Phytochemical Profiles of New Red-Fleshed Apple Varieties

Article pubs.acs.org/JAFC

Phytochemical Profiles of New Red-Fleshed Apple Varieties Compared with Traditional and New White-Fleshed Varieties David Bars-Cortina,† Alba Macià,† Ignasi Iglesias,§ Maria Paz Romero,† and Maria José Motilva*,† †

Food Technology Department, XaRTA-TPV, Agrotecnio Center, Escola Tècnica Superior d’Enginyeria Agrària, University of Lleida, Avinguda Alcalde Rovira Roure 191, 25198 Lleida, Catalonia, Spain § Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Fruitcentre, PCTAL, Parc de Gardeny, 25003 Lleida, Catalonia, Spain S Supporting Information *

ABSTRACT: This study is an exhaustive chemical characterization of the phenolic compounds, triterpenes, and organic and ascorbic acids in red-fleshed apple varieties obtained by different breeding programs and using five traditional and new whitefleshed apple cultivars as reference. To carry out these analyses, solid−liquid extraction (SLE) and ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) were used. The results showed that the red-fleshed apples contained, in either the flesh or peel, higher amounts of phenolic acids (chlorogenic acid), anthocyanins (cyanidin-3-Ogalactoside), dihydrochalcones (phloretin xylosyl glucoside), and organic acids (malic acid) but a lower amount of flavan-3-ols than the white-fleshed apples. These quantitative differences could be related to an up-regulation of anthocyanins, dihydrochalcones, and malic acid and a down-regulation of flavan-3-ols (anthocyanin precursors) in both the flesh and peel of the red-fleshed apple varieties. The reported results should be considered preliminary because the complete phytochemical characterization of the red-fleshed apple cultivars will be extended to consecutive harvest seasons. KEYWORDS: anthocyanins, phenolic compounds, red-fleshed apples, UPLC-MS/MS

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INTRODUCTION

apple peel comes from the anthocyanins. Temperature has a major effect on anthocyanin synthesis, directly affecting the transcriptional levels of MYB10 and other members of the activation complex. This results in a decline in the expression of genes of the anthocyanin biosynthetic pathway and less red color in apple peel. Most candidate apple MYB repressors also decline in expression under high temperatures, because their expression is coordinated by MYB10.13 In addition, although the concentration of phenolic compounds is much greater in the peel of apples than in the flesh,2 a million pounds of peel is thrown away every year (the waste product of applesauce and canned apple manufacture), and also some people (no information was found on that subject in the literature) and cultures discard the peel before eating.14 Moreover, there is a rapidly increasing interest in potential crops for coloring food naturally15 without transgenic or cysgenic programs because consumers’ purchasing behavior regarding genetically modified food is mainly negative.16 Hence, colored fruits and vegetables are attractive for both fresh fruit and juice processing companies to grow and diversify their markets. Regardless of the commercial interest, the enhanced levels of anthocyanins in the apple flesh (apart from the peel) could be an interesting approach to increase consumer acceptance and the potential health benefits17−21 through the ingestion of this popular fruit. In addition to anthocyanins and other phenolic compounds, apples are the main source of dihydrochalcones in the diet.22 Recently,

The apple is consumed worldwide and is one of the most important dietary sources of phenolic compounds. It is available year-round on the market, is low in price, and has a positive “health image”.1−4 Since the discovery of a wild red-fleshed apple (Malus pumila ‘Niedzwetzkyana’) in the hotspot of apple origins (Tian Shan mountain forests of Inner and Central Asia) by the Russian botanist Niedzwetzky and, especially, the works of the two main breeders (Niels Hansen and Albert Etter) who developed the two main red-fleshed apples strains,5 an interest in developing a commercial red-fleshed apple has grown in recent years. In the past few years, the poor taste of the wild red-flesh varieties (e.g., M. pumila ‘Niedzwetzkyana’) has been improved through crossbreeding programs with good-flavored white-fleshed apples to give a commercially viable red-fleshed eating variety.5 Four main good-tasting red-fleshed apples have become available over the past decade. These are the ‘Baya Marisa’ (Germany), ‘Rosette’ (England), ‘Redlove’ (Switzerland), and ‘Weirouge’ cultivars (Germany, Italy) cultivars. These are slightly closer in taste to commercially grown apples but with hints of a “not-quite-apple” flavor and flesh texture, a hint of strawberry, raspberry, or red currant making them very unusual.5 It is well established that consumers generally prefer redpeeled apples as they are perceived to be associated with better taste and flavor, and the high marketability of this fruit is thus important for growers6−9 but also difficult to reach in areas with warm and hot climates. Even so, important progress in terms of the capability to develop high peel color in those climatic conditions has been made by either selecting high-color strains of the original cultivars (‘Gala’, ‘Delicious’, ‘Fuji’, ...) or breeding for high color and eating quality.10−12 The red coloration in © XXXX American Chemical Society

Received: June 30, 2016 Revised: November 11, 2016 Accepted: February 5, 2017

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DOI: 10.1021/acs.jafc.6b02931 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Red- and white-fleshed apple varieties studied.

when selecting for high and attractive peel color is the objective.12 The objective of the present study is to assess the interest of the new red-fleshed apple cultivars, released from different breeding programs, compared with some traditional and new white-fleshed with a significant impact on the fruit industry, through the analysis of the whole profile of phenolic compounds, triterpenes, organic acids, and ascorbic acid, comparing them with commercial white-fleshed apple cultivars. To carry out these analyses, different analytical methodologies were developed and optimized, this being a specific objective of the work. After optimization of these methods, their quality parameters were studied to obtain reliable results. To the best of our knowledge, no study has previously addressed the investigation of the phytochemicals of red-fleshed apples intended for commercial purposes. Nevertheless, this work should be considered a preliminary study because it is based on the results of only one harvest.

research into dihydrochalcones has been intensified due to their exclusive chemotaxonomy and potential benefits for human health based on their antidiabetic effect, preventing dietinduced obesity, hepatic steatosis, and insulin resistance.23,24 Another aspect of interest is that red-fleshed apples could be considered a biofortified crop. Biofortification (food staples eaten widely by the poor with increased vitamins/minerals/ other health compounds through either conventional plant breeding or the use of transgenic techniques or fertilization) can reduce the global incidence of “hidden hunger”, malnutrition caused not by too few calories but by an inadequate intake of essential micronutrients in the daily diet. The main biofortified crops are such staples as rice (e.g., with high β-carotene, zinc, or folate contents), maize and cassava (e.g., with high β-carotene or ascorbate contents or with multiple micronutrients), and wheat (e.g., with zinc biofortification). However, food crops not only can be fortified with vitamins/minerals but can also be designed to contain bioreactive compounds (e.g., a variety of plant seed oils have been developed through metabolic engineering to synthesizing omega-3 fatty acids; a tomato with an anthocyanin content similar to those of blueberries and blackberries through the insertion of two anthocyanin transcription factor genes from the snapdragon plant).25 The red-fleshed apples are an analogous case to these tomatoes but with the difference that they originated from conventional crossbreeding and not through transgenic techniques, with the advantage that apple consumption is widespread and this fruit is cheap and available year-round.1−4 In the case of apples, breeding has mainly concentrated for decades on improving fruit quality, fruit appearance, and disease resistances and has largely ignored the health properties of the fruit. However, a recent breeding target is focusing on developing new red-fleshed cultivars because of the close relationship between many polyphenols and anthocyanin in apple and the potential benefit of these compounds to human health. In particular, in selecting for high red flesh color apples, breeders will want to know which if any other polyphenols are also being altered as a result.26 Our results try to answer this question. Also, when breeding for traditional white-fleshed cultivars, the recent discovery of different molecular markers associated with high peel coloration even in warm climates will be a useful tool for fast breeding

