Seasonal Variability of Phytochemical Composition of New Red

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Bioactive Constituents, Metabolites, and Functions

Seasonal Variability of Phytochemical Composition of New Red-Fleshed Apple Varieties Compared with Traditional and New White-Fleshed Varieties. David Bars-Cortina, Alba Macià, Ignasi Iglesias, Xavier Garanto, Llorenç Badiella, and Maria José Motilva J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03950 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 4, 2018

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Journal of Agricultural and Food Chemistry

Seasonal Variability of Phytochemical Composition of New Red-Fleshed Apple Varieties Compared with Traditional and New White-Fleshed Varieties

David Bars-Cortinaa, Alba Maciàa, Ignasi Iglesiasb, Xavier Garantob, Llorenç Badiellac, Maria-Jose Motilvaa,d*

a

Food Technology Department, XaRTA-TPV, Agrotecnio Center, Escola Tècnica

Superior d’Enginyeria Agrària, Universitat de Lleida, Avinguda Alcalde Rovira Roure 191, 25198 Lleida, Catalonia, Spain. b

Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Fruitcentre, PCiTAL, Parc

de Gardeny, 25003 Lleida, Catalonia, Spain. c

Statistical Consulting Service, Universitat Autònoma de Barcelona, 08193

Bellaterra, Catalonia, Spain. d

Current adress: Instituto de Ciencias de la Vid y del Vino (CSIC-Universidad de la

Rioja-Gobierno de La Rioja), Logroño, La Rioja, Spain

*Corresponding author: Tel.: +34 941 894 980 (ext. 410103); Fax: +34 941 899 728; E-mail: [email protected]

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Abstract

2

The main objective of this study was to evaluate the impact of the

3

season on the apple phytochemical composition (phenolic compounds,

4

triterpenes, and organic and ascorbic acids). For this proposal four red-

5

fleshed and five white-fleshed apple varieties from two consecutive seasons

6

(2015 and 2016) were studied. A significant interaction with the season in

7

some compounds was observed. The total phenolic content in the apple flesh

8

from 2015 was higher than 2016 probably related with the lower rainfall during

9

the harvest period in 2015 that could have favored hydric stress in the apple

10

trees. The impact of the season on the apple skin was different. The 2016

11

season was characterized by higher maximum and minimum temperatures

12

resulting in higher content of flavonols, triterpenes and organic acids.

13

Anthocyanin concentration in both flesh and skin of the red-fleshed apples

14

showed no clear relation to the season and each variety showed an individual

15

pattern.

16 17

Keywords: anthocyanin, cyanidin galactoside, phenolic compounds, red-

18

fleshed apples, season, UPLC-MS/MS

19


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INTRODUCTION

21

Consumption of fruit is largely considered to be good for one’s health.

22

Remembering the Welsh proverb from the 1860s (“An apple a day keeps the

23

doctor away”) demonstrates that the importance of fruit for health has been

24

accepted by both healthcare professionals and the general public.1

25

Nevertheless, the old adage refers specifically to apples. These fruit (with a

26

projected world production of 76.2 million tons for 2017/2018 as stated in the

27

latest report on Fresh Deciduous Fruit published by the USDA in December

28

2017) are consumed worldwide as they are available year-round in markets,

29

have a low cost and a good “health image”.2–5 In addition, since the discovery

30

by the Russian botanist (Niedzwetzky) of the wild red-fleshed apple (Malus

31

pumila ‘Niedzwetzkyana’) in the hotspot for apple origin (the current Xinjiang

32

Uygur region of China and the Republics of Kazakhstan, Uzbekistan,

33

Turkmenistan, Kyrgyzstan and Tajikistan), and especially from the works of

34

the two breeders (Niels Hansen and Albert Etter) who developed the two main

35

red-fleshed apple varieties,6 interest in developing a new commercial red-

36

fleshed apple varieties has grown in recent years. The first commercial

37

cultivars have been evaluated in the main research centers worldwide and

38

some of them planted in commercial orchards in the last decade with the aim

39

of being branded and filling a potential market in the coming years.7 The poor

40

taste of the ancient red-fleshed varieties has been improved through

41

crossbreeding programs with good-flavored white-fleshed apples to get

42

marketable red-fleshed apples .

