 mL of

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methanol/acetic acid 1%, 50/50, V/V. Purified phenolic extracts were concentrated under

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vacuum, frozen at -25 °C and freeze-dried. Extracts were kept in a desiccator under argon

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atmosphere until analysis.

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HPLC-DAD-MS analysis of yellow-orange colored phenolic compounds

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Phenolic extracts were analyzed on a system composed of a SCM1000 degasser (Thermo

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Scientific, San Jose, CA, USA), a 1100 Series binary high-pressure pump (Agilent

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technologies, Palo Alto, CE, USA) and a Surveyor autosampler thermostated at 4 °C (Thermo

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Finnigan, San Jose, CA, USA). Two detectors were connected in series: a UV−visible diode

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array detector (model UV6000 LP, Thermo Finnigan, San Jose, CA, USA) and an ion trap

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mass spectrometer equipped with an electrospray ionization source (model LCQ Deca,

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Thermo Finnigan, San Jose, USA). The reversed-phase chromatography column used in this

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part of the work was a ZORBAX Eclipse XDB-C18 column (2.1 × 150 mm; 3.5 µm, Agilent

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Technologies, Santa Clara, CA, USA) equipped with a ZORBAX Eclipse XDB-C8

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precolumn (2.1 × 12.5 mm, 5 µm, Agilent Technologies, Santa Clara, CA, USA). The

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analysis procedure was adapted from Guyot et al.13. The oven was thermostated at 30 °C.

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Two microliters of sample were injected for analysis. The solvent system was a gradient of A

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(0.1% formic acid in ultrapure water) and B (0.1% formic acid in acetonitrile), which were

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filtered on 0.45 µm cellulose acetate and PTFE filters (VWR, Fontenay sous Bois, France),

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respectively, before use. An elution gradient was applied as follows with a flow rate of 0.2 8

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mL/min: initial, 3% B; 0-5 min, 9% B, linear; 5-15 min, 16% B, linear; 15-45 min, 50% B,

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linear, followed by washing and reconditioning of the column. UV-visible detection covered

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the 240-600 nm wavelength range. Particular attention was paid to signals recorded at 420 nm

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as it corresponds to the response of yellow-orange compounds. The whole effluent was

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injected in an ESI source. Source parameters were negative ion mode, spray voltage (3.7 kV),

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capillary voltage (−70 V), sheath gas (67 arbitrary units), auxiliary gas (5 arbitrary units), and

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capillary temperature (240 °C). The nebulizing gas was nitrogen and the damping gas was

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helium. MS spectra were acquired in full scan, negative ionization mode in the m/z 50−2000

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range. The signals obtained correspond to the deprotonated molecule [M−H]−. A MS/MS

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dependent-scan mode was also performed to obtain the daughter ion spectrum of the main

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molecular ions detected in full scan mode. For the MS/MS experiment, collision energy was

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selected arbitrarily as 35%. Data obtained were processed with 1.2 Xcalibur software

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(Finnigan Corp., San Jose, CA, USA).

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Determination of CIE Lab color parameters

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For apple juices, color was characterized using the Commission Internationale de l’Eclairage

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(CIE – International Commission on Illumination) color parameters. Absorbance spectra were

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measured at 5 nm intervals in the visible wavelength range, from 400 to 700 nm, with a

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spectrophotometer (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany). The

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absorbance data were then converted into CIE L*, a* and b* parameters using mathematical

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processing, considering the illuminant D65 and 10° standard observer. They were also

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expressed and discussed in terms of Lightness (L*), Chroma (C*) and hue angle (h).

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Gelatin fining

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Gelatin is a well-known fining agent, often used to remove part of the condensed tannins

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during beverage production33. Here, a gelatin solution and purified polyphenol extract from 9

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‘Dous Moen’ oxidized juice were combined in 20 mM, pH 4.2 malate buffer. The final

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concentrations of gelatin and phenolic extract were 1g/L. The incubation medium was placed

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at room temperature under mild stirring for the first 30 min then without agitation for 15 min.

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After centrifugation for 30 min (4000 rpm, rotor model JA-10, Beckman Coulter, Villepinte,

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France), the supernatant was recovered and analyzed using HPLC-DAD-MS in the same

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conditions as described previously.

