Phytochemical Composition of Red-Fleshed Apple Cultivar ‘Baya Marisa’ Compared to Traditional, White-Fleshed Apple Cultivar ‘Golden Delicious’

Abstract

We analyzed the red-fleshed apple cultivar ‘Baya Marisa’ and compared it with the traditional, white-fleshed apple cultivar ‘Golden Delicious’. The objective of this study was to compare sugars, organic acids, and phenolic compounds of ‘Baya Marisa’ with the widely known and consumed white-fleshed apple cultivar ‘Golden Delicious’. In addition, flesh firmness, color, and soluble solid content was studied. A total of 46 phenolic compounds were quantified and identified, some for the first time in apples. The study showed that the total analyzed phenolic content (TAPC) was 1.6× higher in the skin of red-fleshed ‘Baya Marisa’ and 1.4× higher in the flesh of red-fleshed ‘Baya Marisa’. Organic acid (citric acid, malic acid, and ascorbic acid) content was higher in the red-fleshed cultivar ‘Baya Marisa’, whereas sugar content (sucrose, glucose, and sorbitol) was similar, except for fructose, which was 1.1× higher in ‘Golden Delicious’. The content of citric acid was 1.6× higher in ‘Baya Marisa’, whereas the content of malic acid was 1.2× higher in ‘Baya Marisa’ and the content of ascorbic acid was 2.8× higher in ‘Baya Marisa’. Among phenolics, total dihydrochalcones in the skin were 3.3× higher in ‘Baya Marisa’ and similar in the flesh compared to ‘Golden Delicious’. Flavonols were 1.4× higher in the skin of ‘Baya Marisa’ and 2.8× higher in the flesh of ‘Golden Delicious’. Anthocyanins were not identified in the white-fleshed cultivar ‘Golden Delicious’. Total hydroxycinnamic acids were 2.0× higher in the skin of ‘Golden Delicious’ and similar in the flesh compared to ‘Baya Marisa’. Total flavanols were 1.8× higher in the skin and 2.2× higher in the flesh of the white-fleshed cultivar ‘Golden Delicious’.

1. Introduction

Apples (Malus domestica Borkh.) account for 12.5% of the fruit consumed worldwide [1]. Due to their year-round availability, easy consumption, and low prices, they are consumed all over the world [2]. Apples are known for their health benefits [3]. Usually, red skinned apples are purchased because they appear tastier in the eyes of consumers and also have a higher content of phenolic compounds in the skin [4]. Therefore, in changed climatic conditions, growers are looking for new technological measures which affect better coloring of the fruit [5].

Five important groups of phenolic compounds are found in apples: dihydrochalcones, flavonols, flavanols, anthocyanins, and hydroxycinnamic acids. The content of these phenolic compounds varies among apple cultivars [6]. In apples, flavanols account for 71% to 90% of the total polyphenolic compounds. Hydroxycinnamic acids account for 4% to 18%, flavonols from 1% to 11%, dihydrochalcones from 2–6%, and anthocyanins 1–3% of the total polyphenolic compounds [7].

The composition and number of anthocyanins are important for the red skin blush of apples [8]. In the red-skinned apples, anthocyanins are restricted to a few hypodermal and epidermal cell layers. The mesocarp usually does not contain anthocyanins and is not pigmented [9]. However, some apple cultivars have transcription factors, which are responsible for anthocyanin biosynthesis in the flesh. During fruit maturation, a strong correlation exists between the expression of apple anthocyanin levels and the MdMYB10 gene, which is overexpressed in red-fleshed apples [10]. Espley et al. [10] reported that white-fleshed apple cultivars have different promoters of MYB10, which is an allele of the MdMYB10 and MYBA genes, which mainly determine skin and flesh color of fruits. Apples with red flesh are attractive to consumers because of the red color and higher content of anthocyanins, which have a positive effect on health [9]. Although there are different consumer demands in quality from the apple fruit chain [11], studies show that almost all red-fleshed apples originate from wild Malus domestica var. niedzwetzkyana (Dieck Langenf), which originated in Central Asia [9].

Phenolic compounds, organic acids, and sugars contribute to the flavor, color, aroma, and metabolic activity of apples and are influenced by climate, storage, and growing season [12].

Previous studies have shown a higher content of anthocyanins, dihydrochalcones, malic acid and lower content of flavanols (proanthocyanidin precursors) in red-fleshed apples compared to white-fleshed apples [13]. Originally, red-fleshed apple cultivars had a poor flavour, which was improved with crossbreeding programs with white-fleshed apples to obtain new, marketable, red-fleshed apples [14].

Anthocyanins are flavonoids found in vegetables, fruits, and flowers that give them a red-to-blue color, hence the name red-fleshed apples. More than 635 anthocyanins have been identified in nature, involving six common aglycones and several types of acylations and glycosylations. The most important aglycones are cyanidin and peonidin, which are responsible for the red color of the skin and flesh of apples. Cyanidin-3-galactoside is the most important anthocyanin for red coloration in apple skin and flesh [10]. Many studies have shown that a higher anthocyanin content in fruits has greater health benefits when consumed [15].

The aim of our study was to, in detail, compare the content of phenolic compounds, sugars, and organic acids in the white-fleshed apple cultivar ‘Golden Delicious’ and the red-fleshed apple cultivar ‘Baya Marisa’. The focus of our study were individual phenolic compounds and phenolic groups in the skin and flesh of the two cultivars. This study is one of the most detailed and prospective studies conducted, although analyses have already been conducted on other red-fleshed apple cultivars. The results are important for nutritionist, breeders, researchers, and scientists. This study is widely applicable, not only for studies on fresh apple fruits, but also for processing.

2. Materials and Methods

2.1. Plant Material

For the study, the red-fleshed apple cultivar ‘Baya Marisa’ and the white-fleshed apple cultivar ‘Golden Delicious’ were collected from an intensive apple orchard in the southeastern part of Slovenia, Zdole (45°98′02″ N; 15°52′07″ E; 307 m.a.s.l.). Planted trees were 4 years old at the time of sampling. Cultivars were grafted on M9 rootstocks and spaced 3.2 m × 1.0 m apart. The orientation of the rows was north–south. The orchard was managed according to standard integrated pest management. Both ‘Baya Marisa’ and ‘Golden Delicious’ were harvested at the time of their technological maturity. ‘Baya Marisa’ was harvested on 23 September 2021, ‘Golden Delicious’ was harvested on 15 September 2021. The extraction was made on 28 September 2021. Fruits were in cold storage at 3 °C.