43

plant breeders to devote so much effort to obtaining commercial red-fleshed

44

apple varieties?.

6

However, what are the main reasons for


3


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It is largely considered that people usually choice red-peeled apples as

46

they are perceived to be associated with better taste and flavor, and the high

47

marketability of this fruit is thus important for growers

48

not easy to grow in areas with warm and hot climates. Even so, important

49

progress in terms of the capability of developing high skin color under those

50

climatic conditions has been done by either selecting high-color strains of the

51

original cultivars (‘Gala’, ‘Delicious’, ‘Fuji’, etc.) or breeding for high color and

52

eating quality.12–16 In addition, apart from consumers preference for red skin

53

and although the concentration of phenolic compounds is much greater in the

54

skin of apples than in the flesh,3 many people discard the peel before eating

55

the apple mainly for cultural reasons.17,18

56

8–11

although these are

Furthermore, there is a heightened public interest in potential crops for 19

57

coloring food naturally

58

consumers’ attitudes towards

59

negative.20 Hence, strongly colored fruit and vegetables are attractive for both

60

fresh fruit and juice processing companies to increase and expand their range

61

of products. Apart from any commercial interest, enhanced levels of

62

anthocyanins in the apple flesh (apart from the peel) could be an interesting

63

approach for increasing consumer acceptance and obtain their anthocyanin

64

health profit

21–23

without transgenic or cysgenic programs due to genetically modified foods are mainly

through of the ingestion of this popular fruit.

65

Nevertheless, for decades, apple breeding has mainly focused on

66

improving fruit quality, appearance and disease resistances and has largely

67

ignored the health properties. However, recent breeding targets include both

68

the selection of high-color varieties even in warm climates13 and the

69

development of new red-fleshed cultivars because of the close relationship


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between many phenolic compounds (including anthocyanins) and human

71

health benefits. In particular, when selecting high red-fleshed apples,

72

breeders will want to know if any other concentration of phenolics is also

73

being altered. In a previous study24 different analytical methodologies were

74

developed and optimized to study the whole profile of phenolic compounds,

75

triterpenes, organic acids, and ascorbic acid of the new red-fleshed apple

76

cultivars, released from different breeding programs, compared with some

77

traditional and new white-fleshed apple cultivars. Results showed an up-

78

regulation of anthocyanins, dihydrochalcones, and malic acid and a

79

downregulation of flavan-3-ols (proanthocyanidin precursors) in red-fleshed

80

apples comparing with white-fleshed apples.

81

The main objective of this work is to extend our previous study

24

82

assessing the impact of the season on the phytochemical composition

83

(phenolic compounds, triterpenes, and organic and ascorbic acids) of red-

84

fleshed apples intended for commercial purposes compared to new and

85

traditional white-fleshed apples studied over two consecutive seasons (2015

86

and 2016). Our results try to contribute to the recent development of

87

functional genetic markers for both high skin14 and flesh coloration and their

88

use in marker-assisted selection (MAS), essential to increasing breeding

89

efficiency and any new range of apple varieties that may appear.

90 91

MATERIALS AND METHODS

92

Plant material. Four different type-1 red-fleshed apple varieties: the ‘RS-

93

1’ (‘Red Sun’) from Red Moon Companie, Italy, and the ‘107/06’, ‘117/06’ and

94

‘119/06’ from Lubera AG, Switzerland, both protected and commercialized in


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the EU under the brand name Redlove Era, were selected. Five white-fleshed

