Polyphenol Profiling of a Red-Fleshed Apple ... - ACS Publications

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Polyphenol Profiling of a Red-Fleshed Apple Cultivar and Evaluation of the Color Extractability and Stability in the Juice Marta Malec,† Jean-Michel Le Quéré,§ Hélène Sotin,§ Krzysztof Kolodziejczyk,† Rémi Bauduin,# and Sylvain Guyot*,§ †

Institute of Chemical Technology Food, Lodz University of Technology, ul. Stefanowskiego 4/10, 90-924 Lodz, Poland UR 1268 Biopolymères Interactions et Assemblages, Equipe Polyphénols, Réactivité & Procédés, INRA, Domaine de la Motte., B.P. 35327, 35653 Le Rheu Cedex, France # Institut Français des Production Cidricoles (IFPC), F-35653 Le Rheu, France §

S Supporting Information *

ABSTRACT: Red-fleshed apples can be used for the production of innovative products such as rosé juices and ciders. Phenolic compounds including procyanidins (i.e., condensed tannins) and anthocyanins were quantified in the fruits and juices of a redfleshed apple cultivar by chromatography coupled to UV−visible and mass spectrometry. Juice color was characterized by colorimetry. The influence of oxygen, pH, sulfites, ascorbic acid, and copper on the color stability of the juice was studied in an experimental design. Fruits were rich in polyphenols (0.5 g/100 g FW), with anthocyanins and procyanidins accounting for 9 and 73% of total polyphenols, respectively. Extractability of anthocyanins in the juice was 26%. Juice storage under air atmosphere at 35 °C resulted in significant browning with the anthocyanin level decreasing up to 86% after 14 days. In contrast, color was stable for storage under argon atmosphere. Sulfites, ascorbic acid, and copper have only a slightly influence on color stability in those conditions. KEYWORDS: Malus domestica, rosé, phenolic compounds, anthocyanin, tannins, cider, LC-MS

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oxidase (PPO).7 Thus, as a consequence of fruit handling, crushing, and pressing, this compound takes part as a precursor in a series of oxidation reduction reactions and oxidative coupling reactions of polyphenols that lead to the formation of neoformed phenolic molecules, some of them responsible for the yellow, orange, or brown color of apples-based products. In France, the yellow-orange color of apple juices and ciders can be considered as a positive criterion contributing to the overall organoleptic quality. However, in the perspective of red-colored apple juice and cider production, these yellow-brown compounds have a negative impact on the color. Flavonoids, the largest group of polyphenols, are represented by flavanols, flavonols, dihydrochalcones, and anthocyanins. In both cider and dessert varieties, the most concentrated in the fruits are flavanols, represented by (+)-catechin, (−)-epicatechin, and their oligomers and polymers (i.e., procyanidins and condensed tannins).8,9 Catechin monomers are also largely involved in apple enzymatic browning reactions.7 Condensed tannins are mainly responsible for astringency and bitterness of apple fruits and their products.10 Procyanidins possess acid labile interflavan linkages that give the possibility to wholly characterize and quantify them in apple and apple products after acidolysis reaction in the presence of an excess of a nucleophile agent, such as phloroglucinol11 or benzyl

INTRODUCTION According to FAO, total world apple production in 2010 crossed 69.5 million tons, from which about 65% were produced in Asia.1 The second world-leading apple producer is Europe, with Poland, Italy, and France as the main producing countries. A significant part of the apple production is destined for processing aims, mainly for apple juice and cider production. Color is the one of the most important factors in the decisions made by today’s customers. It can influence the attitude of consumers and create positive or negative feelings about the products.2 Nowadays, people always expect new, attractive foods and beverages However, they prefer natural and healthier products with low content of artificial additives. Therefore, in this context, red-fleshed apple juices have great potential, provided that every effort ismade to ensure the stability of their unique color. Apples, and in particular cider varieties, are rich in polyphenols that contribute to some of their sensorial criteria such as browning susceptibility, bitterness, and astringency.3 As a whole, polyphenols contribute to quality characteristics of apple-derived foods, such as flavor, appearance, and health benefits including prevention of chronic diseases and slow aging.4 Apple phenolics can be divided into two main categories that are hydroxycinnamic acid derivatives and flavonoids. Hydroxycinnamic acids are mainly represented by 5caffeoylquinic acid (commonly known also as chlorogenic acid) and in a lower level by 4-p-coumaroylquinic acid. These compounds are present in all parts of fruits5,6 Noticeably, 5caffeoylquinic acid is the preferential substrate of polyphenol © XXXX American Chemical Society

Special Issue: International Workshop on Anthocyanins (IWA2013) Received: January 20, 2014 Revised: March 21, 2014 Accepted: March 24, 2014

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dx.doi.org/10.1021/jf500336v | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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mercaptan.12 In those conditions, interflavan linkages are disrupted and the flavanol units corresponding to extension and upper units of condensed tannins are liberated in the medium as flavanyl carbocations immediately converted into stable monomeric derivatives after trapping by the nucleophilic agent. At the same time, terminal flavanol units of condensed tannins are liberated as such in the reaction medium. Then, all of these reaction products can be easily separated and quantified by reversed phase HPLC.13 Apple flavonols are a series of quercetin (Q) glycosides namely: galactoside (hyperoside, generally the most concentrated), Q-3-O-glucoside (isoquercitrin), Q-3-O-rhamnoside (quercitrin), Q-3-O-rutinoside (rutin), Q-3-O-xyloside (reynoutrin), and Q-3-O-arabinoside (avicularin). Flavonols are yellow compounds mainly located in the peel of fruits and are poorly extractable. Therefore, juices and ciders are usually almost deprived of these components.14 Among usually consumed fruits and fruit-derived foods, dihydrochalcones are the only group of flavonoids that can be considered specific for apples, although they can be present in low concentration in other species. One well-known compound of this class, namely, phloridzin (phloretin-2-glucoside), is present in apple tissues and apple juices and was also studied for its potential phytoestrogen activity.15 In addition, phloridzin is considered as a feature compound for the adulteration of beverages with apple juice and hence used for quality control.16 Phloridzin is also widely applied in the food, cosmetic, and pharmaceutical industries.17 Different from popular apple species, red-fleshed ones are characterized by high amounts of anthocyanins.18,19 According to Sadilova et al.,18 anthocyanins of the red-fleshed ‘Weirouge’ apples were mainly located in the peel at a concentration about 2.5 times higher than in the flesh. In both studies concerning ‘Weirouge’18 or ‘Niedzwiedzkyana’19 varieties, the anthocyanin class is mainly represented by cyanidin-3-O-galactoside (namely, ideain), although other cyanidin glycosides such as xyloside, arabinoside, rhamnoside, and glucoside have been also detected and quantified.18 Interestingly, juices from red-fleshed varieties containing high levels of cyanidin-3-O-galactoside were generally the ones showing also a high antioxidant capacity.19 The fact that anthocyanins exist as several forms in equilibrium depending on the pH is well documented.20,21 At low pH (∼3), the colored flavylium cation form of anthocyanins is significantly present and is more stable. As the pH increases, colorless species (i.e., carbinol pseudobase and chalcones) may significantly contribute to this equilibrium. Apple varieties strongly differ in quantitative content of each polyphenol.7,9,22 The same was observed by comparing apples and juices made of them. According to Guyot et al.,23 the extraction rates of hydroxycinnamic acids, dihydrochalcones, and flavanols during juice production differ according to variety. However, in general, apples with higher content of polyphenols gave juices with higher polyphenol concentrations in comparison to low-polyphenol-containing varieties. Interestingly, solid parts of the fruits act as selective barriers influencing the transfer of polyphenols from the fruits to the juices.23 In particular, flavanol oligomers and polymers (i.e., condensed tannins) and especially those having a high degree of polymerization are largely adsorbed and retained on the apple pomace. In the apple juice and cidermaking industries, enzymatic and nonenzymatic oxidations of polyphenols are important phenomena that contribute to the juice and cider quality.3