For analysis, 25 apples of each cultivar were collected and transported to the laboratory of the Department of Agronomy, Biotechnical Faculty, University of Ljubljana (Slovenia) for the analysis of phenolic compounds, sugars, organic acids, flesh firmness, fruit color, and soluble solid content.

2.2. Flesh Firmness, Soluble Solid Content, and Fruit Color

Fruits of the two apple cultivars were measured (fruit firmness, fruit color, and soluble solid content) on 13 October 2021. A digital penetrometer (T.R. Turoni, Forli, Italy) with an 11 mm piston, expressed in Newton [N], was used to measure fruit firmness. Soluble solid content was measured using a digital refractometer (Milwaukee Digital Brix Refractometer MA871, Rocky Mount, NC, USA). The colorimeter CR-300 Chroma (Minolta Co., Osaka, Japan) was used to measure fruit skin color parameters: L* (lightness), where values vary from 0 (black) to 100 (white); a* and b*, where positive a* values represent red and negative green, and positive values of b* represent yellow and negative values represent blue; C* values corresponds to colorfulness, where higher values represent a more intense color, as described by Medic et al. [16].

2.3. Extraction of Sugars, Organic Acids, and Phenolic Compounds

Extraction of sugars, organic acids, and phenolic compounds was as described by Medic et al. [16], Mikulic–Petkovsek et al. [17], and Medic et.al. [18] with minor modifications. Twenty-five apples of each cultivar were used as five biological replicates with five apple fruits each. For each replicate, five apples of each cultivar (‘Baya Marisa’ and ‘Golden Delicious’) were randomly selected, each apple was divided into five parts, and the cores were removed. The samples were then cut into small pieces with a knife and crushed with the flat part of the knife. The skin of the apples was separated from the flesh and sampled separately for the extraction of phenolic compounds. For the extraction, briefly, 2.5 g of skin was extracted with 5 mL of 80% methanol (MeOH; Sigma-Aldrich, Steiheim, Germany) and 3% formic acid in bi-distilled water. For the extraction of phenolic compounds from the flesh, 10 g of the sample was used and extracted in 10 mL of 80% methanol with 3% formic acid in bi-distilled water. The extraction ratio for the skin was 1:2 (w/v) and for the flesh 1:1 (w/v). The samples were then sonicated in iced water for 60 min (Sonis 4 ultrasonic bath; Iskra pio, Sentjernej, Slovenia). They were then centrifuged at 10,000× g for 10 min at 4 °C (5810 R; Eppendorf, Hamburg, Germany). Samples were filtered through 0.2 µm polyamide filters (Chromafil AO -20/25; Macherey-Nagel, Düren, Germany), filled into vials, and stored at −20 °C for further analysis.

For the extraction of the organic acids, ascorbic acid, and sugars, the skin was not separated from the flesh, as was the case with the extraction of the phenolic compounds. The extraction ratio for the sugars and organic acids was 1:5 (w/v). Briefly, 5 g of the sample was extracted with 25 mL of bi-distilled water. The samples were placed in tubes and shaken in a Unimax 1010 shaker (Heidolph Instruments, Schwabach, Germany). They were then centrifuged at 10,000× g for 10 min at 4 °C (5810 R; Eppendorf, Hamburg, Germany). Samples were then filtered through 0.2-µm polyamide filters (Chromafil Xtra MV -20/25; Macherey-Nagel, Düren, Germany) into vials and stored at −20 °C for further analysis.

For the extraction of ascorbic acid, a procedure similar to that described by Bizjak et al. [19] was used; the extraction ratio was 1:5 (w/v), briefly 1 g of the sample was extracted with 5 mL of 2% metaphosphoric acid with bi-distilled water.

2.4. HPLC Analysis of Organic Acids and Sugars

Analysis of the individual organic aids and sugars was performed using Vanquish, a UHPLC system from Thermo Scientific (San Jose, CA, USA). Separation of the individual sugars was performed using the Rezex RCM monosaccharide column from Phenomenex (Torrance, CA, USA) at 65 °C. Bi-distilled water was used as the mobile phase at a flow rate of 0.6 mL/min. The refractive index (RI) detector (Refractomax 520, Idex health and science KK 5-8-6, Nishiaoki Kawaguchi, Japan) was used to monitor the eluted carbohydrates. The method used was as previously described by Medic et al. [16]. The same UHPLC system was used for the analysis of organic acids, equipped with a UV detector at 210 nm and a Rezex ROA column from Phenomenex at 65 °C. The conditions were as described by Medic et al. [16]. The elution solvent used was 4 mM sulfuric acid in bi-distilled water at a flow rate of 0.6 mL/min. Ascorbic acid was analyzed as described by Mikulic-Petkovsek et al. [17]. The content of organic acids and sugars was expressed in mg/kg fresh weight.

2.5. HPLC-Mass Spectrometry Analysis for Phenolic Compounds

The phenolic compounds were analyzed using a UHPLC system (Thermo Scientific; San Jose, CA, USA). The diode array detector for anthocyanins was set at 530 nm, for flavonols at 350 nm, and for the other phenolic compounds at 280 nm. The conditions were as described by Medic et al. [18]. The injection volume was 20 µL. The recorded spectra ranged from 200 nm to 600 nm. A C18 column (Gemini; 150 × 4.60 mm, 3 u; Phenomenex, Torrance, CA, USA) operated at 25 °C was used to separate the compounds. Phenolic compounds were identified by tandem mass spectrometry (MS/MS; LCQ Deca XP MAX; Thermo Scientific, Waltham, MA, USA) with heated electrospray ionization operated in a positive-ion mode for the detection of anthocyanins and in a negative-ion mode for the remaining compounds. The conditions were similar to those described by Medic et al. [16]. For the analysis, complete MS scans were obtained from m/z 50 to 2000. Data acquisition was performed using Xcalibur 2.2 software (Thermo Fischer Scientific Institute, Waltham, MA, USA). External standards were used for quantification and identification of these compounds when available. For identification of unknown compounds, MS fragmentation and literature data were used, whereas quantification was performed using a similar standard.