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HPLC-DAD-MS analysis of native polyphenols

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Native

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hydroxycinnamic acids, dihydrochalcones and flavonols were extracted using 1.2 mL of

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methanol acidified with 1% acetic acid, V/V. For each apple variety, extractions were

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performed in triplicate from 500 µL of freeze-dried juice. After homogenization, the

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extraction medium was placed in ice and ultrasonicated for 15 min. Samples were filtered on

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0.45 µm PTFE filters (VWR, Fontenay sous Bois, France) and analyzed using HPLC-DAD-

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MS. Dilutions with methanol/formic acid 0.5%, 50/50, V/V were used if necessary to avoid

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saturation of the UV-visible detector. The HPLC system was the same as described above, but

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the chromatography column was a Hibar® HR Purospher® STAR RP-18 endcapped column

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(150 mm x 2.1 mm, 3 µm, Merck, Darmstadt, Germany). The elution gradient used was

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initial, 3% B; 0-3 min, 7% B, linear; 3-21 min, 13% B, linear; 21-27 min, 13% B, linear, 27-

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41 min, 20% B, linear; 41-51 min, 45% B, linear, followed by washing and reconditioning of

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the column. Native compounds were identified by comparing the retention times, UV-visible

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spectra and full MS spectra with those of available standards. Quantitation was performed by

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integrating peaks on UV-visible chromatograms at 280 nm for flavan-3-ols, procyanidins and

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dihydrochalcones, 320 nm for hydroxycinnamic acids and 350 nm for flavonols. (+)-Catechin,

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(-)-epicatechin, procyanidin B2, caffeoylquinic acid, phloridzin and hyperoside were

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quantitated according to their own calibration curves. Quantitation of other compounds was

polyphenols,

including

catechins,

low

molecular

weight

procyanidins,

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carried out using a reference compound in the same phenolic class displaying a very similar

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UV-visible spectrum.

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Determination of total procyanidin concentration and average degree of procyanidin

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polymerization using HPLC after phloroglucinolysis

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The phloroglucinolysis method was adapted from Kennedy and Jones35. The reactions were

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performed in triplicate on 500 µL of freeze-dried juice using 400 µL of methanol acidified

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with 0.3N hydrochloric acid. After addition of phloroglucinol (50 g/L) and ascorbic acid (10

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g/L), the extraction medium was incubated at 50 °C for 30 min. The reaction was stopped by

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placing the vials in an ice bath and adding 1200 µL of sodium acetate (0.2 mM). Samples

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were filtered on 0.45 µm PTFE filters (VWR, Fontenay sous Bois, France) and analyzed

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using HPLC-DAD-MS. Dilutions with methanol/formic acid 0.5%, 50/50, V/V were used if

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necessary to avoid saturation of the UV-visible detector. The analysis system was the same as

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described for the native polyphenol analysis. The elution gradient used was initial, 3% B; 0-3

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min, 7% B; linear; 3-21 min, 13% B; linear; 21-27 min, 13% B, linear; 27-30 min, 14.5% B,

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linear; followed by washing and reconditioning of the column. Peaks corresponding to apple

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procyanidin reaction products (i.e. (+)-catechin, (-)-epicatechin and (-)-epicatechin

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phloroglucinol adduct) were integrated on the 280 nm chromatogram and quantitation was

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performed using the corresponding calibration curves.

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RESULTS AND DISCUSSION

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Varietal diversity of cider apple juice color

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The four oxidized juices displayed contrasting colors, which is consistent with the range of

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coloration found in classical French cider products36. In contrast, the four juices deprived of

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oxidation were very bright, as shown by their very high lightness L* values ranging between 11

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99.3 and 100.1. These juices were almost colorless or very slightly colored, as revealed by

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their very low Chroma C* values (Table 1). Their “redness”, expressed by positive a* values,

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was close to zero. The “yellowness”, expressed by positive b* values, was noticeably

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different for ‘Guillevic’ juice, suggesting that the others were slightly yellower (b* values of