96

apple varieties, including the new (‘Brookfield Gala’, ‘Zhen Aztec Fuji’, and

97

‘Story’ (Story (Inoredcov)) and the traditional (‘Golden Smoothee’ and ‘Granny

98

Smith’) varieties, were selected (Figure 1S in Supplementary Material). The

99

apples were harvested from 6-year-old trees grafted onto M9 EMLA rootstock

100

and planted in an experimental plot at the IRTA - Lleida Experimental Station

101

(Mollerussa, Catalonia, Spain) (41:37:7.719N 0:52:7.402E), characterized by

102

a dry and warm summer (see Supplementary Material Figure 2S for more

103

meteorological data). The trees were trained with a central leader system

104

spaced at 4 m x 1.2 m. Fruit size and crop load were very uniform among

105

trees and cultivars. Apples from each variety were harvested from different

106

trees in the autumns of 2015 and 2016 at the same maturation stage, taking

107

into account both their flesh firmness (range 7.0-8.0 kg) and starch index

108

(range 6.5-7.5 on EUROFRU code 1-10),12 and placed in cold storage at 0.5

109

ºC.

110

As soon as the apples arrived in the laboratory, they were washed,

111

cored and peeled. Then, flesh (cut transversely into slices across the equator)

112

and skin from individual apples were separately frozen in liquid nitrogen and

113

subsequently freeze-dried

114

Catalonia, Spain). Each freeze-dried apple sample (flesh and skin) was kept

115

in individual sealed plastic bags at – 80 ºC until the chromatographic analysis.

116

Prior to analysis, a fine powder of the freeze-dried apple samples was

117

obtained with the aid on an analytical mill (A11, IKA, Germany). For each

118

variety and fruit part (flesh and skin), an n=9 with three replicates was

119

analyzed.

(Lyophilizer

Telstar


Lyobeta

15,

Terrassa,

6

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120 121

Chemicals and Reagents. All chemicals and reagents used were detailed in our previous study.24

122

Analytical methods.

123

Sample pretreatment. Solid-liquid extraction process was performed

124

according to Bars-Cortina et al. method. 24

125

Ultraperformance Liquid Chromatography Coupled to Tandem Mass

126

Spectrometry (UPLC-MS/MS). Chromatographic method details, mass

127

spectrometer conditions for identification, and the quantification of each of the

128

compounds studied are described in our previous study.24 Due to the lack of

129

standards of some phenolic compounds they were tentatively quantified using

130

calibration curves of similar phenolic structures.

131 132

Statistical analysis. Statistical treatment was realized with R 3.4.1 software version.25

133

Phytochemical concentration values were indicated as means (n=9) ±

134

standard deviation (SD). On the comparative study of the two harvest

135

seasons, the significance of differences among means was determined by the

136

two-way ANOVA, in order to examine variety * season interaction, followed by

137

Tukey’s test. Response variables were log-transformed if necessary to obtain

138

normally distributed data. If the assumption of homogeneity of variances was

139

violated, ANOVA Welch followed by Games-Howell’s post-hoc tests were

140

used. In all the ANOVA studies, a 5% level of significance was established.

141

The R packages used were the following: xlsx, lsmeans, dplyr, car,

142

userfriendlyscience, multcompView, nlme and agricolae.

143

In order to offer a more pleasant and enjoyable output to interpret the

144

data, a principal component analysis (PCA) and ascending hierarchical


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145

classification of the individuals was included in the both seasons in order to

146

evaluate whether the red-fleshed apple phytochemical components were

147

distributed differently from their white-fleshed counterparts. To perform this

148

exploratory

149

FactoInvestigate R packages were used.

data

analysis,

the

FactoMineR,

factoextra,

corrplot

and

150 151

RESULTS AND DISCUSSION

152

Effect of the harvest season on the apple phenolic profile.

153

Anthocyanins. The four red-fleshed apple varieties studied were Hansen’s

154

type (also known as type-1 red-fleshed apples). Those phenotypes show a

155

red coloration throughout the fruit and also in the plant tissues including the

156

stems, roots, flowers, and developing leaves (Figure 3S in Supplementary

157

Material)6,26,27, in contrast to type-2 red-fleshed apples, which only present

158

the red pigment in the fruit cortex and new green leaves The main

159

anthocyanin identified and quantified in the red-fleshed apple varieties was

160

cyanidin galactoside (Tables 1 and 2) similarly to that indicated in

161

Additionally, other anthocyanins were detected in flesh and skin of the red-

162

fleshed apples (see Table 2S Supplementary Material). Comparing the

163

anthocyanin concentrations in the flesh between seasons, changes occurred

164

in three red-fleshed varieties harvested in September (‘117/06’, ‘119/06’ and

165

‘RS-1’) (Table 1), while no changes were detected between seasons in the

166

early harvested variety (‘107/06’, harvested on August 22nd). For a better

167

observation of the differences between the two seasons, Figure 1 shows the

168

total concentrations of the main phenolic groups (including anthocyanins)

169

quantified in the apple flesh. With regard to the apple skin, an analogous


28,29

.