Those reactions are responsible for the yellow-orange color of apple juices and ciders. After cell wall and cell membrane disruption, which occur when fruits are crushed and pressed, chlorogenic acid and, to a lower extent, catechins that contain a catechol group in their structure are enzymatically converted by the enzyme PPO into their corresponding o-quinones.7 The quinones produced are very reactive species leading to the formation of neoformed polyphenolic structures involving most of the native polyphenols. As far as we know, there is no direct influence on anthocyanins, which are not considered as PPO substrates.24 However, in a red wine model solution, it was found that anthocyanins can be indirectly involved by coupled oxidation reduction reaction with hydroxycinnamic acid oquinones.24 Furthermore, during juice or cider aging, there are series of nonenzymatic reactions involving polyphenol that potentially change the color. They may be favored at higher pH and in the presence of metal ions such as iron or copper.18 Among the different ways of inhibition or controlling of oxidation processes, the use of reducing agents is well documented. For instance, sulfites are highly effective and have been used in many products since antiquity.7 In food technology, sulfite reagent doses are limited and banned for use in fresh fruits and vegetables because of their adverse effects on health.25 In the fermented beverages industry, concentrations of residual sulfites are generally not authorized above 350 and 200 mg/L for wines and ciders, respectively.26,27 Ascorbic acid is also a widespread reducing agent. However, when it comes from the apple itself, it is rapidly consumed during crushing, as a consequence of cell wall disruption and oxidation reduction reaction with enzymatically produced o-quinones leading to the formation of dehydroascorbic acid. Then, dehydroascorbic acid may be degraded into a variety of products (aldehydes, ketones, carboxylic acids). A noticeable advantage of ascorbic acid is that there are fewer limitations for its use in food products (usually 50−150 mg/L in wines). Food chemistry of anthocyanins28 and in particular their sensitivity to oxidation have been well studied in the red wine model.27 Then, pyranoanthocyanins appear as a class of very diverse anthocyanin derivatives that are largely involved in the evolution of wine color during aging. 29 In addition, anthocyanins and probably also their derivatives are known for their high antioxidant activity. In this study, the qualitative and quantitative analyses of polyphenols of a red-fleshed apple cultivar and its clear and cloudy juices are presented. The transfer of polyphenols from fruits to juices, especially for anthocyanins responsible for red color, was measured. In a second part of the experiment, attention was paid to the stability of juice color depending on different parameters including oxygen presence, pH values, and addition of ascorbic acid, sulfites, and copper salt.

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MATERIALS AND METHODS

Chemicals and Phenolic Standards. HPLC gradient grade methanol and acetonitrile were obtained from Carlo Erba reagents (Val de Reuil, France). Formic acid was purchased from NORMAPUR (VWR Prolabo, France). Ultrapure water was prepared with a Milli-Q water purification system (Millipore S.A., Molsheim, France). Ascorbic acid was from Fisher Scientific (Loughborough, UK). Copper(II) sulfate pentahydrate was from VWR (Fontenay Ss Bois, France). Sodium metabisulfite was from Merck (Darmstadt, Germany). Standards of (+)-catechin, (−)-epicatechin, 5-O-caffeoylquinic acid, p-coumaric acid, phloridzin, and avicularin (quercetin-3-O-arabinoside) were obtained from Sigma-Aldrich (Lyon, France). Hyperoside B



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Figure 1. Reversed phase UV−visible chromatograms of the acidified methanol extract of the red-fleshed apple powder. Different wavelengths are used to optimize the detection of the phenolic compounds: (A) 280 nm, all phenolics are detected; (B) 320 nm, more specific for hydroxycinnamic; (C) 350 nm, more specific for flavonols; (D) 510 nm, more specific for anthocyanins. (quercetin-3-O-galactoside), ideain chloride (cyanidin-3-O-galactoside chloride), procyanidin B1, and procyanidin B2 standards were purchased from Extrasynthese (Genay, France). (−)-Epicatechinphloroglucinol, phloretin xyloglucoside, 4-p-coumaroylquinic acid, and procyanidin B5 were available standards purified in the laboratory from apple material by HPLC at the semipreparative scale. Plant Material. A red-fleshed apple hybrid cultivar was selected at INRA Angers (code X4876). It was a cross between F2 parent Malus pumila varieties ‘Jonathan’ and ‘Niedzwetzkyana’. Twenty kilograms of apples was harvested in late September 2012 at maturity stage “Starch 7”30 in the experimental orchard of INRA Angers, Domaine de Beaucouzé. Sample Preparation. Fruit Sample Preparation. Apples were sampled into three batches of 10 fruits. For each batch, fruits were mechanically cut and fruit pieces were randomly selected according to a systematic procedure.42 This fresh material was immediately frozen in liquid nitrogen and freeze-dried. This dried material was crushed into a fine and homogeneous powder (Electrical Crusher Retsch, model YGG, Bioblock Scientific), which was stored at −30 °C until analysis. Apple Juice Preparation. Apple juice was prepared in accordance with the internal procedure. Milling and pressing of the apples were performed in the same extraction instrument based on a modified hydropress 90 (Spiedel, Germany) modified by Demoisy (Baune,