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2.0 for ‘Guillevic’ and ranging from 4.1 to 4.5 for ‘Petit Jaune’, ‘Dous Moen’ and ‘Marie

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Ménard’). As expected, the considerable differences in the color parameters for the oxidized

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juices indicate the crucial role of enzymatic oxidation in the generation of apple juice

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coloration. In a previous study, Falguera et al.37 described CIE Lab parameters of apple juices

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made from six different apple varieties. A comparison with our data highlights considerable

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disparity in the values obtained, particularly for L* and a*. This is probably related to the

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differences in apple processing and the use of dessert apple varieties that have different

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phenolic content and profiles from cider apple varieties. After oxidation, Lightness values

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remained particularly high in our four juices, varying from 84.9 to 97.3, whereas L* values of

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the six juices produced by Falguera et al.37 ranged from 30.3 to 62.7. These low values could

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be explained by the fact that only centrifugation was performed before color measurement.

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Here, juices were micro-filtered leading to very bright, limpid products. In the present work,

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juices were produced in conditions very similar to the technological processing steps used in

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the French cider-making industry. Despite all these variations, two conclusions were similar

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in both studies. First, the color of the juice differed substantially according to the apple

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variety. Second, all the juices had higher yellowness than redness. Indeed, greater variations

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were observed for b* values than a* values for all four oxidized apple juices. CIE L*, a*, b*,

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C* and h values presented in Table 1 indicate that the four oxidized juices had an overall

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yellow hue, according to hue angle values, but highlight significant nuances. The shades are

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described more easily using the hue angle because this polar coordinate directly reflects the

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hue information of the juice whereas a* and b* orthogonal parameters need to be described

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together to provide a complete picture of the color. Consequently, in the present study, the 12

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colors of the juices are described mainly according to the hue angle. The h value of

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‘Guillevic’ juice was 101.4° (which corresponds to the a* negative value of -4.9), higher than

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90° (yellow hue), meaning that ‘Guillevic’ juice became greenish-yellow after oxidation.

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‘Dous Moen’ (h value of 86.0°) and ‘Marie Ménard’ (h of 83.1°) juices had a more orangey-

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yellow hue. The latter is more orange as the hue angle is smaller. ‘Petit Jaune’ juice clearly

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had a dominant yellow hue, as the h value was very close to 90° (91.5°). In this case, with an

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a* value close to 0 (-1.1), the yellow color of this juice was described directly by the positive

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value of b* (40.7). Regarding C* values, the colors of ‘Marie Ménard’ and ‘Dous Moen’

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juices were 3 times more intense than the color of ‘Guillevic’ juice, for which the lightness

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remained close to 100 even after oxidation. Overall, the higher the C* values, the lower the

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lightness values. The varietal differences in terms of hue and color intensity (indicated by

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Chroma C*) were related to the pigment content formed by polyphenol oxidation during fruit

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processing. The structures and relative concentrations of these chromophores are crucial in the

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final color of the juice.

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HPLC-UV/visible profiles of yellow-orange phenolic compounds in juices.

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Yellow-orange compounds absorb in the range 400-450 nm. HPLC-UV/visible profiles of

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purified phenolic fractions of oxidized juices at 420 nm are presented in Figure 1. The four

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apple juices provided contrasting colored compound profiles. Although most polyphenol

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oxidation products formed during apple processing were colorless38, these chromatographic

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profiles clearly highlight two kinds of response at 420 nm: several well-defined peaks

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corresponding to yellow-orange compounds (peaks 1 to 7 on chromatograms A to D, Figure

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1) and a globally unresolved absorbance hump. With regard to the well-resolved compounds,

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two groups of molecules were distinguished by their chromatographic behavior that reflected

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their polarity. Indeed, compounds (1) to (3) eluted early, contrary to compounds (5) to (7).