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170

pattern was detected but adding a significant decay (p < 0.05) in two red-

171

skinned varieties (‘Story’ and ‘107/06’) (Table 1 and Figure 2).

172

One of the reasons for these anthocyanin changes could be the variation

173

between seasons in terms of average temperatures. This variation occurred,

174

as can be clearly observed in Figure 2S Supplementary Material. Both daily

175

maximum and minimum temperatures before harvest were higher in 2016

176

than in 2015. In the literature, it is well described that temperature has a major

177

effect on anthocyanin skin synthesis (compromising anthocyanin biosynthetic

178

pathway genes expression) and therefore leading to apple skin with less red

179

color and therefore lower anthocyanin concentration.12–16 In addition, a recent

180

publication29 has gone one step further and demonstrated this temperature

181

mediated anthocyanin decay in the apple flesh through an approximation in

182

the laboratory (using red-fleshed apples harvested at 110-115 days after

183

blooming (DAB) and treated with different light/oxygen regimens during one

184

week). Nevertheless, although the literature indicated above shows a direct

185

relationship between higher temperatures and anthocyanin decay, based on

186

our findings, we hypothesize that this decay is dependent on the apple variety

187

and that phenomenon occurs first at the skin level12. The early harvested red-

188

fleshed variety (‘107/06’, harvested on August 22nd 2016) only suffered

189

anthocyanin decay in the skin, maybe because the fruit were harvested during

190

a brief period with daily maximum mean temperatures greater than the

191

previous year (see Figure 2S Supplementary Material). The other red-

192

fleshed varieties (‘117/06’, harvested on September 20th; ‘RS-1’, harvested on

193

September 23rd; and ‘119/06’, harvested on September 30th) suggested this

194

hypothesis to us. Specifically, these three varieties suffered a large common


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195

heat peak (from September 2nd to 13th, see Figure 2S Supplementary

196

Material) but the ‘117/06’ and ‘RS-1’ varieties showed they were resistant to

197

anthocyanin decay in contrast to their ‘119/06’ counterpart, which was the

198

only cultivar that underwent significant anthocyanin decay in both the flesh

199

and the skin (p < 0.05).

200

Other Phenolic Compounds. A wide spectrum of phenolic compounds

201

was identified and quantified in the flesh and the skin of red- (Table 1) and

202

white-fleshed (Table 2) apples. In both seasons studied (2015 and 2016), the

203

phenolic acids were the major representative group of the phenolic

204

compounds in the flesh of the red- and white-fleshed apples (50-60% of total

205

phenols). Curiously, only in the ‘Granny Smith’ and in ‘Story’ (Table 2)

206

varieties the total flavan-3-ols concentration was higher than the phenolic acid

207

concentration and this behavior was maintained within seasons. Chlorogenic

208

acid has been the most abundant phenolic acid detected in all the nine apple

209

varieties in the present study, which is in accordance with the literature.5,31–34