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MATERIALS AND METHODS

Chemicals and Reagents. Pelargonidin-3-O-glucoside, cyanidin3-O-glucoside, cyanidin-3-O-galactoside, delphinidin-3-O-glucoside, malvidin-3-O-glucoside, hydroxytyrosol, luteolin, kaempferol, eriodictyol, quercetin, luteolin-7-O-glucoside, kaempferol-3-O-glucoside, quercetin-3-O-rhamnoside, quercetin-3-O-glucoside, isorhamnetin-3O-glucoside, dimer B2, quercetin-3-O-rutinoside (rutin), myricetin, and phloretin-2′-O-glucoside were purchased from Extrasynthese (Genay, France). p-Hydroxybenzoic acid, 3,4-dihydroxybenzoic acid (protocatechuic acid), p-coumaric acid, gallic acid, caffeic acid, ferulic acid, chlorogenic acid, naringenin, catechin, epicatechin, ursolic acid, and butylated hydroxytoluene (BHT) were from Sigma-Aldrich (St. Louis, MO, USA). Vanillic acid and malic acid were from Fluka (Buchs, Switzerland). Ascorbic acid, methanol (HPLC grade), acetonitrile (HPLC grade), and acetic acid were purchased from Scharlab Chemie (Sentmenat, Catalonia, Spain). The water was of Milli-Q quality (Millipore Corp., Bedford, MA, USA). Stock solutions of standard compounds were prepared by dissolving each compound in methanol at a concentration of 1000 mg/L and stored in a dark flask at 4 °C. Ascorbic acid was prepared in Milli-Q water instead of methanol. Plant Material. Four different type-1 red-fleshed apple varieties, ‘RS-1’ (Red Moon Companie, Italy) and ‘107/06’, ‘117/06’, and ‘119/ 06’ (Lubera AG, Switzerland) protected and commercialized in the EU under the mark Redlove Era, were selected. Five white-fleshed apple varieties, including new (‘Brookfield Gala’, ‘Zhen Aztec Fuji’, and B


Article

Journal of Agricultural and Food Chemistry ‘Story’ (Story (Inoredcov)) and traditional (‘Golden Smoothee’ and ‘Granny Smith’) varieties, were selected. All apple varieties were provided by IRTA-FruitCentre (Lleida, Catalonia, Spain) (Figure 1). Apples were harvested from 5-year-old trees, grafted onto M9 EMLA rootstock, and planted in an experimental plot at the IRTA-Estació Experimental de Lleida (Møllerussa, Catalonia, Spain), characterized by a dry and warm summer. The apple trees were trained with a central leader system, spaced at 4 m × 1.5 m. Fruit size and crop load were very uniform among trees and cultivars. Apples from each variety were harvested from different trees in the autumn of 2015 at the same maturation stage, taking into account both their flesh firmness (range 7.5−8.5 kg) and starch index (range 6.5−7.5 on EUROFRU code 1− 10),10 and placed in cool storage at 0.5 °C. Immediately after the apples arrived at the laboratory, they were washed, cored, and peeled. Then, individual apple flesh (cut transversely into slices across the equator) and peel were separately frozen in liquid nitrogen and subsequently freeze-dried (Lyophilizer TELSTAR Lyobeta 15, Terrassa, Spain). Each freeze-dried apple sample (peel and flesh) was kept individually in sealed plastic bags at −80 °C until the chromatographic analysis. Prior to analysis, a fine powder of the freeze-dried apple samples was obtained with the aid of an analytical mill (A11, IKA, Germany). For each variety, three apples were selected for phytochemical analysis (each with separate flesh and peel), and three replicates were carried out by apple (n = 9). Analytical Methods. Sample Pretreatment. For the analysis of the phenolic compounds and triterpenes, lyophilized apple flesh (0.2 g) and peel (0.1 g) were weighed. Then, 5 mL of methanol/Milli-Q water/acetic acid (79.9:20:0.1, v/v/v) as the extraction solvent was added for the analysis of the anthocyanins and the rest of the phenolic compounds, and 20 mL was used for the analysis of the triterpenes. The samples were vortexed for 10 min and then centrifuged at 8784g for 10 min. The supernatant was filtered through a 0.22 μm nylon filter (Scharlab, Barcelona, Spain). For the analysis of the organic acids and ascorbic acid, 0.06 g of lyophilized sample (flesh or peel) was weighed. Then, 240 μL of 0.1% BHT (prepared in methanol) and 180 μL of Milli-Q water were added. The samples were also vortexed for 10 min and centrifuged at 8784g for 10 min. The supernatant was filtered through 0.22 μm PVDF filters (Scharlab), and the filtered samples were diluted 4-fold with Milli-Q water. Ultraperformance Liquid Chromatography Coupled to Tandem Mass Spectrometry (UPLC-MS/MS). LC analyses were carried out on AcQuity Ultra-Performance liquid chromatography and tandem mass spectrometry equipment from Waters (Milford, MA, USA). Four chromatographic methods were used for the analysis of (1) anthocyanins, (2) the rest of the phenolic compounds, (3) triterpenes, and (4) organic acids and ascorbic acid. In all of the methods, the flow rate was 0.3 mL/min and the injection volume, 2.5 μL. For the analysis of phenolic compounds (including anthocyanins) and triterpenes, the analytical column used was an AcQuity BEH C18 (100 mm × 2.1 mm i.d., 1.7 μm) equipped with a VanGuard PreColumn AcQuity BEH C18 (5 × 2.1 mm, 1.7 μm), also from Waters. For the analysis of anthocyanins, the mobile phase was 10% acetic acid (eluent A) and acetonitrile (eluent B). The elution gradient was the same as in our previous study.27 Briefly, this was 0−10 min, 3−25% B; 10−10.10 min, 25−80% B; 10.10−11 min, 80% B isocratic; 11−11.10 min, 80−3% B; 11.10−12.50 min, 3% B isocratic. For the analysis of the rest of the phenolic compounds and triterpenes, the mobile phase was 0.2% acetic acid (eluent A) and acetonitrile (eluent B). The elution gradient for the analysis of the phenolic compounds was 0−5 min, 5−10% B; 5−12 min, 10−12.4% B; 12−18 min, 12.4−28% B; 18−23 min, 28−100% B; 23−25.5 min, 100% B isocratic; 25.5−27 min, 100−5% B; and 27−30 min, 5% B isocratic; and for the analysis of triterpenes, 0−20 min, 50−100% B; 20−23 min, 100% B isocratic; 23−26 min, 100−50% B; 26−30 min, 50% B isocratic. The analytical column for the analysis of the organic acids and ascorbic acid was an AcQuity HSS T3 column (100 mm × 2.1 mm i.d., 1.8 μm,) equipped with a VanGuard Pre-Column AcQuity HSS T3 (5 × 2.1 mm, 1.8 μm) also from Waters. The mobile phase was 0.1% formic acid (eluent A) and methanol (eluent B). The elution gradient