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Consequently, the polarity of the former was probably greater than the latter and produced 13

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wider chromatographic peaks. Compound (4) showed intermediate polarity. Its presence was

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particularly preponderant in ‘Petit Jaune’ juice, although it was also detected to a lesser extent

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in the other juices. The particular greenish-yellow coloration of ‘Guillevic’ juice discussed

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previously, was clearly related to a specific profile of phenolic pigments (Figure 1, A). It

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appeared less complex compared to the HPLC profiles of the other varieties, with the main

300

pigments being polar compounds (1) to (3), and the absorbance of the unresolved hump was

301

lower (figure 1, A). ‘Dous Moen’ and ‘Marie Ménard’ extracts had very similar 420 nm

302

HPLC profiles, in accordance with their rather similar color parameters (Table 1). In contrast

303

with the ‘Guillevic’ colored compound profile, there were less polar compounds (1) to (3) in

304

‘Dous Moen’ and ‘Marie Ménard’ than the more apolar compounds (5) to (7) indicated by

305

intense peaks at 420 nm. Moreover, the unresolved absorbance hump was well represented in

306

these juices, which displayed a more orange hue than ‘Guillevic’ and ‘Petit Jaune’ juices.

307

Consequently, it is suggested that more well-resolved apolar compounds and compounds

308

responsible for the unresolved absorbance hump could be involved in the differences in hue

309

observed. The absorbance maxima in the visible region of compounds (4), (6) and (7) (Table

310

2) shifted slightly (circa 20 nm) to higher wavelengths in comparison with more polar

311

compounds (1), (2) and (3). However, UV/visible spectra were acquired in acidified analytical

312

conditions, meaning that pigments were in a different chemical environment to that of the

313

juices. Consequently, this could alter slightly their optical properties measured by the diode

314

array detector. Unfortunately, except for compound (1), we did not succeed in obtaining clear

315

MS data from the colored compounds displaying well-resolved peaks on 420 nm

316

chromatograms. The structural characterization of compound (1) will be discussed later in a

317

specific paragraph. It must be underlined that colorless compounds were largely more

318

concentrated than colored compounds in the purified phenolic fractions. Although it was not

319

revealed on the 420 nm chromatograms, numerous co-elutions occurred with colorless and

320

UV absorbing compounds. Thus, MS signals detected at retention times of colored 14

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compounds corresponded mainly to native colorless compounds and oxidation products co-

322

eluting at those retention times. Lea27 proposed that some new colored compounds formed

323

after oxidation of apple juices are similar to dehydrodicatechin A, which are yellow dimers

324

resulting from oxidative coupling of two catechin molecules39. However, the extracted ion

325

chromatogram of the deprotonated molecule [M-H]- at m/z = 575, which corresponds to the

326

molecular ion of dehydrodicatechin A, did not indicate clear traces of this compound in the

327

four oxidized juices studied here (data not shown). Indeed, ions at m/z 575 may also

328

correspond to colorless oxidation products of procyanidin dimers or to product ions of

329

procyanidin polymers that can be produced in the source of the mass spectrometer40. Further

330

purification steps and optimized separation will be needed to obtain more information on the

331

structure of these pigments formed in apple juices.

332

Gelatin fining of the polyphenolic extract of ‘Dous Moen’ juice resulted in condensed tannin

333

precipitation, and most of these procyanidins were colorless. However, as illustrated in figure

334

2, the phenolic profile at 420 nm showed a clear decrease in absorbance of the previously

335

described unresolved hump, suggesting that oxidized tannins were also partly responsible for

336

the coloration of apple juices. In contrast, well-resolved peaks at 420 nm were not affected by

337

gelatin fining, suggesting that those colored products do not have tanning properties. In the

338

literature, “brown polymers” were often mentioned as being responsible for browning in

339

crushed apple tissues41. However, to our knowledge, this formation of highly polymerized

340

colored pigments has not been clearly demonstrated. Polymerization mechanisms are not

341

likely to generate more conjugation in molecules so should not lead directly to the formation

342

of chromophores. Oleszek et al.29 considered the role of procyanidin oxidation products in

343

enzymatic browning phenomena. In a model solution, the authors showed that enzymatic

344

oxidation of procyanidins B2 and C1 could lead to intense coloration even if the reaction rate

345

was very slow. Nevertheless, these experiments were conducted in a model solution at pH 5.5,

346

close to the optimal pH for PPO activity. These pH conditions may favor autoxidation of 15

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347

polyphenols since they are much higher than pH values usually encountered in apple juice. In

348

the present study, the colored oxidized tannins highlighted could result from the addition or

349

coupling of some chromophores such as dehydrodicatechin A or dehydrotricatechin A, to

350

procyanidin oligomers and polymers. It could also explain why the deprotonated molecules

351

corresponding to these chromophores were not directly detected in juices.