France). This instrument was specifically designed to limit oxidation during apple milling and pressing (3 bar) by keeping the atmosphere inert using carbon dioxide. The processed batch was approximately 15 kg of apples. A part of the juice was prevented from enzymatic oxidation by adding sodium fluoride (inhibitor of PPO, 0.2 g/L). The rest was left as nonprevented product. Juices were frozen and stored at −30 °C until use. Prevented juice was used for chromatographic characterization of the polyphenol profile, whereas nonprevented juice was used for the experimental design aiming to evaluate the stability of the color. Before analysis, the prevented apple juice was divided into two parts. The first part was centrifuged for 15 min at 3000 rpm on Beckman J2-21 floor model centrifuge. The supernatant, corresponding to “clear” juice, was recovered, whereas the second part was kept cloudy without any change and named “cloudy juice”. Aliquots (0.5 mL) of both juices were freeze-dried overnight in 5 mL hemolysis tubes using a Lyovac GT2 freeze-drier (Leybold Vacuum, France). Freeze-dried samples were kept stored in a desiccator under vacuum at room temperature until analysis. Analytical Procedures. Determination of Simple Polyphenol Compounds in Fruits and Preserved Juice. Simple polyphenols (i.e., hydroxycinnamic acids, monomeric flavanols, flavonols, dihydrochalcones, and anthocyanins) were extracted from freeze-dried apple powders using acidified methanol. About 50 mg of powder was precisely weighed in 5 mL hemolysis tubes and extracted using 1.2 mL C



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Table 1. LC-UV−Visible/MS Identification and Quantificationa of the Main Phenolic Compounds in Red-Fleshed Apple Fruit and the Corresponding Clear Apple Juices peak

Rt (min)

λmax

[M − H]−

1

14.7

279

577

2 3 4 5

16.0 17.0 18.4 21.0

279 325 512 279

289 353 447 577

6

23.2

279

289

7 8 9 10

23.6 25.2 26.3 29.0

312 512 280 280

337 417 865 1153

11 12

34.5 35.4

350 280

463 577

13 14 15 16 17 18 19

35.4 39.4 40.7 45.1 37.0 40.6 12.0

350 350 284 284 350 350 524

463 433 567 435 433 447 737e

425 (100); 451 (32); 407 (18); 289 (16) 289 (100); 245 (22) 191 (100); 179 (6) 285 (100) 425 (100); 451 (32); 407 (18); 289 (16) 289 (100); 245 (51); 205 (9); 179 (6) 173 (100); 163 (15) 285 (100) 739 (100); 695 (79) 1135 (100); 739 (42); 863 (36); 575 (19) 301 (100); 300 (68) 425 (100); 451 (32); 407 (18); 289 (16) 301 (100); 300 (53) 433 (100); 271 (61); 365 (27) 273 (100); 167 (6) 285 (100); 241 (13) 301 (100); 300 (41) 301 (100); 300 (51) 575 (100); 423 (7)

20

23.1

495

517e

355 (100)

MS/MSb

identified compound procyanidin B1d

in apple fruit (mg/100 g FW)

in clear juicec (mg/100 g FW)

7.9 ± 0.2

11.7 ± 0.6

± ± ± ±

(+)-catechind 5-caffeoylquinic acidd cyanidin-3-galactosided procyanidin B2d

11.6 16.6 37.2 34.6

(−)-epicatechind

23.3 ± 0.5

4-p-coumaroylquinic acid10,23 cyanidin pentoside19 flavanol trimer flavanol tetramer

14.1 5.7 13.3 6.7

quercetin 3-O-galactosided procyanidin B5 quercetin 3-O-glucosided quercetin 3-O-arabinoside phloretin-2-xyloglucoside10,23 phloridzind quercetin 3-O-xylosided quercetin 3-O-rhamnosided anthocyanin and flavanol adduct 5-carboxy-pyranocyanidinglucoside19

± ± ± ±

0.2 1.0 1.3 1.1

0.6 0.2 0.7 0.4

3.0 ± 0.7 2.8 ± 0.4 0.9 1.3 5.9 12.7 0.7 0.1 0.3

± ± ± ± ± ± ±

0.3 0.2 0.5 0.7 0.1 0.0 0.1

0.4 ± 0.0

8.4 16.7 10.0 21.3

± ± ± ±

0.4 0.8 0.5 1.0

16 ± 0.9 12.9 1.2 7.5 3.6

± ± ± ±

0.6 0.1 0.3 0.1

0.5 ± 0.0 0.5 ± 0.0 nd 0.1 4.6 2.7 0.1 0.2 0.2

± ± ± ± ± ±

0.0 0.2 0.1 0.0 0.0 0.0

0.4 ± 0.0

a Mean ± standard deviation, n = 3. bEnergy of collision = 35%. cExpressed according to 100 g of fresh juice. nd, not detected. dIdentified according to a commercial standard. eDetected in MS positive mode [M]+.

of pure methanol containing 5% v/v formic acid for 15 min in an ultrasonic bath containing ice (Brasson 2200, USA). Then, samples were filtered on 0.45 μm PTFE filters (Uptidisc, Interchim, France), and the filtrates were ready for HPLC analysis. This procedure was performed in triplicate. For the cloudy and clear preserved juices, freeze-dried samples were extracted by 1.2 mL of acidic methanol in the ultrasonic bath according to the same procedure. This procedure was performed in triplicate. Depolymerization of Procyanidins by Acidolysis in the Presence of the Phloroglucinol. The procedure was adapted from that of Kennedy and Jones.31 Phloroglucinolysis reaction gives access to the subunit composition of procyanidins. The interflavan linkages of procyanidins can be easily disrupted by acidolysis in organic acid medium, leading to the formation of flavanyl carbocations corresponding to the extension units of the procyanidin structure, whereas the terminal units are released in the medium as catechin molecules. Flavanyl carbocations are immediately trapped by nucleophilic phloroglucinol in excess, leading to flavanyl-phloroglucinol adducts. In the case of apple procyanidins, phloroglucinolysis releases (+)-catechin and (−)-epicatechin from the terminal subunits and a (−)-epicatechin−phloroglucinol adduct from the extension subunits. Then, the HPLC analysis of the phloroglucinolysis media gives access to the concentration of each type of terminal and extension catechin unit that initially composed the procyanidin fraction. Therefore, it is possible to estimate the average degree of polymerization (DPn), the average distribution of the catechin units, and the total concentration of procyanidins in the fruit or juice samples.32 Apple powder samples (50 mg in 5 mL hemolysis tubes) or freezedried cloudy and clear preserved apple juices were dispersed in 0.8 mL of methanol containing phloroglucinol (75 g/L) and ascorbic acid (15 g/L). Then, the reaction was started by adding 0.4 mL of HCl (0.3 N in methanol), and media were immediately incubated at 50 °C for 30