210

The concentrations of this phenolic acid in the flesh and skin were similar in

211

red- and white-fleshed apples (Tables 1 and 2). In detail, in the fleshy part,

212

the ‘107/06’, ‘119/06’ and ‘RS-1’ presented the highest chlorogenic acid

213

concentration jointly with the ‘Golden Smoothee’ and ‘Zhen Aztec Fuji’ (Table

214

3S Supplementary Material). Meanwhile, the ‘119/06’ in conjunction with the

215

‘Brookfield Gala’, ‘Granny Smith’ and ‘Story’ presented significantly lower

216

concentrations (p < 0.05). On the other hand, in the skin, the chlorogenic acid

217

concentration was more uniform in the two types of apples and only the

218

‘Granny Smith’ and ‘Story’ presented lower concentrations (Table 3S

219

Supplementary Material). Furthermore, as was also observed in


24

, the

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220

concentrations of other less common phenolic acids were higher and more

221

statistically significant (p < 0.05) in the red-fleshed than in white-fleshed

222

apples. These phenolic acids were vanillic acid hexoside, protocatechuic acid

223

and vanillic acid in the flesh, and hydroxytyrosol and protocatechuic acid in

224

the skin (Table 3S Supplementary Material). Finally, comparing the phenolic

225

acid concentration between the two seasons, this was lower (p < 0.05) in all

226

the apple varieties from 2016, mainly the chlorogenic acid concentration in the

227

flesh and skin (Tables 1 and 2) with the exception of the ‘Granny Smith’ skin,

228

which showed a very low concentration of chlorogenic acid (Table 2).

229

Moving on to the flavan-3-ols, their abundance was higher (p < 0.05) in

230

2016 in the flesh and skin of white-fleshed apple varieties in comparison with

231

their red counterparts (Table 3S Supplementary Material), similarly to other

232

publications.24,32–34 In detail, the total flavan-3-ol contents of the flesh of red-

233

fleshed apple varieties were within a narrow range between 4.2±0.5 and 9.1±1

234

mg/kg (Table 1) and from a wide range in the white-fleshed ones, between

235

32.7±5.4 and 161.3±33 mg/kg flesh (Table 2). The tendency in the flavan-3-

236

ols concentration in apple skin was analogous to that in the flesh but with

237

higher concentrations. The low flavan-3-ol concentration obtained in red-

238

fleshed apples commercialized in the European Union and harvested over two

239

consecutive seasons (2015 and 2016) differs from the limited published

240

studies focused on the study of the phenolic profile of new red-fleshed

241

cultivars, including crab-apples (non-commercial size and low eating quality)

242

nearer to the native Malus niedzewetzkyana.2,31 Similarly, a previous study by

243

Espley et al.,35 developed a red-fleshed ‘Royal Gala’ MYB10 transgenic apple

244

line. Although this new ‘Royal Gala’ apple variety presented a high expression


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245

of anthocyanins, the flavan-3-ol content did not decrease and also the eating

246

quality was close to the standard ‘Royal Gala’. The lower flavan-3-ol

247

abundance observed (in the flesh and skin) in the red-fleshed apples varieties

248

(Tables 1, 2 and Table 3S Supplementary Material) included in the present

249

study could be related to the unspecific crossbreeding techniques used to

250

obtain these apple varieties.

251

Although the next discussion it’s out of our scope, we considered useful

252

to mention briefly because we hypothesize that could be a reasonable further

253

research in order to try to respond for the low values of flavan-3-ols in these

254

red-fleshed apples for commercial purposes, in both seasons. Therefore, no

255

speculative attitude was done, only our humble opinion was described. The

256

phenolic biosynthesis pathways are well known and several studies have

257

highlighted the major enzymes involved.36,37 However the mechanisms

258

involved

259

unclear.37,38 The well-known biosynthetic pathway for flavonoids (Figure 4S

260

Supplementary Material) shares common enzymatic steps, whereas the

261

activity of specific enzymes for proanthocyanidin, anthocyanin or flavonol

262

synthesis leads exclusively to the biosynthesis of the respective molecular

263

structure by competing for common substrates.37,39,40 In persimmons,

264

strawberries and grapevine, transcription factors have been detected which

265

regulate proanthocyanidin accumulation, and these findings suggest that

266

proanthocyanidin accumulation in apples is probably regulated at the

267

transcriptional level, although no transcriptional factors have been identified

268

that are involved in regulation of proanthocyanidin biosynthesis in apples until

269

recently.38,41 In tobacco,38 it is claimed that the apple MdLAR1 gene

in

the

proanthocyanidin

(flavan-3-ols)