Phenolic compounds are expressed as mg/kg. The sum of all identified phenolics is represented as total analyzed phenolic content (TAPC), expressed in mg/kg fresh weight.

2.6. Chemicals

The standards used for the analyses are as follows: chlorogenic acid, cryptochlorogenic acid, quercetin-3-O-rutinoside, peonidin-3-O-galactoside, shikimic acid (Sigma-Aldrich Chemie GmbH, Steinheim, Germany); p-coumaric acid, ferulic acid, procyanidin B1, cyanidin-3-O-galactoside, (-) epicatehin, caffeic acid, quercetin-3-O-glucoside, quercetin-3-O-galactoside, quercetin-3-O-rhamnoside, phloridzin, citric acid, malic acid, fumaric acid, fructose, glucose, sorbitol, and sucrose (Fluka Chemie GmbH, Buchs, Switzerland); cyanidin-3-O-arabinoside, quercetin-3-O-arabinofuranoside, quercetin-3-O-xyloside, and quercetin-3-O-arabinopyranoside (Apin Chemicals, Abingdon, UK).

Bi-distilled water purified with Milli-Q water purification system (Millipore, Bedford, MA, USA) was used for all sample preparations and analyses. The chemicals used for the mobile phases of the gradients for MS analysis (formic acid, acetonitrile) were from HPLC-MS (Fluka Chemie GmbH, Buchs, Switzerland). The methanol used for the extractions was HPLC grade (Sigma-Aldrich, Steinheim, Germany). Metaphosphoric acid used for ascorbic acid extraction was from Sigma-Aldrich (Steinheim, Germany).

2.7. Statistical Analysis

The collected data were gathered in Microsoft Excel 2016. A further statistical analysis was performed using the R commander program. Five repetitions of the two apple cultivars studied were performed. Data presented are means ± standard errors (SE). For further analysis of the data, t-test was used to determine the differences between the data. The confidence level was 95% to calculate the significance of the differences.

3. Results and Discussion

3.1. Flesh Firmness, Fruit Color, and Soluble Solid Content

The average flesh firmness of 25 fruits of red-fleshed ‘Baya Marisa’ was 57.0 N and had a soluble solid content of 14.9 °Bx. The white-fleshed cultivar ‘Golden Delicious’ had an average flesh firmness of 59.0 N, and an average soluble solid content of 17.3 °Bx in 25 fruits. The red overcolor of the red-fleshed ‘Baya Marisa’ was: L*, 26.5; a*, 24.7; b*, 11.3; C*, 27.1; and the ground color was: L*, 50.2; a*, 18.1; b*, 33.3; C*, 38.2. In the white-fleshed cultivar ‘Golden Delicious’, only the ground color was measured due to its yellow color, as shown in Figure 1: L*, 63.8; a*, 4.7; b*, 46.1; C*, 46.4. As reported by Contessa & Botta [20], red-fleshed cultivars had a soluble solid content of 14.4 °Bx and flesh firmness of 78.0 N, whereas white-fleshed ‘Golden Delicious’ had a soluble solid content of 13.9 °Bx and flesh firmness of 67.0 N. Flesh firmness, fruit color, and soluble solid content appear to be important to consumers, as reported by Espley et al. [21], who compared wild-type ‘Royal Gala’ with ‘Royal Gala’ transformed by a binary vector containing MYB10 cDNA, where the wild-type white-fleshed ‘Royal Gala’ had a similar flesh firmness (78.0 N) compared to the transformed red-fleshed ‘Royal Gala’ (79.0 N). As reported by Espley et al. [21], fruits with higher anthocyanin content ripen faster, resulting in perceived softness and reduced stiffness of cell walls. In our study, there was no significant difference in firmness between the white-fleshed cultivar ‘Golden Delicious’ and red-fleshed cultivar ‘Baya Marisa’, which indicates that the difference in flesh firmness is due to a different cultivar characteristic. As reported by Contessa & Botta [20], flesh firmness, soluble solid content (°Bx), and acidity are one of the most important criteria to determine fruit maturity. Soluble solid content is one of the most important factors for the taste of apples, whereas flesh firmness is important for choosing the right harvest time for apple cultivars. ‘Golden Delicious’ in our study had a higher soluble solid content, compared to red-fleshed ‘Baya Marisa’, whereas ‘Golden Delicious’ in Contessa and Botta’s [20] study had a slightly lower soluble solid content (13.9 °Bx), compared to the red-fleshed cultivar (14.4 °Bx).

3.2. Content of Organic Acids and Sugars

The sourness and sweetness of apple fruit influence consumer acceptance. For this reason, an analysis of these components was performed. We identified three different organic acids and four sugars.

Table 1. Individual organic acids and sugars (mean ± SE, in g/kg FW) in fruits of red-fleshed ‘Baya Marisa’ and white-fleshed ‘Golden Delicious’.

Compounds	‘Baya Marisa’	‘Golden Delicious’
Organic acids
Citric acid	0.898 ± 0.034 a	0.570 ± 0.047 b
Malic acid	9.685 ± 342.2 a	7.801 ± 0.153 b
Ascorbic acid	0.026 ± 0.005 a	0.009 ± 0.002 b
Sugars
Sucrose	43.674 ± 1.445 a	42.561 ± 0.593 a
Glucose	15.958 ± 1.445 a	17.035 ± 0.696 a
Fructose	51.234 ± 1.311 b	57.896 ± 0.847 a
Sorbitol	6.669 ± 0.518 a	5.376 ± 0.248 a

Same letters in a row following mean values are not significantly different.

shows the content of sugars and organic acids in the flesh and skin of red-fleshed ‘Baya Marisa’ and white-fleshed ‘Golden Delicious’.