min. The reaction was stopped by placing the tubes in an ice bath and by adding 1.2 mL of 0.2 M aqueous sodium acetate. Samples were filtered on 0.45 μm PTFE filters (Uptidisc, Interchim). The filtrates were ready for HPLC analysis. All phloroglucinolyses were performed in triplicate. HPLC-UV−Vis and Mass Spectrometry Analysis. Analysis was carried out using a system composed of a thermostated autosampler (model Surveyor, Thermo Finnigan, San Jose, CA, USA), a binary high-pressure pump (model 1100, Agilent Technologies, Palo Alto, CA, USA), a UV−vis diode array detector (model UV6000 LP, Thermo Finnigan), and an ion trap mass spectrometer equipped with an electrospray source (model LCQ Deca, Thermo Finnigan). The column was a ZORBAX Eclipse XDB-C18 column (Agilent Technologies, 2.1 × 150 mm; 3.5 μm) equipped with a precolumn (2.1 × 12.5 mm) of the same material and thermostated at 30 °C. Solvents were (A) acidified pure water (0.1% formic acid) and (B) acidified acetonitrile (0.1% formic acid), both degassed by flushing with helium. The flow rate was 0.2 mL/min and the gradient was as follows: initial, 3% B; 0−40 min, 20% B, linear; 40−55 min, 35% B, linear followed by washing and equilibrating the column. The UV−vis detection was performed in the 240−600 nm range. The ESI source was used in the negative mode with the following parameters: spray voltage (−5 kV), capillary voltage (−70 V), sheath gas (67 arbitrary units), auxiliary gas (4 arbitrary units), and capillary temperature (240 °C). The MS detection was carried out with the following parameters: MS spectra were acquired in full scan negative ionization mode in the m/z 50−2000 range to obtain the signals corresponding to the deprotonated [M − H]− molecular ions. The method also included the MS/MS dependent scan mode, which was used to obtain the product ion spectrum of the main molecular ions detected on the chromatogram in the full scan mode. The collision energy was optimized in the range of 25−35% (arbitrary units) to clearly observe the production of both parent and main daughter ions. Data were collected and processed by XCalibur software (version 1.2, Thermo D



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Finnigan). By comparison with available standards, the retention times, UV−vis spectra, full MS spectra, and MS/MS spectra were used for complete identification. When the standard was not available, those criteria were used for only a partial identification. Quantifications were carried out by integration of the peaks on UV−vis chromatograms at 280 nm for flavanols and dihydrochalcones, at 320 nm for hydroxycinnamates, at 350 nm for flavonols, and at 510 nm for anthocyanins (Figure 1). (+)-Catechin, (−)-epicatechin, procyanidin B2, caffeoylquinic acid, phloridzin, hyperoside, and ideain were quantified according to their own calibration curves, whereas other compounds were quantified “as equivalent” according to a reference compound belonging to the same polyphenol class and showing a very similar UV−vis spectrum (see Table 1). Experimental Design for Color Stability Evaluation. In addition to oxygen, four factors, namely, the pH of the juice (pH), ascorbic acid concentration (AA), sulfite concentration (SO2), and cupric ion concentration (Cu), were considered as they were suspected to have an effect (main and quadratic) on color evolution with potential interactions between them. To evaluate these effects, a central composite design (fixed to be rotable and orthogonal) was chosen with factors pH, AA, SO2, and Cu at five levels for each. The design matrix was defined using the tutorial of Stagraphics software: the factor levels were encoded −2, −1, 0, 1, and 2, and the center value was replicated 12-fold, giving 36 total experiments. The range of the factors was chosen to correspond to those encountered in cider and dessert apple juices (from pH 3.0 to 4.4) or usually added in apple juice or cider processing (from 0 to 200 mg/L for ascorbic acid and from 0 to 80 mg/L for SO2) or measured in the juices and ciders (from 0 to 400 μg/L for copper). This design was applied to both conditions, with oxygen (under air saturation at the starting of the experiment) and without oxygen (under argon for the duration of the experiment), so that oxygen was considered as a two-level factor. The crude juice (ca. 600 mL) was first thawed at 10 °C in the presence of CO2 to prevent it from oxidation. Then, the juice was clarified by the addition of 80 μL/L of pectinase (Rapidase C80 Max, DSM, The Netherlands) to hydrolyze pectins and protect the juice from undesirable residues during experiment. After a whole night, the juice was centrifuged for 20 min (Beckman J2-21 floor model centrifuge), and the clear juice corresponding to the supernatant was used for the experimental design. The initial pH of the juice was 3.4 (model 827 pH Lab pHmeter, Metrohm, France). The juice was divided into five equal volumes for which the acidity was modified by addition of malic acid or potassium hydroxide. Then, the juice was distributed into 10 mL aliquots in glass flasks and mixed with different doses of ascorbic acid (0, 50, 100, 150, or 200 mg/L final), sodium metabisulfite Na2S2O5 (0, 20, 60, 40, and 80 mg/L in SO2 equivalent), and CuSO4 (0, 100, 200, 300, or 400 μg/ L in Cu equivalent) according to the design matrix (Table 2). Finally, 36 flasks were placed under argon atmosphere, and another 36 flasks were prepared under air-saturated atmosphere. Preparation of those samples was carried out with appropriate microbiological conditions to maintain low contamination risk: Vessels were sterilized in steam (120 °C, 20 min), laboratory tools were kept in alcohol, and the additions were done in sterile conditions. All flasks were capped hermetically with inert caps. The storage experiment was carried out for 2 weeks. Samples were stored in the absence of light for 10 days at 35 °C and for 4 days at 50 °C. Samples under air and argon atmospheres were analyzed by HPLC-coupled to UV−vis and mass spectrometry detection on the first and last days of the experiment. Spectrophotometric Measurements of the Juices and Data Conversion. The native clear juice and the clear juices from the experimental design were analyzed using a spectrophotometer to collect the data necessary for the calculation of the colorimetric parameters. Aliquots (200 μL) of the juices stored under air atmosphere were taken daily from each flask to measure the absorbance corresponding to the visible spectrum in the 400−700 nm region at room temperature using a microplate spectrophotometer (model SPECTRAmax PLUS 384, Molecular Devices). Samples with argon were analyzed only at the last day of storage. Mathematical