352

Flavanol monomers, hydroxycinnamic acids and dihydrochalcones: the main phenolic

353

classes containing color precursors

354

The diversity of apple juice colors is based on the diversity of colored oxidation products that

355

can coexist in juices. These pigments are derived from native colorless polyphenols. Using

356

multiple linear regression analysis, Goupy et al.22 established that CIE L*, a*, and b* of an

357

apple phenolic solution were correlated to both initial and oxidized amounts of phenolic

358

classes. Here, native phenolic content of oxygen-deprived and oxidized juices were compared

359

by discussing the impact of oxidation within different phenolic classes (Figure 3 and Table 3).

360

Details concerning specific compounds in these classes can be found in the supplementary

361

data. First of all, some varietal discrepancies were clearly noted in accordance with results of

362

previous studies42. For example, the original colored profile of ‘Guillevic’ juice can be related

363

to a distinctive native polyphenol composition. The amount of total polyphenols in this juice

364

was low in comparison with the other varieties (Table 3), as it contained nearly twenty times

365

less polyphenols than ‘Marie Ménard’ oxidized juice. Moreover, as shown in figure 3B, the

366

‘Guillevic’ juices did not contain any flavanol monomers (i.e. (-)-epicatechin and (+)-

367

catechin), which are known to act as precursors of oxidation products developing intense

368

coloration23,24. In addition, ‘Guillevic’ juices contained very low concentrations of

369

procyanidins (Figure 3, B), which were shown to be involved in color formation, as revealed

370

by the gelatin fining of Dous Moen oxidized juices (Figure 2). Therefore, the absence of

371

flavanols in ‘Guillevic’ juice could explain the considerable difference observed between its 16

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weakly saturated greenish-yellow hue and the orange-yellow hue of ‘Dous Moen’ and ‘Marie

373

Ménard’ juices. This oxidized juice was also the only one with a CQA/ pCoQA ratio below 1

374

(Table 3). This particular fraction of the hydroxycinnamic acid class could also be a factor of

375

“color limitation”. Indeed, p-coumaroylquinic acids could act as potential inhibitors of PPO as

376

they are derived from p-coumaric acid, molecule known to be an o-diphenolase inhibitor43.

377

Concerning the other juices, oxidation phenomenon strongly influenced flavanol content,

378

particularly monomers. There were up to 94% less monomers in oxidized ‘Petit Jaune’ juice

379

than in O2-deprived juice (Figure 2, A). The procyanidin content was also significantly lower,

380

which may firstly be due to part of the oxidized tannins being adsorbed on parietal material

381

during processing. Secondly, after oxidation, some procyanidins can become partly resistant

382

to phloroglucinolysis, which could suggest an underestimation of the procyanidin content.

383

Most of the oxidized polyphenols were colorless, but major classes impacted by oxidation

384

were probably involved in the generation of oxidized colored compounds. Hydroxycinnamic

385

acids were also well represented in cider apple juices. This class is clearly impacted by

386

oxidation, but not to the same extent as flavanol monomers, as illustrated by the differences

387

between O2-deprived and oxidized juices (Figure 3C). These compounds seem to be more

388

“protected” against oxidation, which can be explained by coupled oxidation-reduction

389

reactions. Indeed, several authors22–24,26,44 have mentioned the crucial role of the balance

390

between hydroxycinnamic derivatives and flavanols in browning susceptibility. As the best

391

PPO substrate, 5-O-caffeoylquinic acid, the major compound in this phenolic class in most

392

apple varieties, generates o-quinones that can co-oxidize flavanol monomers. Through these

393

reactions, caffeoylquinic acid o-quinones are regenerated as o-diphenolic forms with

394

simultaneous oxidation of monomers leading to new compounds, some of which are highly

395

colored products. Consequently, the data shown here seems consistent with results in the

396

literature.