Malic acid is the major organic acid detected in both apple cultivars, followed by citric acid, as shown in Table 1. The content of malic acid was higher in ‘Baya Marisa’ (9.685 g/kg FW) than in ‘Golden Delicious’ (7.801 g/kg FW). The content of citric acid was also significantly higher in ‘Baya Marisa’ (0.898 g/kg FW) compared to ‘Golden Delicious’ (0.570 g/kg FW). The results of Bureau et al. [22], who measured the organic acids in different apple cultivars (‘Golden Delicious’, ‘Fuji’, ‘Gala’, ‘Granny Smith’, ‘Canada’, ‘Chantecler’, and ‘Pink Lady’) with different techniques of sample preparation, show that the content of organic acids ranges from 2800 to 7300 mg/kg FW for malic acid and from 0.0 to 180.0 mg/kg FW for citric acid. The results of Bars–Cortina et al. [13] showed a higher content of malic acid in red-fleshed apple cultivars compared to white-fleshed cultivars, with the exception of ‘Granny Smith’, which had a similar malic acid content compared to red-fleshed apple cultivars. Other organic acids did not differ between red-fleshed and white-fleshed apple cultivars. In our study, where malic acid and citric acid were higher in the red-fleshed apple cultivar ‘Baya Marisa’, we suggest that the reason for this is due to genetic differences between the two apple cultivars, as reported by Jing et al. [23]. The results of Jing et al. [23] suggest that organic acids, especially ascorbic acid and citric acid, may be natural antioxidants, i.e., anti-browning agents that protect metabolic reactions and structure, and are also known to reduce microbial contamination of fresh-cut vegetables and fruits.

Of all four sugars identified, only fructose was statistically significant (p < 0.05), as shown in Table 1. The measured content of fructose was 51.2 g/kg FW in ‘Baya Marisa’ and 57.9 g/kg FW in ‘Golden Delicious’. The sucrose content is similar, where ‘Baya Marisa’ contained 43.7 g/kg FW and ‘Golden Delicious’ 42.6 g/kg FW. Glucose content is also similar, where ‘Baya Marisa’ contained 15.9 g/kg FW and ‘Golden Delicious’ contained 17.0 g/kg FW. Sorbitol was measured at 6.7 g/kg FW in ‘Baya Marisa’ and 5.4 g/kg FW in ‘Golden Delicious’. Similar results for sugars in apple fruit were obtained by Ticha et al. [24], where the sugar content in different apple cultivars (‘Golden Delicious’, ‘Selena’, ‘Red Delicious’, ‘Ontario’, ‘Opal’ and ‘Melrose’) ranged from: fructose, from 48.0 to 81.0 g/kg FW; glucose, from 9.0 to 30.0 g/kg FW; and sucrose, from 21.0 to 72.0 g/kg FW. As described by Hudina and Stampar [25], sugar content highly correlates with plant genotype, horticultural practices, and environmental factors. In our study, the location for both apple cultivars was the same, and both cultivars had similar horticultural practices, suggesting that genetic variability could be the reason for the different sugar synthesis of these two apple cultivars. The study conducted by Begic–Akagic et al. [26] showed variations in total and individual sugar content in all twelve white-fleshed apple cultivars studied, leading to the conclusion that sugar content depends on genetic variability.

The sugar–acid ratio in ‘Baya Marisa’ was 11.1, compared to ‘Golden Delicious’, which was 14.7. As previously reported by Begic–Akagic et al. [26], sugar/acid ratio ranged from 5.4 (‘Granny Smith’) to 189.9 (‘Prijedorska zelenika’).

The sweetness index for ‘Baya Marisa’ was 192.8 g/kg and the total sweetness index was 132.7 g/kg. The sweetness index for ‘Golden Delicious’ was 207.7 g/kg, whereas the total sweetness index was 142.4 g/kg. As previously reported by Ticha et al. [24], the sweetness of different apple cultivars ranged from 127.0 g/kg (‘Ontario’) to 195.0 g/kg (‘Opal’).

Ascorbic acid was higher in red-fleshed ‘Baya Marisa’ (0.026 g/kg FW) compared to ‘Golden Delicious’ (0.009 g/kg FW). Results obtained by Bars–Cortina et al. [14], showed that ascorbic acid content in red-fleshed apple cultivars ranged from 0.011 to 0.021 g/kg FW in the flesh and from 0.011 to 0.092 g/kg FW in the skin, and ascorbic acid in white-fleshed apple cultivars ranged from 0.004 to 0.016 g/kg FW in the flesh and from 0.008 to 0.097 g/kg FW in the skin. The higher levels of ascorbic acid could be the reason for the absence of polyphenol oxidation causing excessive browning of apple juice, for which ascorbic acid is usually added to prevent polyphenol oxidation, as reported by Wagner et al. [27]. However, as reported by Farr and Giusti [28], oxidation in apple juices from the addition of ascorbic acid or ascorbate should be avoided because anthocyanins are readily degraded by the addition of ascorbic acid.

3.3. Identification of Individual Phenolic Compounds

A total of 46 different phenolic compounds were identified in the flesh and skin of apple fruit, based on the literature and mass spectra. Of these, 3 anthocyanins, 2 dihydrochalcones, 23 hydroxycinnamic acids, 11 flavanols, and 7 flavonols were identified. Phenolic compounds were identified on the basis of the mass-to-charge ratio (m/z) of the characteristic fragmentation ions and molecular ions. When standards were not available for the compounds, the compounds were tentatively identified based on their pseudomolecular ions (i.e., M − H]⁻ and [M + H]⁺ ions) and by specific fragmentation patterns (i.e., MS², MS³). Where possible, a comparison with authentic standards were made.

Table 2. Tentative identification of the 47 phenolic compounds of Malus domestica Borkh. cultivars ‘Baya Marisa’ and ‘Golden Delicious’ and used standards.