Table 2. Doses of Additives in the Solutions Used in the Experimental Design pH of juice

ascorbic acid (mg/L)

SO2 (mg/L)

Cu (μg/L)

no. of samples

3

100

40

200

1

3.35 3.35 3.35 3.35 3.35 3.35 3.35 3.35

150 150 150 150 50 50 50 50

20 20 60 60 20 20 60 60

300 100 300 100 300 100 300 100

1 1 1 1 1 1 1 1

3.7 3.7 3.7 3.7 3.7 3.7 3.7

0 100 100 100 100 100 200

40 0 40 40 40 80 40

200 200 0 200 400 200 200

1 1 1 12 1 1 1

4.05 4.05 4.05 4.05 4.05 4.05 4.05 4.05

150 150 150 150 50 50 50 50

20 20 60 60 20 20 60 60

300 100 300 100 300 100 300 100

1 1 1 1 1 1 1

4.4

100

40

200

1

processing of the visible spectra was used to convert the absorbance data into colorimetric CIE L, a, and b parameters. Then, colorimetrical values including lightness (L), saturation (C*), and hue (h) were used for color characterization. The correctness of conversion was checked before by comparison of data from colorimetrical measurement and spectrophotometric data conversion of the same samples. Statistical Analysis. Statistical analyses were done by Statistical Analysis and Data Visualization software, Statgraphic (Sigma Plus, France). The experimental design was analyzed by the general linear model.

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RESULTS AND DISCUSSION Phenolic Compounds in the Red-Fleshed Apple Fruits. The polyphenol profile of the fruit and the juice of the red-fleshed apple variety ‘Jonathan’ × ‘Niedzwetzkyana’ were characterized by reversed phase HPLC coupled to UV− vis and MS detection. Figure 1 shows the chromatograms of the fruit methanol extract at four different wavelengths to have a better observation of the different polyphenol classes: all polyphenols including more or less polymerized procyanidins are detected at 280 nm. For this reason, the chromatogram appears very crowded with significant baseline deviation corresponding to coelution of several compounds. Hydroxycinnamates were more selectively detected at 320 nm, and flavonols are clearly observable at 350 nm between 34 and 42 min. Finally, the wavelength 520 nm was suitable for the specific detection of anthocyanins. Most of the compounds were numbered and unambiguously identified on the basis of their chromatographic (RT) and spectrometric (UV−vis, MS/ E



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Table 3. Concentration of Phenolic Compounds Present in Apple Fruits and Apple Juices from Red-Fleshed Apples and Juice Extraction Ratea,b total flavanols

total HA

total flavonols

total anthocyanins

total DHC

total polyphenols

392.1 ± 23.1 165.1 ± 7.5 164.4 ± 5.6 42

30.7 ± 1.7 29.4 ± 5.2 30.8 ± 1.3 100

5.0 ± 1.3 1.0 ± 0.2 0.9 ± 0.0 16

43.6 ± 1.6 11.7 ± 2.0 12.3 ± 0.6 26

18.6 ± 1.2 7.3 ± 1.3 7.6 ± 0.3 40

490.0 ± 28.9 214.5 ± 16.2 216.0 ± 7.8 40

apple fruit (mg/100 g of FW) cloudy juice (mg/100 mL) clear juice (mg/100 mL) extraction rate (%) a

Mean ± standard deviation, n = 3. bPCA, procyanidins; HA, hydroxycinnamic acids; DHC, dihydrochalcones.

Table 4. Concentrationsa of Flavanols and Percentage of Terminal and Extension Procyanidin Subunits in Apple Fruits and Apple Juices procyanidins

apple fruit (mg/100 g of FW of the whole fruit) cloudy juice (mg/100 mL of juice) clear juice (mg/100 mL of juice) a

total flavanols

DPnb of flavanols

(+)-catechin t.suc (%)

(+)-catechin

(−)-epicatechin

total procyanidins

11.6 ± 0.2

23.3 ± 0.5

357.1 ± 16.0

392.0 ± 16.7

3.6 ± 0.1

6.8

14

79.2

8.1 ± 1.4

15.1 ± 2.7

135.7 ± 4.0

158.9 ± 8.1

2.49 ± 0.1

13

16.8

70.2

8.4 ± 0.4

16 ± 0.9

133.6 ± 2.9

158.0 ± 4.2

2.47 ± 0.0

13.2

16.5

70.3

(−)-epicatechin t.suc (%)

(−)-epicatechin e.sud (%)

Mean ± standard deviation, n = 3. bDPn, average degree of polymerization. ct.su, terminal subunits. de.su, extension subunits.