for the analysis was 0−10 min, 0% B isocratic; 10−10.1 min, 100% B; 10.1−15 min, 100% B isocratic; 15−15.1 min, 100−0% B; and 15.1− 20 min, 0% B isocratic. Tandem MS analyses were carried out on a triple-quadrupole detector (TQD) mass spectrometer (Waters) equipped with a Z-spray electrospray interface. Ionization was achieved using the electrospray (ESI) interface operating in the positive mode [M − H]+ for the analysis of anthocyanins and in the negative mode [M − H]− for the other compounds. The data were acquired through selected reaction monitoring (SRM). The ionization source parameters were as follows: capillary voltage, 3 kV; source temperature, 150 °C; desolvation gas temperature, 400 °C, with a flow rate of 800 L/h. Nitrogen (99.99% purity, N2LCMS nitrogen generator, Claind, Lenno, Italy) and argon (≥99.99% purity, Aphagaz, Madrid, Spain) were used as the cone and collision gases, respectively. Two SRM transitions were studied, selecting the most sensitive transition for quantification and a second one for confirmation purposes. The SRM transition for quantification and the individual cone voltage and collision energy for each phenolic compound are shown in Table 1 of the Supporting Information. The dwell time established for each transition was 30 ms. Data acquisition was carried out with MassLynx 4.1 software. Validation of the Chromatographic Method. The instrumental quality parameters of the developed methods, such as the linearity, calibration curve, precision, accuracy, detection limits (LODs), and quantification limits (LOQs), were determined by using a serial dilution of a stock solution of the standards in methanol/Milli-Q water/acetic acid (79.9:20:0.1, v/v/v), except for organic acids and ascorbic acid, for which Milli-Q water was used. These quality parameters were in line with U.S. Food and Drug Administration (FDA) guidelines28 (data not shown). Table 1 of the Supporting Information also shows how each phenolic compound was quantified or tentatively quantified. Statistical Analysis. Concentration values were reported as means (n = 9) ± standard deviation (SD). All data were analyzed by JMP Software, version 12.0 (SAS Institute Inc., Cary, NC, USA). One-way analysis of variance (ANOVA) and Tukey’s test were used to determine the significance of differences among apple varieties at a level of 0.05.

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RESULTS AND DISCUSSION Although different analytical methods have been reported in the literature for the determination of anthocyanins (cyanidin galactoside)29 in red-fleshed apple cultivars or phenolic compounds30−33 or triterpenes34−36 in white-fleshed apple cultivars, in the present study these methods were optimized to select a single extraction solution able to extract all of these phytochemicals. Then, after optimization of the sample pretreatment (extraction solution), the chromatographic conditions were also improved in terms of elution time and peak efficiency. Analysis of Phenolic Compounds and Triterpenes: Sample Pretreatment. The initial experiments for extracting phenolic compounds and triterpenes from apple samples were the ones reported in our previous study for the extraction of phenolic compounds from Arbutus unedo fruit.37 Then, to obtain the maximum efficacy, different parameters that affect the extraction of these compounds were studied and optimized. These parameters were the sample weight, nature of the extraction solution, and volume used. Different sample weights of lyophilized apple (flesh and peel), from 0.04 to 1 g; extraction solutions, such as ethanol, methanol, Milli-Q water, methanol/water mixtures with and without acetic acid, and the solution acetone/Milli-Q water/acetic acid (70:29.5:0.5, v/v/ v);38 and extraction solution volumes, from 5 to 20 mL, were tested. C


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Figure 2. Extracted ion chromatogram of ascorbic acid and malic acid isolated from flesh ‘Golden’ apple when different percentages of 0.1% BHT in Milli-Q water were used as the extraction solution: (A) 10%; (B) 30%; (C) 60%; (D) 80%; (E) 100%. The extraction solution of 0.1% BHT/Milli-Q water (60:40, v/v) with the analytes was diluted (F) 2-fold and (G) 4-fold with Milli-Q water. The solutions were filtered through PVDF filters.

The best results were obtained with 0.2 and 0.1 g of lyophilized apple flesh and peel, for the analysis of the phenolic compounds and triterpenes, respectively. The methanol/MilliQ water/acetic acid (79.9:20:0.1, v/v/v) and acetone/Milli-Q water/acetic acid (70:29.5:0.5, v/v/v) extraction solutions gave the best results for the extraction of these compounds. Although the results obtained for the extraction of these compounds were similar, the methanol/Milli-Q water/acetic acid (79.9:20:0.1, v/v/v) solution was chosen because acetone absorbed in UV, and it could interfere with the compounds that eluted at the first times in the UPLC-PDA chromatogram. Five milliliters of methanol/Milli-Q water/acetic acid (79.9:20:0.1, v/v/v) was enough for the complete extraction of phenolic compounds from the apple samples (flesh and peel). In the case of triterpenes, a higher volume of extraction solution was necessary to complete extraction from the apple peel, and this was 20 mL. Analysis of Organic Acids and Ascorbic Acid: Sample Pretreatment. For the extraction of organic acids and ascorbic acid from the apple samples, Milli-Q water was the most effective extraction solvent related with the high polarity of these compounds. However, ascorbic acid was very unstable in the aqueous solution. To overcome this drawback, the addition of different stabilizing agents to the extraction solution, such as

EDTA and BHT, has been reported when MS is used as the detector system.39−41 Therefore, the addition of BHT to the aqueous solution was evaluated in the present study. BHT was prepared in methanol at a concentration of 0.1%, because it is not soluble in the aqueous solution. BHT (0.1%) with different proportions of Milli-Q water, from 90 to 0%, was tested for the extraction of ascorbic acid and organic acids from the apple samples. The extracted ion chromatograms for the analysis of ascorbic acid and malic acid are shown in Figure 2. When the extraction solution was 10−30% of BHT (0.1%) in Milli-Q water (Figure 2A,B), ascorbic acid was not detected, probably due to its high instability in this extraction solvent. The stability of ascorbic acid was significantly improved when the extraction solution contained a proportion of BHT (0.1%) above 40% (Figure 2C−E). Unfortunately, when the amount of the BHT (dissolved in methanol) in the extraction solution was increased, the peak efficiency (peak shape) of ascorbic acid and malic acid decreased. Under these conditions, the chromatographic peaks were highly distorted, showing doubled and broader peaks, and tailing peaks. This could be explained by differences in the viscosity and polarity between the injected sample (sample matrix), which contains a high percentage of methanol, and the mobile phase, which is an aqueous solution.42,43 D


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Figure 3. (A) MS spectrum of ursolic acid (10 mg/L) obtained in daughter scan mode by applying different collision energies, from 5 to 50 eV. (B) Extracted ion chromatogram of ursolic acid (10 mg/L) obtained in (i) SIM mode in the first quadrupole, (ii) SIM mode in the second quadrupole, and (iii) SRM mode.

On the basis of the efficacy of the BHT for the stabilization of ascorbic acid in the sample extract, a mixture of 60% methanolic solution of BHT (0.1%) and 40% Milli-Q was selected (Figure 2C) as the extraction solvent. To improve the efficiency of the chromatographic peaks, the sample extract was diluted 4-fold with Milli-Q water (Figure 2G), thus improving the peak efficiency. Before the chromatographic analysis, the samples were filtered through PVDF filters instead nylon filters because this material retained the organic acids, thus reducing the instrumental response. This was also previously reported by other authors.44 Tandem MS for the Chromatographic Analysis of Triterpenes. To identify the phenolic compounds in the nine apple varieties studied (in both the peel and flesh), different MS analyses were carried out. These were based on the full-scan mode in MS mode and the daughter scan mode in MS/MS mode. Nevertheless, in the analysis of the triterpenes, no fragments were generated or these were not dominant in the MS spectrum when high collision energies were applied. This is shown in Figure 3A corresponding to the analysis of the ursolic acid standard (as the representative triterpene) was analyzed.