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397

Interestingly, the significance of the dihydrochalcone class in the oxidation phenomenon is

398

also highlighted in figure 2. Indeed, even if the concentrations of these compounds are lower

399

than flavanols and hydroxycinnamic derivatives, the dihydrochalcone content decreased by

400

the same order of magnitude as hydroxycinnamic acids in oxidized juices, except for ‘Petit

401

Jaune’ in which the content decreased less. Previous studies in model solutions have already

402

shown that these molecules can result in yellowish products. Some synergistic effects on the

403

browning rate have even reported29,30 when dihydrochalcones are in the presence of

404

chlorogenic acids or flavanol monomers. In previous studies31,32, we characterized a yellow

405

colorant consisting in a phloridzin oxidation product (POPj). The variations in

406

dihydrochalcone content in the four juices indicated that the role of compounds in this class in

407

the formation of color in apple juices must be particularly considered in the present study.

408

Finally, we noted that flavonols (i.e. a series of quercetin glycosides), localized mainly in the

409

apple epidermis and known to be the only phenolic class displaying native yellow

410

compounds, were found in very low quantities in juices (about 1 to 7 mg/L, Figure 3, E). They

411

did not appear to be the main compounds responsible for the color of apple juices.

412

Identification of a yellow compound formed in real apple juices: yellow pigment

413

phloretin xyloglucoside oxidation product (PXGOPj) as xyloglucose analogue of POPj

414

colorant previously identified by Le Guernevé et al., (2004)31

415

As previously specified, the juice obtained from the ‘Guillevic’ variety exhibited a singular

416

native phenolic composition. Logically, it led to a particular profile of colored compounds and

417

consequently, to an oxidized juice with a very specific hue and saturation level. The greenish-

418

yellow coloration of this juice appeared to be related mainly to the presence of three

419

compounds (peaks 1, 2 and 3, Figure 1), also present in lower concentrations in the other

420

juices studied. The MS signal corresponding to peak 1 revealed the existence of a product

421

with a molecular weight of 598 Da ([M-H]- at m/z = 597, see Figure 4, A). Three main

422

MS/MS product ions of the deprotonated molecule were detected at m/z = 553, 259 and 303. 18

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423

Interestingly, this molecular weight and this fragmentation pattern were strictly in accordance

424

with the xyloglucose analogue of the POPj molecule32. Indeed, the first product ion (m/z =

425

553) corresponded to the loss of the free carboxylic group (- 44 amu). The MS/MS ion at

426

m/z=259 (losses of 162 and 44 amu) could be obtained after the loss of the sugar moiety and

427

the carboxylic group. Finally, the product ion at m/z=303 corresponded to the remaining

428

phenolic part of the molecule after the loss of the sugar moiety. This new compound was

429

named PXGOPj and its proposed structure is illustrated in Figure 4, B. Fromm et al.45

430

previously suggested the formation of a POPj analogue pigment derived from phloretin

431

xyloglucoside during the enzymatic oxidation of apple seed extracts. However, this compound

432

was not clearly detected and the authors concluded that it was co-eluted with POPj in their

433

analytical conditions. PXGOPj could be generated following the same reaction pathway as

434

already described for POPj32. Thus, phloretin xyloglucoside could be oxidized by apple PPO

435

involving

436

rearrangement, ring re-aromatization and addition of water32. As expected, the UV/visible

437

spectrum of this xyloglucose analogue of POPj was very similar to the POPj spectrum (Figure

438

4, C). Only the sugar moiety differs between both structures, which should not modify the

439

electronic delocalization, and consequently, the chromophore is not modified. Theoretical

440

calculations could be used to confirm this assertion. Interestingly, although phloridzin was

441

significantly present in the oxygen-deprived juice, the extracted ion chromatogram at

442

m/z=465, which corresponds to the POPj deprotonated molecule, only revealed the presence

443

of traces of POPj in the oxidized ‘Guillevic’ juice (data not shown). This preferential

444

formation of PXGOPj could be related to a higher affinity of polyphenol oxidase for phloretin

445

xyloglucoside than for phloridzin. This would be in accordance with the observations of

446

Oleszek et al29. Indeed, these authors showed that in a model solution phloretin xyloglucoside

447

was more readily oxidized by PPO than phloridzin, even if both were PPO substrates. They

both

cresolase

and

catecholase

activities

coupled

with

intramolecular

19

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448

also noticed that enzymatic oxidation of these native dihydrochalcones resulted in yellowish

449

products.