Table 5. Literature Data for Concentrations of Phenolic Compounds in Apples and Apple Juices ref

total polyphenols

total PCAa

5-CQAb

present study

490 ± 29 214−216 104−544 55−631 104−699 66−1360 26−97 25−104

283 ± 16 134 47−468

16.6 ± 1.0 16−17 0.3−59 3−116 15−119 17−110 8−49 8−13

type and no. of apple varieties red-fleshed X4876 apple 67 varieties from Poland 19 English cider apples 14 French cider apples 31 Basque cider apples 7 German cider apples 2 Polish dessert apples a

fruit (mg/100 g of FW) juice (mg/100 mL) fruit (mg/100 g of FW) fruit (mg/100 g of FW) fruit (mg/100 g of FW) juice (mg/100 mL) juice (mg/100 mL) juice (mg/100 mL)

23 38 10 45 37 15

51−473 35−351 6−76

b

Procyanidins. 5-Caffeoylquinic acid.

compound corresponding to peak 19 ([M]+ = 737, Table 1) likely corresponded to an (epi)catechin-cyanidin-hexoside. Phloroglucinolysis reaction directly applied on apple powder followed by HPLC analysis was used to define the total content of procyanidins and the average degree of polymerization of flavanols. The total concentration of polyphenols in fruits assayed by HPLC amounted to 490 mg/100 g of fresh weight (Table 3). In accordance with previously published data for which thiolysis or phloroglucinolysis reaction was used to quantify condensed tannins in apple fruits also considering their polymerized forms,34,35 major constituents present in samples were flavanols, with >70% content of procyanidins in apples. Among them, procyanidin B2 was the most abundant molecular form, whereas within monomers, (−)-epicatechin prevailed. As generally observed for apple condensed tannins, procyanidin extension subunits were only (−)-epicatechin, whereas (+)-catechin was found as a small proportion of the terminal units. The average degree of polymerization (DPn) of the flavanol class in the apples equaled about 3.6 (Table 4). The second dominant group in apples was anthocyanins, with 9% of average total polyphenol content (Table 3). The major anthocyanin was ideain, namely, the cyanidin-3-Ogalactoside (Figure 1D, peak 4) with a concentration close to 37 mg/100 g FW (Table 1) accounting for 85% of total anthocyanins in the fruit. This can be compared to other studies on red-fleshed apples. For instance, this concentration

MS) features by comparison with authentic standards when they were available. In other cases, they were only partially identified on the basis of their UV−vis and MS/MS spectra and by comparison with previously published data. Thus, considering the phenolic compounds usually found in common apple fruits, seven flavanols: ((+)-catechin (2), (−)-epicatechin (6), procyanidins B1 (1), B2 (5), and B5 (12), one trimer (9), and one tetramer (10)), five flavonols (avicularin (14), hyperoside (11), isoquercitrin (13), quercitrin (18), and reynoutrin (17)), two hydroxycinnamic acids (5caffeoylquinic acid (3) and 4-p-coumaroylquinic acid (7)), and two dihydrochalcones (phloridzin (16) and phloretin-2xyloglucoside (15)) were detected and identified in the fruit (Figure 1). For the particular class of anthocyanins that are responsible for the red color, four peaks were clearly observable on chromatograms at 510 nm (Figure 1D). The main peak (4) was unambiguously identified as ideain (i.e., cyanidin-3-O-galactoside), and peak 8 was identified as cyanidin pentoside, likely corresponding to cyanidin-3-O-xyloside, already described as the second most concentrated anthocyanins after ideain in redfleshed apples.33 Two red-colored compounds corresponding to peaks 19 and 20 were also detected. Referring to a previously mentioned publication, compound 20 with [M]+ ion at m/z 517 and a main product ion at m/z 355 could be 5-carboxypyrano-cyanidin-galactoside, but the information was not confirmed.18 According to its MS and MS/MS spectra, F



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Figure 2. Pictures and colorimetric data of (A) the native clear red-fleshed apple juice without any additives and (B) four selected juices at the end (+14 days) of the color stability experiment: on the right, corrected pH 4.4; on the left, corrected pH 3.0; upper pictures, juices under argon atmosphere; lower pictures, juices under air-saturated atmosphere. All juices were diluted 4 times before spectrophotometric measurements.

of ideain in studied fruits was about 3 times higher than in ‘Weirouge’ apples studied by Sadilova et al., about 9.3 mg/100 g in unpeeled fruits. Hydroxycinnamic acids, namely, 5-O-caffeoylquinic and 4-pcoumaroylquinic acid, represented >6% of total phenolic compounds (Table 3). These two compounds were present approximately at the same concentration, close to 15 mg/100 g FW (Table 1). Interestingly, in comparison to other apple varieties (Table 5), the red-fleshed cultivar was from 2 to 6 times less concentrated in 5-caffeoylquinic acid. Because this compound is the preferential substrate of PPO,7 its presence at a very low concentration could be linked to a weak sensitivity of this apple variety to enzymatic oxidation. Dihydrochalcones represented by phloridzin and phloretin-2xyloglucoside accounted for only 3.8% of total phenolic compounds (Table 3), with phloridzin predominating at a concentration close to 13 mg/100 g FW, which is about 2 times higher than the level of phloretin-2-xyloglucoside (5.9 mg/100 g FW) (Table 1). Flavonols accounted only for 1% of total phenolic compounds (Table 3), which represented 5.0 mg/100 g FW. They were represented by quercetin 3-O-galactoside (3.0 mg/ 100 g of fresh weight) and quercetin 3-O-arabinoside (1.3 mg/ 100 g of fresh weight). Other flavonols (quercetin glucoside, xyloside, and rhamnoside) were quantified in the range from 0.1 to 0.9 mg/100 g FW (Table 1). Quercetin 3-O-rutinoside (i.e., rutin), which is usually present in most apple varieties, was detected as only traces by mass spectrometry. Thus, it was not considered for UV−vis quantification in the present study. The studied red-fleshed apples were rich in polyphenols. In comparison to other apple varieties, their phenolic quantity is included in the upper level (Table 5). They are characterized by a high concentration of procyanidins and a low amount of 5caffeoylquinic acid. Red-colored apple juices are also rich in polyphenols but with a relatively low concentration of 5-CQA (Table 5). Phenolic Compounds in the Juice and Their Extractability from the Fruits. The fruits for which polyphenol composition was described in the preceding paragraphs were used to produce a red-colored juice according to a standardized procedure. Distinction was made between the crude cloudy juice directly obtained after pressing and the clear juice obtained after centrifugation of the cloudy juice. Most of the