Therefore, in the SRM mode of our study, the collision energy applied in the collision cell was set at a low value (5 eV) to minimize fragmentation. Under these conditions, the precursor ion isolated in the first quadrupole (Q1) passed through the collision cell (Q2) without fragmentation, and the same ion was monitored in the third quadrupole (Q3).45,46 The instrumental response of the ursolic acid obtained under these SRM conditions were also compared with the one obtained in the SIM mode (using the first or third quadrupole) (Figure 3B). As can be observed, the SRM mode (Figure 3Biii) showed much higher sensitivity and selectivity than the SIM mode (Figure 3Bi,Bii). This was previously observed by other authors45 for the determination of ursolic acid in human plasma by UPLCMS/MS. Therefore, the SRM mode with one transition that reported the same ions as both precursor and daughter was selected to quantify the triterpenes (Table 1 in the Supporting Information). After the optimization and validation of the analytical methods, these were applied for the analysis of the “phytochemical profiling” of the red-fleshed apple varieties using the commercial white-fleshed varieties as reference. For this proposal, a wide range of compounds was studied in the E


F

1.30 0.85 0.18 0.08

± 0.52 ± 0.29 ± 0.08 ± 0.06ab 1.02 0.55 0.22 0.11

± 0.54 ± 0.46 ± 0.05 ± 0.04ab

298 ± 92.6cde

± 2.18ab ± 2.24a ± 0.02a ± 0.02a ± 2.18abc ± 0.10b

± 11.3a ± 9.37a ± 28.6b ± 27.9b ± 0.38a ± 0.05a ± 0.77a ± 19.1b ± 22.1ab ± 3.82c ± 2.09c ± 1.84c ± 0.29c

119/06

0.28 0.09 0.07 0.03

± 0.06 ± 0.03 ± 0.02 ± 0.01ab

336 ± 67.2bcde

49.3 44.0 184 168 3.26 0.32 9.88 66.9 51.6 22.0 7.00 11.5 2.40 nd c 12.6 9.26 0.13 0.13 0.98 0.59 ± 0.24a ± 0.27ab

± 2.19b ± 2.22ab

± 8.33b ± 7.56a ± 11.0a ± 10.8a ± 0.35a ± 0.07a ± 0.60a ± 49.9a ± 48.6a ± 2.00c ± 0.48cd ± 0.81d ± 0.20c

RS-1

2.67 1.07 0.50 0.56

± 2.32 ± 1.11 ± 0.41 ± 0.46a

522 ± 73.7a

16.9 14.9 328 306 3.24 0.22 5.80 152 141 15.8 4.56 8.12 2.09 nd c 8.24 5.95 nd b nd c 1.57 1.01 ± 0.12bc ± 0.12ab

± 0.30c ± 4.80cd ± 4.16cd ± 25.2a ± 19.4a ± 6.12a ± 2.19a ± 0.09b ± 1.65ab ± 0.56b ± 0.01ab

± 0.06c ± 0.01c ± 13.8ab ± 13.0ab

0.39 0.19 0.04 0.06

± 0.14 ± 0.08 ± 0.02 ± 0.03ab

447 ± 45.6ab

0.73 0.03 217 210 nd b nd b 1.67 23.5 16.5 197 81.7 86.1 15.31 1.48 8.80 2.75 0.01 nd c 0.79 0.79

Brookfield Gala ± 0.11c ± 0.09bc ± 10.5b ± 10.1b ± 0.06a ± 0.05ab ± 0.99b ± 13.2bc ± 13.9bcd ± 6.82a ± 4.04a ± 4.14a ± 2.06a ± 0.24a ± 3.56a ± 4.27a ± 0.01a ± 0.01ab ± 0.41ab ± 0.41a

0.35 0.14 0.05 0.08

± 0.05 ± 0.03 ± 0.00 ± 0.01ab

399 ± 34.6abc

1.23 0.11 164 156 1.04 0.03 3.20 46.3 28.9 167 58.4 84.4 15.4 2.17 18.8 11.9 0.06 0.06 1.35 1.34

Golden Smoothee

± 0.81bc ± 7.27bc ± 4.35cd ± 9.10a ± 6.09a ± 7.53a ± 1.62a ± 0.28a ± 1.48b ± 1.48b

± 0.06c ± 0.02c ± 21.7ab ± 21.1ab ± 0.47b

0.21 0.08 0.03 0.04

± 0.10 ± 0.01 ± 0.03 ± 0.03ab

390 ± 39.7abcd

0.60 0.03 202 192 0.31 nd b 2.33 32.4 14.2 149 50.0 76.4 14.3 2.11 6.11 2.55 nd b nd c nd d nd c

Zhen Aztec Fuji

white-fleshed apples

0.21 0.08 0.01 0.02

± 0.16 ± 0.02 ± 0.01 ± 0.00b

267 ± 45.2de

± 0.30d ± 4.11d ± 4.02d ± 27.2a ± 10.5a ± 17.1a ± 3.63a ± 0.59a ± 2.40ab ± 2.53ab ± 0.20ab ± 0.20bc ± 0.09c ± 0.05b

± 0.10c ± 0.07bc ± 11.1c ± 11.5c

Granny Smith 0.74 0.07 60.7 52.3 nd b nd b 0.94 16.1 11.4 179 54.1 90.4 17.0 2.55 9.82 5.38 0.14 0.14 0.56 0.51

Story

± 2.13ab ± 1.58ab ± 0.01a ± 0.01ab ± 0.15abc ± 0.15ab

± 0.06ab ± 0.25bc ± 6.54cd ± 5.70bcd ± 10.9b ± 4.91b ± 5.80b ± 1.05b

± 0.10c ± 0.10b ± 20.6c ± 20.3c

1.71 0.40 0.75 0.29

± 0.80 ± 0.22 ± 0.54 ± 0.17a

228 ± 41.2e

0.99 0.34 90.4 80.4 nd b 0.06 2.43 32.3 25.1 92.3 29.6 51.0 7.65 nd c 9.21 5.82 0.06 0.05 1.03 1.01

For each row, values not displaying the same letter are significantly different (one-way ANOVA, Tukey’s test between all means, p < 0.05). nd, not detected. bQuercetin derivatives: quercetin arabinoside, quercetin rhamnoside, quercetin glucoside.

a

triterpenes (total) ursolic acid hydroxyursolic acid euscaphic acid

391 ± 50.6abcd

total phenolics

± 6.70ab ± 7.31a ± 0.05a ± 0.06a ± 0.55a ± 0.38ab

± 3.57b ± 3.34a ± 63.4ab ± 64.7ab ± 0.60a ± 0.05a ± 1.12a ± 14.6bc ± 12.7bcd ± 3.69c ± 0.46d ± 1.09d ± 0.19c

± 1.98ab ± 1.87a ± 0.05a ± 0.06ab ± 0.28a ± 0.15ab

117/06

± 6.55a ± 6.09a ± 36.1ab ± 35.7ab ± 0.54a ± 0.10a ± 0.99a ± 4.67bc ± 5.00bc ± 0.96c ± 0.22cd ± 1.02c ± 0.16c

anthocyanins (total) cyanidin glalactoside phenolic acids (total) chlorogenic acid protocatechuic acid vanillic acid vanillic acid hexoside dihydrochalcone (total) phloretin xylosyl-glucoside flavan-3-ols (total) epicatechin dimer trimer tetramer flavonol (total) quercetin derivativesb flavone (total) luteolin glucoside flavanone (total) eriodictyol glucoside