450 451

To conclude, the present paper proposed an initial exploration of yellow-orange compounds in

452

apple juices produced in conditions very similar to those in the cider-making industry. The

453

oxidized juices showed contrasting color parameters related to very contrasting phenolic

454

profiles of colored compounds. The relative proportions of the different classes of native

455

polyphenols appear to be important in relation to the final color. Flavanol monomers seem to

456

play an essential role in oxidation, probably via direct enzymatic oxidation, but also with

457

coupled oxidation-reduction reactions involving hydroxycinnamic acids. The significance of

458

the dihydrochalcone class as a precursor of yellow oxidation products was clearly highlighted

459

and a pigment derived from phloretin xyloglucoside was identified.

460

The study of colored phenolic compounds encountered in apple juices is far from being

461

accomplished. Further purification steps and attempts to optimize MS detection are still

462

necessary to gain further insight into pigment structures. Moreover, this study focused on the

463

initial phenolic composition of the juices to discuss color generation, but the physico-

464

chemical parameters of a beverage may also strongly influence the expression and stability of

465

juice color. Indeed, factors such as pH, presence of metallic ions or co-pigmentation

466

phenomena must not be neglected to understand the final coloration of a beverage.

467

ABBREVIATIONS USED

468

PPO: Polyphenol Oxidase; UV: Ultraviolet; DAD: Diode Array Detector; MS: Mass

469

Spectrometry; POPj: Phloridzin Oxidation Product (j for yellow); PXGOPj: Phloretin

470

Xyloglucoside Oxidation Product (j for yellow); CQA: caffeoylquinic acids; pCoQA: p-

471

coumaroylquinic acids

472 473

SUPPORTING INFORMATION DESCRIPTION 20

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Detailed table of the different native phenolic compounds quantitated using HPLC-UV/visible

475

in French cider apple O2-deprived and oxidized juices. This material is available free of

476

charge via the Internet at http://pubs.acs.org.

477

478

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procyanidin B2 oxidation products in an apple juice model solution and confirmation of

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their presence in apple juice by high-performance liquid chromatography coupled to

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electrospray ion trap mass spectrometry. J. Mass Spectrom. 2011, 1186–1197.

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Importance; Lozano, J. E., Ed.; Springer: New York, USA, 2006; pp 163–174.

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(42) Guyot, S.; Marnet, N.; Sanoner, P.; Drilleau, J.-F. Variability of the Polyphenolic

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Composition of Cider Apple ( Malus domestica ) Fruits and Juices. J. Agric. Food

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Chem. 2003, 51 (21), 6240–6247.

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595

browning susceptibility of various apple and pear cultivars at maturity. Groupe

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Polyphenols Bulletin de Liaison, 1992, 16 (2), 48–51.

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(45) Fromm, M.; Loos, H. M.; Bayha, S.; Carle, R.; Kammerer, D. R. Recovery and

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603

Note

604

This work was supported by Région Bretagne, Région Pays de la Loire and Pôle

605

Agronomique Ouest, as part of an interregional CICHROM project. 26

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FIGURE CAPTIONS

608 609 610 611

Figure 1 – Reversed-phase UV/visible chromatograms at 420 nm of the four apple juice polyphenolic extracts. Fruit varieties used to produce oxidized juices are specified in bold. Numbers (1) to (7) designate colored compounds producing well-resolved peaks in HPLC.

612 613 614

Figure 2 – Reversed-phase UV/visible chromatograms at 420 nm of ‘Dous Moen’ phenolic extract before (dotted line) and after (solid line) gelatin fining.