simple phenolics found in the fruit were also found in the juice (Table 3). However, contrarily to what has been observed in some previous work,14 no significant difference concerning the polyphenol composition was observed between the cloudy and centrifuged juices. For this reason, the following discussion will concern only the centrifuged clear juice. Total polyphenol content in juice equaled 216 mg/100 mL (Table 3), which corresponds to an extraction rate of about 40%. This extraction rate was calculated by the ratio between the amount of polyphenol in the juice and the amount in the fruits that was processed to obtain this juice. As the juice yield has a high impact on this rate, we will use it only to compare the different phenols in a juice. Total anthocyanins content was 12.3 mg/100 mL (Table 3), which corresponded to about 6% of the total polyphenol content and an extractability rate close to 26%. Logically, the main compound found in this class was cyanidin-3-Ogalactoside, the one that is predominant in fruit flesh. Its extraction rate was close to 26%. Cyanidin pentoside was extractable in about 19%, whereas compound 19 (Table 1) corresponding to a putative anthocyanin−flavanol adduct and compound 20 corresponding to the tentatively identified 5carboxy-pyrano-cyanidin-glucoside were both almost completely extracted in the juice. As previously observed for apple juice when thiolysis or phloroglucinolysis coupled to HPLC was used for procyanidin oligomer and polymer characterization and quantification,14,23 the main polyphenols found in red-fleshed apple juices were flavanols. Procyanidins were predominating with a concentration close to 135 mg/100 mL (Table 4). The extractability of flavanols in the juice was close to 42%, and their average degree of polymerization was close to 2.5. Total content of hydroxycinnamic acids equaled 30.8 mg/ 100 mL in the clear juice. Interestingly, this value is very low in comparison to previously published data concerning apple juices. This fact can be mainly imputed to the very low concentration of 5-caffeoylquinic acid, which is almost at the same level of concentration as 4-p-coumaroylquinic acid (Table 1). Considering that 5-caffeoylquinic acid is the preferential substrate for PPO, this red-fleshed variety should not be sensitive to enzymatic oxidation and browning. Nevertheless, we noted that hydroxycinnamic acids were the most extractable polyphenol group with an extraction rate close to 100%. G



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(from 0 to 400 μg/L) were tested for their effect (main and quadratic effects) on the color parameters: saturation (C*), hue (h), and lightness (L*). Furthermore, the effects of combinations of all these factors (interaction effect) were also estimated. For oxygen, only the main effect and interaction are available as only two modalities were used. Data were treated using the general linear model in Statgraphics software. Figure 2B presents pictures of the samples under argon or air atmosphere at two different pH values (3.0 and 4.4) at the last day of the experimental design. For both pH values, oxygenprotected juices were redder and brighter. Samples that contained oxygen became brown-orange and less bright than those kept under argon atmosphere. Figure 4 shows the value of the coefficients of the statistical model corresponding to the factors and interactions between factors that have a significant influence on the juice color parameters h, C, and L*. A negative coefficient indicates a decreasing effect, whereas a positive one corresponds to an increasing effect. As all of the factors were coded from −2 to +2 corresponding to the range of encountered conditions, this

Dihydrochalcones represented 3.5% of the phenolic content (Table 3) with an extraction rate close to 40%. Total dihydrochalcones were close to 7 mg/100 mL of juices (Table 3), with a predominance of phloretin-2-xyloglucoside compared to phloridzin (Table 1). Flavonols were the least concentrated phenolic compounds in juices, with 0.4% of the total phenolic content (Table 3) and an extraction rate close to 16%. Quercetin 3-O-galactoside was the most concentrated flavanol, whereas the least abundant one was quercetin 3-O-arabinoside. Quercetin 3 -O-glucoside was not detected in the juices (Table 1). Color of the Pure Juice. Figure 2A presents the picture of the pure, centrifuged juice at room temperature, directly before starting experimental design. Color was described in the CIE Lab scale by three orthogonal parameters, L*, which represent the lightness, and a* and b*, which are related to redness and yellowness, respectively. It can be more convenient to express the two-dimensional coordinate system defined by a* and b* by the polar coordinates that are easier to understand in terms of color: the angle (h) in degrees representing the hue (0° for red and 90° for yellow) and the distance (C*) from the center giving the saturation of the color (0° for gray, i.e., black to white depending on L* and >60° for an intense color). Figure 3 is used to indicate the hue position of different fruit juices or ciders in the CIE Lab representation. In comparison to

Figure 3. Position of different fruit juices in the CIE Lab color space: (▲) the studied red-fleshed apple juice; (●) four dessert apple juices;37 (★) cranberry juice;38 (×) raspberry juice;38 (■) strawberry juice,38 (+) grape juice;39 (◆) French ciders.36

French ciders36 or classical clear apple juices37 made from dessert varieties, the studied red-fleshed juice was very different, characterized by a much higher a* value (redness) and a lower b* value (yellowness). Strawberry,38 raspberry,38 and grape39 juices were slightly lower on the yellow scale, but showed about 2 times lower a* values, giving a much paler red color than the red-fleshed apple juice. The closest juice on a color basis was cranberry juice38 (Figure 3). When the juices were compared by using h and C* values, the red-fleshed apple juice had the lowest hue (tend to pure red) and one of the highest saturation values, close to cranberry juice.38 Experimental Design Approach to Color Stability. Statistical analysis of the colorimetric data of 36 samples on the first day of the experiment (“start”) and 72 samples on the last day (‘”finish”) was performed to evaluate the stability of the juice color. The influences of oxygen (presence or absence of air in the flask), pH (3.0, 3.35, 3.7, 4.05, 4.4), ascorbic acid (from 0 to 200 mg/L), SO2 (from 0 to 100 mg/L), and copper

Figure 4. Coefficients of the statistical model describing the effect of the studied parameters (i.e., oxygen (O2), pH, sulfites (SO2), ascorbic acid (AA), and copper ions (Cu)) and their main interactions on the color saturation (A), hue (B), and lightness (C) of the clear redfleshed apple juice. H



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Figure 5. Effect of pH on the hue (A), saturation (B), and lightness (C) of the red-fleshed apple juice at the end of the storage period under air (solid line) or argon (dotted line) atmosphere.