9.34 8.44 214 196 4.34 0.23 6.34 44.3 31.9 15.6 3.65 6.96 1.95 nd c 13.0 10.5 0.18 0.14 1.64 0.91

107/06

49.2 45.4 257 238 4.09 0.31 8.07 47.8 39.1 21.9 5.67 12.0 2.39 nd c 13.1 9.85 0.12 0.12 1.68 1.08

phenolic compounds and triterpenes

red-fleshed apples

Table 1. Concentration of the Main Phenolics and Triterpenes in the Flesh Samples (mg/kg Flesh) from Different Apple Varieties (Mean ± Standard Deviation) (n = 9)a

Journal of Agricultural and Food Chemistry Article


G

± 2.56a ± 0.77a ± 2.05a ± 516abc ± 136ab ± 79.5ab ± 21.3 ± 27.2a ± 5.51 ± 11.3ab

± 176 ± 195ab ± 0.26ab ± 0.22a ± 0.10b ± 2.55a ± 1.29a ± 1.51ab ± 361bc ± 75.0ab ± 9.71ab ± 16.0 ± 23.0abc ± 6.51 ± 16.1a

± 21.1bc ± 17.5bc ± 1.36bc ± 61.5ab ± 58.2bc ± 44.0a ± 3.82a ± 85.4bcd ± 34.9b ± 52.5ab ± 14.5d ± 12.4e ± 4.39e ± 0.75d

± 357 ± 328ab ± 0.11ab ± 0.11a

117/06

± 40.7ab ± 31.8ab ± 3.47ab ± 58.9ab ± 16.2bc ± 42.5a ± 2.39a ± 43.3abc ± 19.4b ± 38.1ab ± 13.7d ± 6.89de ± 8.28d ± 1.32d 81.3 66.9 6.68 257 101 90.1 9.41 298 142 151 70.4 32.4 21.1 5.73 nd c 453 407 0.59 0.53 0.07 10.7 6.30 4.40 1170 330 178 48.9 44.2 15.4 29.0

107/06

197 167 8.10 271 110 100 8.29 375 171 182 117 54.4 45.2 9.42 nd c 526 480 0.44 0.44 nd b 14.0 6.90 7.11 1499 519 263 71.7 127 24.1 22.5

± 44.3a ± 39.5ab ± 1.10ab ± 24.3b ± 3.42c ± 22.2a ± 1.66ab ± 260ab ± 87.3b ± 175a ± 59.0c ± 35.5bc ± 20.8c ± 5.22c ± 0.33b ± 122 ± 147ab ± 0.17ab ± 0.15a ± 0.04a ± 1.22a ± 0.74b ± 0.66a ± 511ab ± 61.8b ± 23.2a ± 29.2 ± 2.92bc ± 5.56 ± 2.29ab

119/06 312 268 8.83 197 46.2 86.6 5.98 509 193 311 248 119 94.4 18.8 3.66 603 547 0.73 0.48 0.25 12.6 5.49 7.14 1881 496 309 93.1 28.8 29.6 13.9

52.6 39.3 3.38 434 284 82.0 7.46 887 653 211 69.4 30.9 23.8 6.27 nd c 399 333 0.79 0.54 0.25 10.8 6.47 4.35 1854 261 132 43.8 45.7 14.1 19.0 ± 129 ± 133b ± 0.26ab ± 0.26a ± 0.06a ± 2.01a ± 1.38a ± 1.31ab ± 434ab ± 50.9b ± 24.3b ± 7.63 ± 9.82abc ± 1.38 ± 7.85ab

± 24.5c ± 19.0c ± 0.72c ± 101a ± 70.9a ± 26.9a ± 1.66a ± 172a ± 146a ± 32.1a ± 5.73d ± 2.41e ± 3.95e ± 0.49d

RS-1 68.0 47.5 6.71 215 143 32.4 4.24 124 88.6 32.8 702 298 312 62.8 6.26 697 593 0.99 0.60 0.39 3.77 1.56 2.21 1810 247 140 45.1 36.6 12.5 2.53

± 14.53c ± 13.2c ± 1.90bc ± 14.1b ± 13.2ab ± 9.27ab ± 0.48abc ± 38.3de ± 23.2bc ± 20.8cd ± 147a ± 77.5a ± 60.3a ± 11.3a ± 0.84a ± 255 ± 271ab ± 0.43a ± 0.34a ± 0.11a ± 0.65b ± 0.23b ± 0.44bc ± 470abc ± 26.6b ± 13.5b ± 4.42 ± 8.22abc ± 2.52 ± 0.84c

Brookfield Gala 7.46 0.10 5.23 209 165 7.21 2.13 176 93.0 79.5 467 173 233 41.9 6.06 425 362 0.42 0.13 0.28 3.20 2.20 1.01 1288 269 184 42.7 14.1 17.2 2.38

± 0.42d ± 0.07d ± 0.24bc ± 25.5b ± 24.7ab ± 0.60cd ± 0.51c ± 18.7bcde ± 10.6bc ± 18.8abcd ± 29.0ab ± 17.8abc ± 28.2ab ± 6.95ab ± 0.72a ± 62.7 ± 75.1a ± 0.20ab ± 0.20ab ± 0.05a ± 1.09b ± 0.71b ± 0.48c ± 138abc ± 47.9b ± 33.3ab ± 6.14 ± 3.27cd ± 3.86 ± 0.77c

Golden Smoothee 36.2 24.1 4.07 244 177 21.4 2.56 171 48.8 120 412 154 202 37.8 5.65 534 463 0.21 nd b 0.21 2.54 1.55 0.99 1400 391 214 75.2 62.7 22.6 5.89 ± 0.13a ± 0.82b ± 0.13b ± 0.81c ± 408abc ± 105ab ± 37.8ab ± 26.8 ± 30.4ab ± 10.6 ± 0.78bc

± 6.57c ± 5.46c ± 0.98bc ± 41.9ab ± 41.8ab ± 7.22bc ± 0.83c ± 128cde ± 28.6c ± 101abc ± 38.5abc ± 27.3abc ± 11.5ab ± 6.16ab ± 0.51a ± 191 ± 192bc ± 0.13b

Zhen Aztec Fuji

white-fleshed apples

± 3.74d ± 0.07d ± 1.69bc ± 11.4c ± 2.08d ± 3.03d ± 0.64bc ± 20.6e ± 6.96c ± 14.2d ± 87.4bc ± 35.2cd ± 57.3bc ± 8.25b ± 0.17a ± 267 ± 230bc ± 0.73ab ± 0.62ab ± 0.15a ± 1.39b ± 0.42b ± 1.02bc ± 392c ± 142ab ± 100ab ± 20.5 ± 6.36d ± 6.44 ± 6.68ab

Granny Smith 9.62 0.12 4.78 45.0 6.68 6.31 2.86 71.5 46.8 23.3 292 97.9 136 37.1 5.18 626 466 1.16 0.75 0.41 4.19 1.99 2.20 1050 380 268 56.5 9.18 14.2 19.6

389 315 15.0 148 12.5 50.0 2.54 169 114 48.8 469 215 200 33.2 5.21 863 743 0.62 0.16 0.46 9.32 2.56 6.76 2048 590 326 83.0 105 27.7 6.25