615 616 617 618 619 620 621 622 623 624

Figure 3 – Comparison of content of different phenolic classes according to the oxidation conditions used to produce the French cider apple juices. A, flavanol monomers; B, procyanidins; C, hydroxycinnamic acids; D, dihydrochalcones; E, flavonols. White bars correspond to O2-deprived juices and black bars to oxidized juices. For each phenolic class, the difference in polyphenol content (expressed in %) between O2-deprived and oxidized conditions is indicated above the black bars. Figure 4 – Identification of PXGOPj, a new yellow compound derived from phloretin xyloglucoside in cider apple juice. A, LC-MSn features; B, Hypothetical structure; C, Comparison of UV/visible spectra of POPj (dotted line) and PXGOPj (solid line).

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Table 1 – CIE Lab Colorimetric Parameters Corresponding To The Different Apple Juices Produced In Absence And In Presence Of Oxygen. Variety Guillevic

Petit Jaune Dous Moen Marie Ménard a

Oxidation type

L*c

a*d

b*e

C*f

hg

O2 - a O2 + b O2 - a

100.1 0.2 -0.36 0.05 1.99 0.09 2.03 0.09

1

97.3

0.7

-4.86 0.06 24.2

99.8

0.1

-0.49 0.06 4.11 0.06 4.13 0.07

96.8

0.8

O2 + b

93.5

0.3

-1.09 0.08 40.7

0.5

91.5

0.1

O2 - a O2 + b O2 - a

99.6

0.2

-0.68 0.04 4.38 0.10 4.43 0.10

98.8

0.5

86.6

0.4

5.55

86.0

0.2

99.3

0.3

-0.80 0.07 4.45 0.04 4.52 0.05 100.1 0.8

84.9

0.2

9.44

O2 + b b

c

0.37

0.40

0.3

0.5

79.0

0.9

77.6

0.9 d

24.7

100

40.8

79.2 78.1

0.3

1.0

1.0

101.4 0.2

83.1

0.2

e

O2 -: oxygen-deprived juice; O2 +: oxidized juice; L*: Lightness; a*: green-red axis value; b*: blueyellow axis value; fC*: Chroma (saturation); gh: hue value; values in italics correspond to standard deviation (n=3).

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Table 2 – Maximal Absorbance In The Visible Region (400-700nm) Of The Yellow Compounds Producing Well-Resolved Peaks On 420 Nm Chromatograms. Compound RT (min) λmax (nm) in the visible region (1)

13.17

417

(2)

13.95

418

(3)

14.73

419

(4)

17.10

437

(5)

21.20

424

(6)

22.80

437

(7)

23.88

433

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Table 3 – Qualitative Parameters And Total Polyphenol Content For The Characterization Of The Polyphenolic Profile In The Four French Cider Apple Juices Produced In Absence (O2 -) And In Presence (O2 +) Of Oxygen.

Variety

a

Guillevic

Petit Jaune

Dous Moen

Marie Ménard

Oxidation type

O2 -

O2 +

O2 -

O2 +

O2 -

O2 +

O2 -

O2 +

PLZa/XPLb

0.55

0.72

0.64

0.79

0.72

0.74

0.41

0.42

EPIc/CATd

_

_

4.38

5.33

2.32

2.68

15.76

18.65

FAe/HCAf

0.30

0.09

0.92

0.33

1.33

0.72

1.67

1.19

CQAg/pCoQAh

1.55

0.98

14.85

9.43

6.30

3.92

18.09

14.06

DPni PCAj

4.17

1.21

3.28

2.15

4.38

3.55

4.14

4.34

Total polyphenol content (mg/L of juice)

460

140

1242

504

1881

1021

4204

2593

PLZ: Phloridzin; bXPL: Phloretin Xyloglucoside; cEPI: (-)-Epicatechin; dCAT: (+)-Catechin; eFA: Flavanols; fHCA: Hydroxycinnamic Acids; gCQA: caffeoylquinic acids; h pCoQA: p-coumaroylquinic acids; iDPn: average Degree of Polymerization of Procyanidins; jPCA: Procyanidins

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Figure 1

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Figure 2

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Figure 3

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A RT (min)

λmax (nm)

[M-H]-

Main product ions on MS/MS spectrum*

13.17

417

597

553 (100); 259 (79); 303 (26)

*Values in parentheses correspond to relative MS intensity B

O

O

OH

C

O

HO O O

Figure 4

Xyloglucose

Figure 4

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For Table of Contents Only

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