Figure 6. Impact of pH and oxygen on percentage of residual anthocyanins (A) and on formation of yellow compounds (B). * Corresponding to the total area of the chromatogram in the 400−460 nm region.

figure allows a comparison of the effects within the usual apple juice conditions. In general, the highest values of the coefficients were observed for saturation and hue, and only small values were observed for lightness. This indicated that lightness of the juice was not greatly modified during storage. In contrast, hue and saturation were strongly changed during the storage, and highest values were observed for the factors oxygen and pH, underlying the great impact of these factors on color change. Figure 5 gives a more detailed illustration of the effects of these two factors on the three color parameters. The presence of oxygen and the higher pH values always increased the hue angle (Figure 5A): when extreme conditions are accumulated (high pH in the presence of oxygen), the color of the initial juice changed from red (h = 20) to orange-yellow (h = 70), and the interaction that was observed between these factors was not high enough to change the sign of the global effect of each factor. These evolutions of the hue went together with changes of saturation and lightness. However, for these parameters, there were strong interactions between oxygen and pH so that the resulting effects of pH on saturation and lightness were inverted when oxygen was present (Figure 5B,C): in the oxygen-containing juices, an increase of pH from 3 to 4.4 led to an increase of saturation and a decrease of lightness. On the contrary, when the juice was under argon, an increase of pH reduced the saturation and enhanced the lightness. In addition, a quadratic effect of pH was observed for these three parameters, showing that the effect of pH is not linear. However, within the pH range that was tested, the quadratic effect was not high enough to give rise to a maximum or a minimum in the parameters. The other factors also had significant effects on the color parameters, but their coefficients were lower: the effects of ascorbic acid and sulfites were in opposition to those of oxygen so that, as expected, these factors partially tend to prevent the evolution due to oxidation. A low effect of copper was observed

on lightness (main effect P = 0.0296, interaction with pH P = 0.0120) but not on hue and saturation. The high effect of oxygen on hue was not surprising because oxidation of anthocyanin is known to modify the color from pink/red toward yellow/orange, thus increasing the hue (Figures 4B and 5A). The concomitant increase of the saturation suggests that the new pigments that were produced by oxidation significantly contributed to the color saturation and, to a lesser extent, slightly reduced the lightness. The effect of pH was more complex due to the strong interactions with oxygen mainly observed for saturation (Figure 4A). However, those results were consistent with known mechanisms: On the one hand, a high pH favored autoxidation of anthocyanins that may produce new colored compounds such as pyranoanthocyanins27 as already observed in wine models. Note that those neoformed colored compounds are not necessarily oxidation products of anthocyanins. For instance, they may result from nonenzymatic oxidation of flavanols leading to the formation of yellow compounds as previously observed in model solutions.40,41 However, this oxidation occurs only when oxygen was present, which explains the interaction between the two factors pH and oxygen. On the other hand, a low pH is known to enhance red color due to a highest content of stable and redcolored flavylium cation forms of anthocyanins.21 This effect reinforced the interaction (pH × oxygen) on saturation and lightness resulting in this inversion of the effect of pH depending of oxygen (Figure 5B,C). An effect of sulfites and ascorbic acid was expected, as these reagents are well-known reducing agents used in food industries. Their effect was actually in conflict with those of oxygen, which was consistent with their properties, but the reduction of the oxidation was rather low. This can be due to the small quantities of juice compared to an equivalent volume of air and also to the high diffusion rate of gases in such a small vial. The protective effect was probably limited by excessive oxygen availability in these I



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neoformed products responsible for color degradation. The present study was more particularly devoted to the color that happens during juice storage. However, it would also be essential to study the color degradation potentially occurring during fruit crushing and pressing when maximum PPO activity is available and also during apple juice fermentation for a particular use of this red juice in the cider industry.

conditions. An effect of copper was also expected, but the results showed that, at the tested concentrations, the role of copper can be neglected compared to the other factors. Taking advantage of the diode array UV−vis detection, HPLC analysis of selected juices at the end of the storage experiment allowed us to complete the information concerning color stability of the red-fleshed apple juice. As seen in Figure 6A, total anthocyanin content strongly changed during the storage. As observed for color stability, oxygen was the most important factor influencing the “red” compounds, so those two evolutions seem to be highly related. The level of decrease was clearly higher in oxygen-containing juices (from 75 to 86%), whereas under argon no more than 50% of the anthocyanins were degraded. The second factor influencing the stability of anthocyanins was the pH. The lowest amounts of anthocyanins were observed at pH 4.4 both at the beginning and at the end of the experiment, whereas at pH 3.0, more “red” compounds were detected on chromatograms. The decrease of anthocyanins in highly acidic conditions was around 74.5%, whereas at pH 4.4 it was up to 85.6%. This indicated that the level of decrease was highly related to the pH of the juice (Figure 6A). The concentration of yellow compounds in the juices at the end of the storage period was estimated by measuring the global area between 5 and 45 min on the average chromatogram in the 400−460 nm range that reveals the absorbance due to yellow-orange (Figure 6B). For all pH values tested, the juices kept under argon atmosphere did not contain high amounts of yellow-orange compounds. In contrast, larger concentrations of yellow-orange compounds were found in the samples containing oxygen, and this concentration was much higher (close to 3.5 times more) for the juices at pH 4.4. Comparison with the 400−460 nm chromatograms of the juices at the beginning of the experiment (data not shown) revealed that most of the yellow-orange compounds found in the juices at the end of the storage period were not present in the fresh juices. These compounds likely corresponded to polyphenol oxidation products as already discussed, and further work is needed to characterize these neoformed yellow-orange compounds. To conclude, the INRA red-fleshed X4876 apple cultivar was shown to be a raw fruit material producing an attractive redcolored clear apple juice. Interestingly, for several reasons, biochemical characteristics and in particular the polyphenol composition and distribution are favorable to red juice production with limitation of browning reactions during processing: (i) anthocyanins that are located in the flesh and responsible for the red color are highly extractable in the juice during pressing; (ii) compared to many other varieties, the juice is relatively acidic, which allows limiting the PPO activity and subsequent browning reactions; and (iii) the low concentration of chlorogenic acid, which is the preferential substrate of PPO, may also contribute to its low sensitivity to browning. The study of the stability of the color revealed that the presence of oxygen in the juice was the main factor highly detrimental to color preservation, and this instability is enhanced when the pH increases. In those conditions, the juice turns orange-brown and HPLC analysis revealed as a consequence both anthocyanin degradation and the formation of yellow compounds likely corresponding to PPO products. In the conditions tested, the use of reducing compounds was only weakly effective for limiting the color degradation. In contrast, the reduction of oxygen level in the juice by using argon was highly effective. Further works would aim to identify those

■

ASSOCIATED CONTENT

S Supporting Information *

ANOVA tables for the colorimetric parameters C*, h, and L* and statistical prediction models for the three parameters C*, h, and L* describing the color of the red-fleshed juices after storage. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*(S.G.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Bernard Petit from INRA, IRHS, Beaucouzé, France, for providing the apple fruits. We also thank Sylvain Hingant from IFPC and Gildas Le Bail from INRA for their contribution to the apple fruit sampling and juicemaking.

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REFERENCES

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