± 49.1a ± 40.1a ± 1.24a ± 14.5b ± 3.79d ± 7.94ab ± 0.33c ± 19.4bcd ± 10.8b ± 5.62bcd ± 53.4ab ± 19.5ab ± 30.6ab ± 3.63b ± 0.51a ± 187 ± 228a ± 0.25ab ± 0.24ab ± 0.12a ± 0.89a ± 0.90b ± 0.80a ± 325a ± 73.7a ± 41.9a ± 11.7 ± 11.0a ± 5.49 ± 2.74bc

Story

For each row, values not displaying the same letter are significantly different (one-way ANOVA, Tukey’s test between all means, p < 0.05). nd, not detected. bQuercetin derivatives: quercetin arabinoside, quercetin rhamnoside, quercetin glucoside.

a

anthocyanins (total) cyanidin galactoside delphinidin rhamnoside phenolic acids (total) chlorogenic acid protocatechuic acid hydroxytyrosol dihydrochalcone (total) phloretin xylosyl-glucoside phloridzin flavan-3-ols (total) epicatechin dimer trimer tetramer flavonol (total) quercetin derivativesb flavones (total) luteolin glucoside apigenin glucoside flavanone (total) eriodictyol glucoside naringenin glucoside total phenolics triterpenes (total) ursolic acid hydroxyursolic acid euscaphic acid maslinic acid betulinic acid

phenolic compounds and triterpenes

red-fleshed apples

Table 2. Concentration of the Main Phenolics and Triterpenes in the Peel Samples (mg/kg Peel) from Different Apple Varieties (Mean ± Standard Deviation) (n = 9)a



Article

Journal of Agricultural and Food Chemistry apple flesh and peel: phenolic compounds (anthocyanins, phenolic acids, flavan-3-ols, dihydrochalcones, flavonols, flavones, and flavanones), triterpenes, organic acids, and ascorbic acid. Apple Phenolic Profile. Anthocyanins. The four redfleshed apple varieties included in the present study were Hansen’s type, equivalent to type-1 red-fleshed apples. These phenotypes show a red coloration throughout the fruit and also in plant tissues, including the stems, roots, flowers, and developing leaves, in contrast to type-2 red-fleshed apples, which presented the red pigment only in the fruit cortex and green new leaves.5,47,48 Different anthocyanins were identified and quantified in the flesh from the red-fleshed apples (Table 1), cyanidin galactoside being the most abundant (around 98% of the total anthocyanin content), as reported in the literature.29 Additionally, a wide range of anthocyanins, such as pelagornidin, peonidin, delphinidin, petunidin, and malvidin derivatives, were identified and quantified at low concentrations (Table 2 in the Supporting Information). The total anthocyanin content in the red-fleshed apples ranged from 9.34 ± 3.57 to 49.3 ± 11.3 mg/kg apple flesh in comparison with the values for white-fleshed apples, which were from 0.60 ± 0.06 to 1.23 ± 0.11 mg/kg apple flesh (Table 1), the ‘107/ 06’ and ‘119/06’ cultivars being the most interesting for their anthocyanin content. With regard to the apple peel, the most surprising fact was related to the white-fleshed ‘Story’ variety. This was the cultivar with the highest intensity of red peel color (Figure 1). The peel of this apple variety showed the highest anthocyanin content (cyanidin galactoside) (Table 2), although not sufficient to be statistically significant from the two red-fleshed varieties ‘107/ 06’ and ‘119/06’. Moreover, taking into account the proportion of flesh and peel (considering the peel as an edible part), which corresponds to the ingestion of one piece of apple, the dose of anthocyanins ingested through an apple of the ‘119/06’ cultivar would be equivalent to 11.67 mg, followed by ‘107/06’ (8.71 mg) and ‘Story’ (6.06 mg) (data not shown). Other Phenolic Compounds. A wide range of phenolic compounds was identified and quantified in the flesh and the peel of red- and white-fleshed apples. Tables 1 and 2 show the concentrations of the main phenolic compounds, which are significantly (p < 0.05) different in the red-fleshed apples from the white-fleshed ones. Additionally, the concentrations of the minor compounds are shown in Tables 3 and 4 of Supporting Information. In general, no qualitative differences in the phenolic composition were observed between apple varieties, although significant quantitative differences were seen. The phenolic acids were the most abundant compounds in the flesh of red- and white-fleshed apples (Table 1), representing >50% of the total phenols. In accordance with the literature,4,30−32 chlorogenic acid was the most abundant phenolic acid detected in all nine apple varieties. The concentrations of this phenolic acid in the flesh (Table 1) and peel (Table 2) of the red-fleshed fruit were similar to the levels in the white-fleshed varieties. Exceptions were the ‘Granny Smith’ and ‘Story’ varieties, in which the content of chlorogenic acid was statistically lower (p < 0.05). Nevertheless, the concentrations of the other minor phenolic acids were higher and statistically different (p < 0.05) in the red-fleshed than in white-fleshed apples. These phenolic acids were protocatechuic acid, vanillic acid, and vanillic acid hexoside in the flesh (Table 1) and protocatechuic acid and hydroxytyrosol in the peel (Table 2).

Dihydrochalcones, a closely related group of chalcones, are present almost exclusively in apple fruit.4 Apart from apple, few other plant species are known to contain phloridzin (the main dihydrochalcone), and these belong mainly to the Rosaceae and Ericaceae families. The specificity of dihydrochalcones (in particular, of phloridzin) is curious because the pear (a closely related genus to M. pumila) does not contain phloridzin. For this reason, phloridzin has been used for chemotaxonomic differentiation of the rosaceous plant species and the identification of fraudulent admixture of apple juices to other fruit juices.22 In the present study, phloretin-2′-O-glucoside (phloridzin) was the predominant dihydrochalcone quantified in the flesh (Table 1) and peel (Table 2) jointly with xylosylglucoside. Apart from the detection of these two major dihydrochalcones, we detected a third type of dyhidrochalcone (hydroxyl phloretin xylosyl glucoside) at a low concentration level in both the flesh and peel (Table 3 in the Supporting Information). As expected, the dihydrochalcone concentration in the apple peel was much higher than in the flesh for all of the apple varieties studied, and their concentrations were statistically different (p < 0.05). In general, the concentration of dihydrochalcones in red-fleshed apples was higher than in conventional cultivars (white-fleshed apples), mainly in the peel (Table 2), ‘RS-1’ and ‘119/06’ being the most interesting varieties as source of dihydrochalcones. In relation to flavan-3-ols, the concentration of this subclass of flavonoids was high in the flesh (Table 1) and peel (Table 2) of white-fleshed apple varieties, as previously reported in the literature.31−33 Epicatechin and its polymerized forms (dimer, trimer, and tetramer) were the main flavan-3-ols detected, mainly in the apple peel (Table 2) with a high variability between apple cultivars. The lower concentration of flavan-3-ols in the flesh and, to a lesser extent, in the peel of the red-fleshed varieties in comparison with the white-fleshed apple is notable. For example, total flavan-3-ol content in the flesh was between 15.6 ± 3.69 and 22.0 ± 3.82 mg/kg in the red-fleshed varieties and from 92.3 ± 10.9 to 197 ± 25.2 mg/kg in the white-fleshed varieties (Table 1). These concentrations were statistically different. Flavan-3-ol concentration was higher in the peel samples than in the flesh samples, and the same behavior was observed between the red- and white-fleshed cultivars. Contrarily to what was observed in the present work, the data in the literature for concentrations of flavan-3-ols in redfleshed apples achieved by breeding programs (variety Jonathan × Niedzwetzkyana)49 or genetic modification50 showed that the major phenol constituents were flavan-3-ols, with >70% content of procyanidins in apples. The lower concentration of flavan-3-ols detected in redfleshed apples, mainly in the flesh, compared with the whitefleshed varieties could be explained by a competitive synthesis between anthocyanins and proanthocyanidins, because these phenolic subclasses are produced by closely related branches of the flavonoid pathway and utilize the same metabolic intermediates.51,52 A competitive interaction from the substrate between anthocyanidin reductase and anthocyanidin synthase enzymes could be responsible for the lower proanthocyanidin (flavan-3-ols) concentration detected in the red-fleshed apples (which contain high amounts of anthocyanins) in comparison with the white-fleshed apples. Nevertheless, more experiments and studies needed to be performed to explain the lower flavan3-ol concentration in red-fleshed apple cultivars in comparison to white-fleshed apple cultivars, and studies will be discussed as future work. H


Article


Figure 4. Total content of the different phenolic subclasses in (a) the flesh and (b) the peel of the red- and white-fleshed apples varieties.