biosynthesis

remain

12

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270

suppresses the expression of the anthocyanin pathway genes in flowers

271

including CHI, F3’H, DFR, ANS and UFGT, leading to a significant loss of

272

anthocyanin. This fact could suggest that pathway flux tends to be shifted

273

away

274

transcription of LAR, ANR and ANS might be regulated by a feedback

275

mechanism38. Therefore, in the case of the overexpression of the enzymes

276

involved in proanthocyanidin synthesis, anthocyanin synthesis is down-

277

regulated. Another plausible explanation is the Henry-Kirk proposal,42 which

278

suggests the presence of degradation processes for proanthocyanidins during

279

the ripening of the apples. The proanthocyanidin biosynthesis has been

280

studied recently in two commercial crab apples (Royalty with red leaves due

281

to the anthocyanins and Flame with green leaves) and it has been seen a

282

gradual decrease in the anthocyanin content in Royalty leaves during their

283

development

284

concentrations41. In addition, up-regulation in the expression of the

285

McMYB12b significantly enhanced the accumulation of proanthocyanidins,

286

and inhibited the biosynthesis of anthocyanins, indicating a metabolic balance

287

between

288

Therefore, these mechanisms mentioned above could explain the lower levels

289

of flavan-3-ols obtained in the red-fleshed cultivars analysed, although we

290

must be prudent because these results refer to the leaves instead of the fruit

291

and to date, no study of the metabolic pathways involved in the

292

proanthocyanidin and anthocyanin biosynthesis has been performed on red-

293

fleshed apple fruit.

from

the

in

anthocyanins

parallel

flavonols

with

towards

the

proanthocyanidins.

increase

(proanthocyanidins)

and

in

the

the

proanthocyanidin

anthocyanin


Then,

biosynthesis.

13


Page 14 of 41

294

The main flavonols encountered in all varieties studied, in both the

295

flesh and skin, were quercetin derivatives, as reported in the literature.28,31

296

The contents of quercetin derivatives were higher in flesh of the red-fleshed

297

fruit than in the white-fleshed ones (Table 3S Supplementary Material).

298

Regarding the differences between the two consecutive seasons (2015 and

299

2016), no differences were detected in the concentrations of quercetin

300

derivatives in the skin of the red-fleshed apples (Table 1 and Figure 2). By

301

contrast, the quercetin derivative concentrations in the white-fleshed apples

302

were significantly changed in 2016 (Table 2 and Figure 2). An analogous

303

pattern was observed in the flesh (Figure 1).

304

Dihydrochalcones are practically exclusive to apple fruit. Apart from

305

apples, few other plant species are known to contain phloretin-2’-O-glucoside

306

also known as phloridzin (the main dihydrochalcone), and these belong

307

mainly to the Rosaceae and Ericaceae families.43 Phloridzin jointly with

308

phloretin xylosyl-glucoside were the prevalent dihydrochalcones identified in

309

the red- (Table 1) and white-fleshed (Table 2) apples being their

310

concentrations higher in the skin than in the flesh for all the varieties and

311

seasons studied. In addition, in both parts of the fruit, the concentration of

312

dihydrochalcones in red-fleshed apples were higher (without taking the ‘RS-1’

313

into account) than in white-fleshed apples (Table 3S Supplementary

314

Material). Comparing the dihydrochalcone content in the flesh (Tables 1 and

315

2, and Figure 1) between the two seasons, a general decrease in

316

dihydrochalcone was detected in 2016 (‘RS-1’ having the strongest decay).

317

By contrast, the season effect was more pronounced in the skin which the


14

Page 15 of 41


318

dihydrochalcone concentration being higher in apples from 2015 with the

319

exception of the ‘Granny Smith’ variety (Tables 1 and 2, and Figure 2).

320

Regarding the minor phenolics, flavanones and flavones were the

321

phenolic compounds determined at scarce levels in all the apple varieties

322

studied (Table 3S Supplementary Material) and in both seasons (Tables 1

323

and 2). As observed in previous studies, apples are not a good source of

324

these phenolic groups.32 Considering the total phenol content as the sum of

325

all the quantified phenolic compounds, in general the flesh from the 2015

326

season showed higher concentrations (p

Seasonal Variability of Phytochemical Composition of New Red

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