Phenolic Compounds	Rt (min)	[M − H]− (m/z)	[M + H]+ (m/z)	MS2 (m/z)	MS3 (m/z)	Expressed as	‘Baya Marisa’		‘Golden Delicious’
							Skin	Flesh	Skin	Flesh
Anthocyanins
Cyanidin-3-O-galactoside	8.9		449	287		cyanidin-3-O-galactoside	X	X
Cyanidin-3-O-arabinoside	11.9		419	287		cyanidin-3-O-arabinoside	X	X
Peonidin-3-O-galactoside	11.0		463	301		peonidin-3-O-galactoside	X	X
Dihydrochalcones
Phloridzin	23.9	481		435,273		phloridzin	X	X	X	X
Phloretin-2-O-xyloside	22.2	567		273,167		phloridzin	X	X	X	X
Hydroxycinnamic acids
p-coumaric acid hexoside derivative 1	10.1	371		325,163		p-coumaric acid	X	X	X
p-coumaric acid hexoside derivative 2	12.3	371		325,163		p-coumaric acid	X
p-coumaroyl hexoside derivative	13.8	363		325,235,119		p-coumaric acid			X
p-coumaric acid hexoside	13.1	325		265,235,163		p-coumaric acid			X
p-coumaric acid hexoside 1	15.3	325		265,235,163		p-coumaric acid	X	X
p-coumaric acid hexoside 2	16.6	325		265,235,163		p-coumaric acid	X	X
Caffeic acid derivative	15.8	335		179,135		caffeic acid		X	X
Caffeic acid derivative 2	12.9	311		179		caffeic acid	X	X
Dicaffeic acid derivative	19.4	403		279,179,135		caffeic acid				X
Dicaffeic acid derivative 1	10.8	457		179,135		caffeic acid	X	X
Dicaffeic acid derivative 2	19.3	403		233,179,135		caffeic acid	X	X
Dicaffeic acid derivative 3	22.6	429		249,205,179,135		caffeic acid		X		X
Caffeic acid hexoside 1	11.8	341		179,135		caffeic acid		X	X
Caffeic acid hexoside 2	11.9	341		179,135		caffeic acid			X
Dihydrodicaffeic acid derivative	20.4	405		225,181		caffeic acid	X	X		X
4-O-p-coumaroylquinic acid	16.0	337		191,173,163		chlorogenic acid			X	X
5-O-p-coumaroylquinic acid	16.5	337		191,163,119		chlorogenic acid		X	X
Chlorogenic acid (5-caffeoylquinic acid)	13.4	353		191,179		chlorogenic acid	X	X	X	X
Caffeoylferuoylquinic acid	14.2	563		385,205	191,193	chlorogenic acid		X		X
Feruloylquinic acid gallate	11.0	658		385,272,193		chlorogenic acid		X		X
Ferulic acid hexoside	14.3	355		193		ferulic acid			X
Ferulic acid hexoside derivative	11.6	401		355	265,235,193	ferulic acid		X	X
Cryptochlorogenic acid (4-caffeoylquinic acid)	14.7	353		191,179		cryptochlorogenic acid			X	X
Flavanols
(-)epicatehin	15.8	289		271,245,205,179		(-) epicatechin	X	X	X	X
(Epi)catechin derivative 1	18.6	583		289,271	271,245,205,179	(-) epicatechin			X
(Epi)catechin derivative 2	18.9	493		331,330,316,289	316,289,271,209	(-) epicatechin			X
(Epi)catechin derivative 3	21.1	477		331,330,316,289	316,289,271,209	(-) epicatechin			X
Flavanol monomer	22.0	289		245,205,179		(-) epicatechin		X
Procyanidin dimer 1	10.1	577		451,425,407	289,245	procyanidin B1			X
Procyanidin dimer 2	11.0	577		451,425,407	289,245	procyanidin B1			X
Procyanidin dimer 3	12.7	577		451,425,407	289,245	procyanidin B1			X
Procyanidin dimer 4	14.6	577		451,425,407	289,245	procyanidin B1	X	X	X	X
Procyanidin trimer	16.9	865		739,695,577		procyanidin B1	X		X
Dihydroprocyanidin dimer	21.3	579		289,245,203	271,245,205,179	procyanidin B1			X
Quercetin-3-O-arabinofuranoside	22.9	433		301,300		quercetin-3-O-arabinofuranoside	X	X	X
Quercetin-3-O-arabinopyranoside	22.5	433		301,300		quercetin-3-O-arabinopyranoside	X
Quercetin-3-O-galactoside	21.2	463		301,300		quercetin-3-O-galactoside	X	X	X
Quercetin-3-O-glucoside	21.4	463		301,300		quercetin-3-O-glucoside	X	X	X
Quercetin-3-O-rhamnoside	23.0	447		301,300		quercetin-3-O-rhamnoside	X	X	X	X
Quercetin-3-O-rutinoside	20.4	609		301,300		quercetin-3-O-rutinoside	X		X
Quercetin-3-O-xyloside	22.2	433		301,300		quercetin-3-O-xyloside	X		X

Rt, retention time; [M − H]⁻, pseudomolecular ion identified in a negative ion mode; [M + H]⁺, pseudomolecular ion identified in a positive ion mode; x, presence of the identified compound.

summarizes the data for all identified compounds for both the flesh and skin of apple fruit.

3.4. Quantification of Individual Phenolic Compounds and Total Analyzed Phenolic Content

Phenolic profiles were utilized in both red-fleshed and white-fleshed apple cultivars (

Table 3. Individual and total analyzed phenolic compounds (TAPC) in the skin and flesh of the two apple (Malus domestica Borkh.) cultivars ‘Baya Marisa’ and ‘Golden Delicious’ (mean ± SE, in mg/kg FW).