As a consequence, the concentration of the flavan-3-ols (epicatechin, dimer, trimer, and tetramer) in the flesh (Table 1) of the red-fleshed varieties was lower and statistically different from that in the white-fleshed fruit. An exception was the ‘Story’ variety, in whose flesh tetramer was not detected. Similarly, epicatechin, dimer’ and trimer were the main flavan3-ols quantified in the apple peel, and the concentration of trimer and tetramer in the red-fleshed apples was lower and statistically different (p < 0.05) from white-fleshed samples. The main flavonols detected in all nine apple varieties, in both the flesh and peel, were quercetin derivatives, such as quercetin arabinoside, quercetin rhamnoside, and quercetin glucoside (Tables 1 and 2), as reported in the literature.29,30,53 The concentrations of these phenolic compounds in the flesh were lower than in the peel. Depending on the apple cultivar, the total flavonol concentration ranged from 6.11 ± 1.48 to 18.8 ± 3.56 mg/kg in the flesh (Table 1) and from 399 ± 129 to 863 ± 187 mg/kg in the peel (Table 2). No differences in the concentration of these phenolic compounds were observed between the red- and white-fleshed cultivars. Apart from these quercetin derivatives, minor flavonols were also determined, and some differences between red- and white-fleshed apples could be reported (Table 4 in the Supporting Information). Flavones and flavanones were the phenolic compounds determined at low concentration levels in all of the apple varieties studied, in both the flesh (Table 1) and peel (Table 2). As reported in the literature, apples are not a good source of flavones and flavanones.31 Luteolin glucoside was the main flavone determined in the flesh and peel. Exceptions were the ‘Golden Smoothee’, ‘Zhen Aztec Fuji’, and ‘Story’ peel samples, in which apigenin glucoside was the main flavone. With regard to flavanones, eriodictyol glucoside was the main compound quantified in the flesh samples (Table 1). On the other hand, eriodictyol glucoside and naringenin glucoside were the main flavanones detected in the peel samples (Table 2). The concentrations of these two flavonones in the red-fleshed and ‘Story’ varieties (from 9.32 ± 0.89 to 14.0 ± 2.56 mg/kg) were higher and statistically different (p < 0.05) than the other whitefleshed varieties (from 2.54 ± 0.82 to 4.19 ± 1.39 mg/kg).

To summarize the phenolic profile of the nine apple cultivars studied, panels a and b of Figure 4 show the total contents of the different phenol subclasses quantified in the flesh and peel, respectively. As shown, the concentration of total phenols is higher in the apple peel than in the flesh. The main phenolic compounds in the red-fleshed apples were phenolic acids, dihydrochalcones, and anthocyanins and in white-fleshed apples, phenolic acids and flavan-3-ols. With regard to apple varieties, the red-fleshed ‘RS-1’ (522 ± 73.7 mg/kg flesh) showed the highest total phenol content in the flesh and the white-fleshed ‘Story’ (228 ± 41.2 mg/kg flesh) the lowest (Figure 4a), and these concentration values were statistically different (Table 1). With regard to the apple peel (Figure 4b), the main phenolic compounds in the red-fleshed varieties were flavonols and dihydrochalcones, and in the white-fleshed varieties these were flavonols and flavan-3-ols. The richest apple peel variety in phenolic content was the white-fleshed ‘Story’ (2048 ± 325 mg/kg peel), and the lowest, the redfleshed ‘117/06’ (1170 ± 361 mg/kg peel) and the whitefleshed ‘Granny Smith’ (1050 ± 392 mg/kg peel). These concentration values were statistically different (p < 0.05) (Table 2). Triterpene Composition. Triterpenes are one of the largest classes of natural plant products, which, jointly with phenolic compounds, are the two major groups of secondary plant metabolites in apples.34 Triterpenes are mainly found in such plant surfaces as the bark of the stem, leaves, and fruit waxes, where they protect the plant from biotic and abiotic stress factors. Therefore, the cuticular wax of apple peel is an important dietary source of these natural compounds. For this reason, in the present work the triterpenes have been quantified in the peel (Table 2) rather than in the flesh (Table 1) samples, which presented very low concentration. No significant differences in triterpene concentrations were observed between the red- and white-fleshed apple varieties. The total triterpene content in the apple peel for all of the apple varieties was from 247 ± 26.6 to 590 ± 73.7 mg/kg peel sample (Table 2), ‘107/ 06’, ‘119/06’, and ‘Story’ being the richest varieties. Ursolic acid was the most abundant triterpene, similar to what was observed I


a

J

nd b 2.61 7565 2.52 1.61 0.22 9.31 76.4 7657 35.1

lactic acid succinic acid malic acid α-ketoglutaric acid tartaric acid shikimic acid quinic acid citric acid total organic acids ascorbic acid

± 0.94bcd ± 677a ± 0.53ab ± 0.54a ± 0.01fg ± 1.71b ± 18.5bc ± 680a ± 21.0c

± 0.10ab ± 372ab ± 0.12 ± 0.01 ± 0.01bcd ± 3.20b ± 13.7abc ± 385ab ± 1.04c 0.23 1.12 350 0.64 13.1 0.33 47.7 133 3688 92.2

nd b 0.92 6337 0.31 nd 0.22 145 198 6680 11.1 nd b 7.52 4168 4.34 0.93 0.22 1114 68.8 4261 41.1

± 0.01cd ± 30.4a ± 43.6ab ± 801ab ± 3.01c ± 0.21ab ± 0.54de ± 666bcd ± 0.23cd ± 0.42ab ± 0.14ef ± 17.7a ± 49.6ab ± 716bcde ± 9.60d ± 3.80a ± 582abcd ± 0.54a ± 0.14abc ± 0.01g ± 3.55b ± 12.6bc ± 601abcd ± 12.0c

± 1.81a ± 708b ± 0.30 ± 0.01 ± 0.04de ± 4.90bc ± 15.8cd ± 751b ± 4.11c

119/06 nd b 4.22 5933 1.11 0.11 0.24 34.6 89.3 6063 12.9

± 0.14bc ± 725ab ± 0.01

117/06

0.63 6.22 5553 1.40 nd d 0.42 8.92 240 5810 11.3

nd b 4.04 9117 0.50 nd 0.23 12.5 235 9370 20.1

Flesh

± 0.09de ± 1.43b ± 32.8a ± 532ab ± 5.02b

± 0.01de ± 1.90bcd ± 46.7a ± 427a ± 4.51d Peel ± 0.13a ± 0.70ab ± 629ab ± 0.08bc

± 0.92a ± 634a ± 0.08

RS-1

0.31 0.81 725 0.53 0.55 1.32 31.0 114 874 nq a

nd b 0.92 1834 0.30 nd 1.20 141 108 2086 nq a ± 0.03a ± 0.09e ± 144e ± 0.14d ± 0.14c ± 0.31a ± 9.61a ± 17.5abc ± 360f