	Skin		Flesh
Compunds	‘Baya Marisa’	‘Golden Delicious’	‘Baya Marisa’	‘Golden Delicious’
Hydroxycinnamic acids
p-coumaric acid hexoside derivative 1	2.4 ± 0.1 a	0.9 ± 0.1 b	0.60 ± 0.1	nd
p-coumaric acid hexoside derivative 2	5.6 ± 0.8	nd	nd	nd
p-coumaroyl hexoside derivative	nd	1.2 ± 0.1	nd	nd
p-coumaric acid hexoside	nd	0.3 ± 0.0	nd	nd
p-coumaric acid hexoside 1	2.6 ± 0.0	nd	0.5 ± 0.0	nd
p-coumaric acid hexoside 2	2.1 ± 0.1	nd	0.2 ± 0.0	nd
Caffeic acid derivative	nd	1.2 ± 0.1	1.1 ± 0.1	nd
Caffeic acid derivative 2	8.5 ± 1.2	nd	7.3 ± 1.1	nd
Dicaffeic acid derivative	nd	nd	nd	0.4 ± 0.0
Dicaffeic acid derivative 1	5.5 ± 0.8	nd	0.9 ± 0.1	nd
Dicaffeic acid derivative 2	1.5 ± 0.1	nd	0.3 ± 0.0	nd
Dicaffeic acid derivative 3	nd	nd	0.4 ± 0.1 a	0.4 ± 0.0 a
Caffeic acid hexoside 1	nd	0.8 ± 0.1	0.2 ± 0.0	nd
Caffeic acid hexoside 2	nd	1.2 ± 0.3	nd	nd
Dihydrodicaffeic acid derivative	1.4 ± 0.2	nd	0.2 ± 0.0 a	0.3 ± 0.0 a
4-O-p-coumaroylquinic acid	nd	27.4 ± 2.4	nd	10.2 ± 0.6
5-O-p-coumaroylquinic acid	nd	17.4 ± 2.5	2.5 ± 0.3	nd
Chlorogenic acid (5-caffeoylquinic acid)	43.0 ± 5.3 b	85.8 ± 4.0 a	41.2 ± 4.9 b	59.0 ± 3.1 a
Caffeoylferuoylquinic acid	nd	nd	5.6 ± 0.1 a	1.7 ± 0.1 b
Feruloylquinnic acid gallate	nd	1.3 ± 0.2	2.4 ± 0.3	nd
Ferulic acid hexoside	nd	1.1 ± 0.1	nd	nd
Ferulic acid hexoside derivative	nd	nd	0.2 ± 0.1	nd
Cryptochlorogenic acid (4-caffeoylquinic acid)	nd	7.8 ± 0.5	nd	0.8 ± 0.0
Dihydrochalcones
Phloridzin	137.6 ± 8.0 a	28.8 ± 2.3 b	4.0 ± 0.5 a	5.7 ± 0.4 a
Phloretin-2-O-xyloside	47.7 ± 3.0 a	27.4 ± 2.5 b	5.4 ± 0.8 a	4.2 ± 0.3 a
Flavonols
Quercetin-3-O-arabinofuranoside	49.1 ± 4.1 a	30.6 ± 2.3 b	0.9 ± 0.1	nd
Quercetin-3-O-arabinopyranoside	15.5 ± 1.5	nd	nd	nd
Quercetin-3-O-galactoside	87.6 ± 11.1 a	58.6 ± 6.4 b	1.1 ± 0.0	nd
Quercetin-3-O-glucoside	14.7 ± 2.0 a	18.7 ± 1.4 a	0.8 ± 0.1	nd
Quercetin-3-O-rhamnoside	23.0 ± 2.5 b	37.4 ± 2.3 a	1.9 ± 0.2 a	1.7 ± 0.2 a
Quercetin-3-O-rutinoside	37.7 ± 2.7 a	23.7 ± 1.9 b	nd	nd
Quercetin-3-O-xyloside	23.1 ± 1.8 a	9.7 ± 0.8 b	nd	nd
Flavanols
(-)epicatehin	80.0 ± 6.1 a	54.1 ± 1.7 b	4.8 ± 0.2 a	15.6 ± 1.2 a
Epicatechin derivative 1	nd	1.7 ± 0.2	nd	nd
Epicatechin derivative 2	nd	9.4 ± 1.5	nd	nd
Epicatechin derivative 3	nd	26.8 ± 1.8	nd	nd
Flavanol monomer	nd	nd	1.1 ± 0.2	nd
Procyanidin dimer 1	nd	6.7 ± 0.7	nd	nd
Procyanidin dimer 2	nd	40.8 ± 2.8	nd	nd
Procyanidin dimer 3	nd	64.9 ± 6.3	nd	nd
Procyanidin dimer 4	99.6 ± 9.0 b	142.1 ± 9.7 a	16.6 ± 1.2 a	33.8 ± 4.8 a
Procyanidin trimer	59.4 ± 5.8 a	41.2 ± 2.6 b	nd	nd
Dihydroprocyanidin dimer	nd	35.5 ± 1.6	nd	nd
Anthocyanins
Cyanidin-3-galactoside	526.3 ± 77.0	nd	80.6 ± 5.4	nd
Cyanidin-3-arabinoside	14.3 ± 0.6	nd	4.8 ± 0.2	nd
Peonidin-3-galactoside	24.60 ± 4.0	nd	1.7 ± 0.1	nd
Total hydroxycinnamic acids	72.6 ± 8.2 b	146.5 ± 8.6 a	69.3 ± 6.3 a	74.7 ± 3.8 a
Total dihydrochalcones	185.3 ± 9.6 a	56.2 ± 4.5 b	9.4 ± 1.2 a	9.9 ± 0.8 a
Total flavonols	250.7 ± 21.6 a	178.6 ± 14.2 b	4.7 ± 0.3 a	1.7 ± 0.2 b
Total flavanols	238.9 ± 20.4 b	423.2 ± 24.2 a	22.5 ± 1.4 b	49.4 ± 5.9 a
Total anthocyanins	565.2 ± 81.5	nd	87.1 ± 5.6	nd
TAPC	1312.7 ± 117.4 a	804.5 ± 39.3 b	193.0 ± 12.6 a	135.6 ± 8.4 b

Presented data are means ± standard error. Means followed by different letters in skin or flesh, respectively (within rows), are significantly different (p < 0.05); nd, not detected.

). The major phenolic compounds were anthocyanins, dihydrochalcones, hydroxycinnamic acids, flavanols, and flavonols. Previously, similar phenolic compounds had been reported in apple cultivars by Tsao et al. [29] and Chinnici et al. [30].