± 0.21a ± 11.6a ± 10.8bcd ± 369d

± 0.12bc ± 367d ± 0.01

Brookfield Gala

0.54 3.22 2620 0.82 0.75 0.74 6.20 184 2814 10.3

nd b 1.80 2808 0.30 0.01 0.40 25.8 257 3093 12.0 ± 0.11a ± 0.82abc ± 352cd ± 0.14cd ± 0.24bc ± 0.23bc ± 1.70bc ± 73ab ± 457cde ± 4.52b

± 0.44ab ± 248c ± 0.11 ± 0.01 ± 0.13b ± 6.20bc ± 64.5a ± 291c ± 1.44c

Golden Smoothee

0.24 1.22 2583 0.74 1.74 1.40 6.44 83.7 2678 7.91

nd b 0.82 3140 0.22 0.14 0.74 5.20 69.1 3216 4.11 ± 0.03ab ± 0.34de ± 524cd ± 0.13cd ± 0.33a ± 0.13ab ± 1.71bc ± 12.8abc ± 538de ± 3.81b

± 0.33bc ± 301c ± 0.09 ± 0.01 ± 0.13a ± 1.22de ± 8.72d ± 306c ± 1.21b

Zhen Aztec Fuji

0.31 1.31 4942 0.44 1.52 0.31 9.60 60.3 5015 97.1

nd b 1.61 6318 0.31 nd 0.12 10.8 107 6438 14.9 ± 0.08a ± 0.11cde ± 875abc ± 0.13d ± 0.12a ± 0.01def ± 1.40b ± 6.30bc ± 881abc ± 16.1d

± 0.01e ± 3.40cd ± 6.22bcd ± 237ab ± 2.11c

± 0.33abc ± 233ab ± 0.11

Granny Smith

0.21 1.02 2124 0.90 nd d 0.54 2.80 41.5 2171 68.7

0.41 0.74 3330 0.31 nd 0.30 2.62 73.1 3407 16.1

0.03a 0.12c 250c 0.05

0.02ab 0.06de 463d 0.12cd ± 0.02cd ± 0.44c ± 4.00c ± 463e ± 10.4cd

± ± ± ±

± 0.01bc ± 0.05e ± 11.3d ± 662c ± 2.70cd

± ± ± ±

Story

For each row, values not displaying the same letter are significantly different (one-way ANOVA, Tukey’s test between all means, p < 0.05). nd, not detected; nq, not quantified (trace levels).

nd b 1.91 8557 0.92 0.14 0.22 36.1 156 8752 13.0

107/06

lactic acid succinic acid malic acid α-ketoglutaric acid tartaric acid shikimic acid quinic acid citric acid total organic acids ascorbic acid

compound

Table 3. Concentrations of Organic Acids and Ascorbic Acid in Apple Flesh (mg/kg Flesh) and Peel (mg/kg Peel) in Different Varieties (Mean Value ± Standard Deviation) (n = 9)a



Article

Journal of Agricultural and Food Chemistry in previous studies.34−36 Other minor triterpenes were tentatively identified in the present work, and these were hydroxyursolic acid, euscaphic acid, maslinic acid, and betulinic acid. These triterpene compounds and their concentration patterns were also reported by He et al.36 for the analysis of gold-red apples (Table 5 in the Supporting Information). Organic Acids and Ascorbic Acid. The acidity of apples, related to their organic acid content, influences the perception of both sourness and sweetness and, thus, consumer acceptance.54 Table 3 shows the concentrations of organic acids detected in the flesh and peel of each of the nine apple varieties studied, with malic acid being the main organic acid quantified in all apple samples. In the flesh samples, the concentration of malic acid in the red-fleshed apple cultivars was higher than in the white-fleshed ones, and these values were statistically different (p < 0.05) except for ‘Granny Smith’, which was statistically similar to the red-fleshed cultivars. The organic acids were analyzed for the first time in red-fleshed apples. The higher content of organic acids, mainly malic acid, in flesh from red-fleshed apples could reduce consumer acceptance, although their acidity level is similar to that of the well-known ‘Granny Smith’ variety. With regard to the organic acid content of the peel samples, the concentration varied according to apple variety, but no significant differences were observed between the red- and white-fleshed apple varieties. Apart from malic acid, other minor organic acids were also identified, but their concentrations were not statistically different. These organic acids were citric acid and quinic acid, among others, and this pattern is in accordance with the studies reported in the literature.55,56 With regard to ascorbic acid, its concentration was generally higher in the peel than in the flesh (Table 3), and no significant differences were observed between red- and white-fleshed varieties. In fact, the red-fleshed varieties included in this study were not submitted by the plant breeder to a specific program to increase the ascorbic acid content. An exception was ‘Brookfield Gala’, for which ascorbic acid was detected at trace levels (nq) in both the flesh and peel (Table 3). Another interesting point is that dehydroascorbic acid (DHA), the oxidative product of ascorbic acid (biologically active as vitamin C), was not detected in any of the apple samples analyzed. In conclusion, this study presents an exhaustive chemical characterization and quantification of the phenolic compounds, triterpenes, and organic and ascorbic acids in red-fleshed apple varieties obtained by crossbreeding programs and, therefore, without the aid of genetic engineering techniques. In addition, the results were compared with traditional and new whitefleshed varieties. Results showed that the most abundant phytochemicals in red-fleshed apples are organic acids, mainly malic acid, but their acidity level is similar to that of the marketed ‘Granny Smith’. Apart from organic acids, red-fleshed apples are rich in phenolic acids, dihydrochlacones, and anthocyanins, whereas the white-fleshed apples are richer in phenolic acids and flavan-3-ols. From all our data obtained, in red-fleshed apples, we detected mainly an up-regulation of anthocyanin, dihydrochalcones, and malic acid and a downregulation in flavan-3-ols (proanthocyanidin precursors), in both the flesh and peel, in comparison to traditional and new white-fleshed apples. No significant differences in the triterpene concentration and ascorbic acid content were observed between the red- and white-fleshed apple varieties. The reported results represent a good starting point to define the phytochemical profiling of the red-fleshed apple cultivars, which

should be extended to consecutive harvest seasons. For this reason we consider that even though the season factor could be important in peel color development, it has less importance when comparing red-fleshed versus white-fleshed cultivars, so we assume that flesh color has a stronger effect on phenolic compounds content than the season factor.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b02931. Additional tables (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(M.J.M.) E-mail: [email protected]. Phone: +34 973 702817. Fax: +34 973 702596. ORCID

Maria José Motilva: 0000-0003-3823-3953 Funding

This work was supported by the Spanish Ministry of Education and Science financing the project AGL2016-76943-C2-1-R and by the Spanish Ministry of Education, Culture and Sport through the “Formación Profesorado Universitario” grant awarded to D.B.-C. (FPU014/02902). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to IRTA (Research & Technology Food & Agriculture) for providing the apples and for a revision from the agronomic point of view. We thank the companies providing plant material of the red-fleshed varieties tested: Grubber Genetti & Lubera (Italy and Switzerland) and Escande & Red Moon Companie (France and Italy, respectively). We also thank Xavier Garanto from IRTA for his collaboration in fruit harvest and sample preparation.

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REFERENCES

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