The major hydroxycinnamic acid in both the skin and flesh of the two apple cultivars was chlorogenic acid (Table 3). Similar results were reported by Tsao et al. [29] for white-fleshed apple cultivars (‘Golden Delicious’, ‘Red Delicious’, ‘McIntosh’, ‘Empire’, ‘Ida Red’, ‘Northern Spy’, ‘Mutsu’, and ‘Cortland’), where the total hydroxycinnamic acid content ranged from 132.2 to 328.0 mg/kg FW in the flesh and from 33.5 to 247.4 mg/kg FW in the skin of these apple cultivars. In our study, the white-fleshed ‘Golden Delicious’ (146.5 mg/kg FW) had a higher total hydroxycinnamic acid content in the skin than the red-fleshed ‘Baya Marisa’ (72.6 mg/kg FW) and a similar content in the flesh (‘Baya Marisa’ 69.3 mg/kg FW, ‘Golden Delicious’ 74.4 mg/kg FW). This result differs from that of Wang et al. [31], in which all red-fleshed apple cultivars (‘No.1 Hongxun’, ‘Hongrouguo’, ‘Roberts Crab’) except ‘Xiahongrou’ had significantly higher levels of chlorogenic acid compared to the white-fleshed apple cultivars (‘Golden Delicious’ and ‘Gale Gala’), in which the only hydroxycinnamic acid identified was chlorogenic acid.

The dihydrochalcones found in this study were phloridzin and phloretin-2-O-xyloside. The content of dihydrochalcones ranged from 56.2 mg/kg FW in the skin of ‘Golden Delicious’ to 185.3 mg/kg FW in the skin of ‘Baya Marisa’. Dihydrochalcone content in the flesh ranged from 9.4 mg/kg FW in ‘Baya Marisa’ to 9.9 mg/kg FW in ‘Golden Delicious’. Red-fleshed ‘Baya Marisa’ is known to be scab resistant, which could be the reason of higher content of dihydrochalcones, since phloridzin is a functional antioxidant, which lowers oxidative stress in the leaves and fruits of apple trees, as reported by Gosch et al. [32]. Chalcone synthase activity could also be the reason for higher content of dihydrochalcones in red-fleshed ‘Baya Marisa’, as it increases with production of anthocyanins during fruit maturation, as reported by Ju et al. [33].

The only flavonols found in our study were quercetin glycosides. Their content ranged from 178.6 mg/kg FW (‘Golden Delicious’) to 250.7 mg/kg FW (‘Baya Marisa’) in the skin and from 1.7 mg/kg FW (‘Golden Delicious’) to 4.7 mg/kg FW (‘Baya Marisa’) in the flesh. ‘Baya Marisa’ had higher levels of flavonols in the skin and similar levels in the flesh. The most prominent flavonol monomer in the skin of both apple cultivars was quercetin-3-O-galactoside, which was 87.6 mg/kg FW in ‘Baya Marisa’ and 58.6 mg/kg in ‘Golden Delicious’. Quercetin-3-O-rhamnoside was the most prominent flavonol monomer in the flesh of ‘Golden Delicious’, quercetin-3-O-arabinofuranoside in the flesh of ‘Baya Marisa’. Higher content of quercetin gylcosides in red-fleshed apple cultivars compared to white-fleshed ones is also reported by Ceci et al. [34], where red-fleshed (‘Bay 3484’, ‘Y103’, ‘Y102’, ‘R201’, ‘RS-1’) showed higher contents compared to white-fleshed apple cultivars. The results reported by Wang et al. [31] showed higher content of flavonols in red-fleshed apple cultivars: ‘No.1 Hongxun’, ‘Hongrouguo’, ‘Roberts Crab’ and ‘Xiahongrou’ compared to white-fleshed apple cultivars: ‘Gale Gala’ and ‘Golden Delicious’. The results of Wolfe et al. [35] showed similar levels of flavonols in the skin and flesh of ‘Golden Delicious’ (167.4 mg catechin equivalents/100 g FW of skin, 42.5 mg catechin equivalents/100 g FW of flesh). The total flavonol content was higher in the red-fleshed and red-skinned apple cultivar ‘Baya Marisa’, compared to white-fleshed and yellow-skinned ‘Golden Delicious’. The reason for the higher content of flavonols was previously described by Wang et al. [36], who reported that enzyme dihydroflavonol reductase is crucial in flavonoid biosynthesis in apples. Reduction of dihydroflavonols to leucoanthocyanidins is catalyzed by dihiydroflavonol reductase, which leads to conversion to anthocyanins and anthocyanidins. Anthocyanins in apple fruits are responsible for the red color of the skin, which synthetize higher amounts of cyanidin glycosides compared to yellow or green apple cultivars. Lower activity of dihydroflavonols in presence of glucosyltransferase and flavonol synthase leads to conversion to higher amounts of quercetin glycosides in red apple cultivars.

Another important group of phenolic compounds in apples are flavanols, which are a product of the flavonoid pathway [37]. Among the flavanols, (-)epicatechin, procyanidin dimer 4, and procyanidin trimer had the highest contents. The values for total flavanol content differed between the skin of ‘Baya Marisa’ and ‘Golden Delicious’ (p < 0.05). The total flavanol content in the skin of ‘Golden Delicious’ was 423.2 mg/kg FW and 238.9 mg/kg FW in ‘Baya Marisa’ (Table 3). Total flavanol content in the flesh was higher in white-fleshed ‘Golden Delicious’ (49.4 mg/kg FW) compared to red-fleshed ‘Baya Marisa’ (22.5 mg/kg FW). The results are similar to those reported by Ceci et al. [34] in some comparisons of red-fleshed (‘Bay 3484’, ‘Y103’, ‘Y102’, ‘R201’, ‘RS-1’) and white-fleshed apple fruits. The total flavanols content was higher in white-fleshed ‘Golden Delicious’ in our study. The reason for the higher content of flavanols in ‘Golden Delicious’ compared to red-skinned and red-fleshed apple cultivars was previously described by Bars–Cortina et al. [13] and Henry–Kirk et al. [37], who reported a negative correlation in the biosynthesis of phenolic compounds, a competing pathway between anthocyanins and flavanols. Flavanols and anthocyanins share the same precursor, namely leucoanthocyanidins. Therefore, a competitive interaction between two different enzymes, anthocyanidin synthase and anthocyanidin reductase, with the substrate appears, which could lead to a different biosynthesis of anthocyanins and flavanols, respectively.

Of the two apple cultivars tested (‘Baya Marisa’ and ‘Golden Delicious’), we found no traces of anthocyanins in ‘Golden Delicious’, which was expected since the skin of this cultivar is green–yellow and the flesh is white, whereas anthocyanins are associated with the red color. As previous studies by Joshi et al. [38] had shown, and our study also suggests that the conversion of flavanols to anthocyanins is different in different apple cultivars, since the content of flavanols was higher in ‘Golden Delicious’ and no anthocyanins were detected. The study by Wang et al. [31] obtained similar results for some red-fleshed apple cultivars (‘No.1 Hongxun’, ‘Hongrouguo’, ‘Roberts Crab’, and ‘Xiahongrou’), whereas no anthocyanins were found in the flesh of white-fleshed apple cultivars (‘Golden Delicious’ and ‘Gale Gala’). It has been reported that anthocyanin accumulation in red-fleshed apples is genotype-dependent [9].

Three anthocyanins were found in the red-fleshed ‘Baya Marisa’: cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, and peonidin-3-O-galactoside. The total anthocyanin content in ‘Baya Marisa’ was measured 565.2 mg/kg FW in the skin and 87.1 mg/kg FW in the flesh. The most prominent anthocyanin in both, skin (526.3 mg/kg FW) and flesh (80.6 mg/kg FW), was cyanidin-3-O-galactoside. The results reported by Wang et al. [31] showed a similar content of anthocyanins in red-fleshed apple cultivars, ranging from 295.3 to 1758.4 mg/kg in the skin and from 12.1 to 559.7 mg/kg FW in the flesh. Ban et al. [4] reported that the MdMYB1-1 gene is dominant for the apple skin color, where apples that are homozygous or heterozygous for this gene are red skinned, whereas apple cultivars without the MdMYB1-1 gene copy have a yellow or green color of the skin. As reported by Umemura et al. [39], MdMYB110a, MdMYB10, and MYB TFs are important for generating the color of the flesh of apple fruits. MdMYB110a, located on chromosome 17, is responsible for the red flesh, known as type 2, where the flesh of fruits is red and other organs are same as other white-fleshed apple cultivars, whereas the allele of MdMYB1, MdMYB10, located on chromosome 9 generates red-flesh and skin, leaves, stems, and flowers, which is known as type 2 red-fleshed apples, as reported by Chagné et al. [40]. Therefore, we suggest, that there is a similar situation, described above, with red-fleshed ‘Baya Marisa’ and ‘Golden Delicious’, although no detailed study for described situation has been conducted on ‘Baya Marisa’.

The TAPC (total analyzed phenolic content) values differed significantly between the red-fleshed cultivar ‘Baya Marisa’ and the white-fleshed cultivar ‘Golden Delicious’. The TAPC of ‘Golden Delicious’ skin was 804.5 mg/kg FW and 1312.7 mg/kg FW in ‘Baya Marisa’ skin. In the flesh, TAPC values ranged from 135.6 to 193.0 mg/kg FW. The TAPC of ‘Golden Delicious’ was 135.6 mg/kg FW and of ‘Baya Marisa’ was 193.0 mg/kg FW. The red-fleshed cultivar ‘Baya Marisa’ contained the highest TAPC levels in both skin and flesh. These results are comparable to those of Sadilova et al. [41], where it was reported that the total analyzed phenolic content of red-fleshed ‘Weirouge’ apples was 1684.0 mg/kg FW in the skin and 379.0 mg/kg FW in the flesh. This difference in our study suggests that red-fleshed ‘Baya Marisa’ is a better source of phenolic compounds compared to white-fleshed ‘Golden Delicious’.

Wang et al. [31] reported similar results, with the total analyzed phenolic content in red-fleshed (‘No.1 Hongxun’, ‘Hongrouguo’, ‘Roberts Crab’, ‘Xiahongrou’) and white-fleshed (‘Gale Gala’, ‘Golden Delicious’) apples ranging from 1641.8 to 4727.8 mg/kg FW in the skin and from 160.3 to 1056.0 mg/kg FW in the flesh. The red-fleshed cultivar ‘Roberts Crab’ had the highest total phenolic content in both skin and flesh whereas ‘Gale Gala’ had the lowest total phenolic content in both skin and flesh, which is consistent with our results comparing white and red-fleshed cultivars.

Overall, the TAPC content, total flavonols, total anthocyanins, and total dihydrochalcones content were significantly higher in the red-fleshed cultivar ‘Baya Marisa’, whereas the total hydroxycinnamic acid and flavanol content was higher in the white-fleshed cultivar ‘Golden Delicious’. Anthocyanins in the skin and flesh of red-fleshed ‘Baya Marisa’ and flavanols in the skin and hydroxycinnamic acids in the flesh of white-fleshed ‘Golden Delicious’ were the most important groups of phenolic compounds with the highest total relative content, as shown in Figure 2. As expected, ‘Baya Marisa’ had higher total analyzed phenolic contents in the skin and flesh samples, compared to ‘Golden Delicious’.

This study is consistent with the studies described by Bars–Cortina et al. [13], which reported that the abundance of flavanols was significantly higher in white-fleshed apple cultivars than in red-fleshed apple cultivars in both flesh and skin. It is reported that this is not reported in all cases, as red-fleshed ‘Royal Gala’, a transgenic apple line, does not have lower flavanol content due to high expression of anthocyanins [21].

4. Conclusions

We identified 46 different phenolic compounds in the skin and flesh of the apple cultivars studied. Some of these phenolic compounds have not been previously identified in apple fruit.

Our results show that different apple cultivars, in our case, the red-fleshed ‘Baya Marisa’ and the white-fleshed ‘Golden Delicious’, contain several classes of phenolic compounds, which could be consumed. ‘Baya Marisa’ had a higher content of anthocyanins, flavonols, and dihydrochalcones, whereas the white-fleshed ‘Golden Delicious’ had higher levels of flavanols and hydroxycinnamic acids. Anthocyanins were not identified in the skin or flesh of ‘Golden Delicious’.

In terms of sugar content, there was a difference only in fructose content, which was higher in the white-fleshed apple cultivar ‘Golden Delicious’.

Organic acids (citric acid, malic acid, and ascorbic acid) were higher in red-fleshed ‘Baya Marisa’.

We suggest that after this detailed analysis of both apple cultivars, further analysis should be carried out on fresh and processed fruits of white-fleshed ‘Golden Delicious’ and red-fleshed ‘Baya Marisa’.