Metabolite Diversity in Pulp Segments, Peel, Leaves, and Bark of a Red-Fleshed ‘Baya Marisa’ Apple Cultivar

Abstract

This study investigated the composition of sugars, organic acids, individual and total phenolic compounds in the pulp, peel, leaves, and bark of a red-fleshed ‘Baya Marisa’ apple cultivar. As the fruit is known for its red pulp color, the study focused on comparing the profiles of primary and selected secondary metabolites in three sections along the equatorial fruit plane. The analyses were carried out using HPLC–MS system, and compositional differences were compared among fruit segments. Inner fruit segments accumulated higher levels of sorbitol and the sum of sugars and lower levels of citric acid. However, no differences in the sum of organic acids could be determined among segments. The phenolic composition differed among pulp (hydroxycinnamic acids > dihydrochalcones ≈ anthocyanins ≈ flavanols > flavonols), peel (flavanols > anthocyanins > dihydrochalcones > flavonols > hydroxycinnamic acids), leaves (flavonols > dihydrochalcones > hydroxycinnamic acids > anthocyanins), and bark (dihydrochalcones > flavonols > flavanols > anthocyanins > hydroxycinnamic acids). The greatest phenolic diversity was detected in bark (25), followed by leaves (18), peel (17) and finally, pulp (11). Three anthocyanins (cyanidin-3-O-galactoside > cyanidin-3-O-arabinoside > peonidin-3-O-galactoside) were determined in all ‘Baya Marisa’ tissues with their content highest in the peel. The innermost sections of the fruit were characterized by higher levels of dihydrochalcones and lower levels of most flavanols, flavonols and anthocyanins. These were predominant in the apple pulp nearest to the peel, with cyanidin-3-O-galactoside being the prevalent representative. Accumulation of anthocyanins in pulp is a rare trait in apples, and therefore, the distinct distribution and diversity of metabolites in this cultivar highlights its potential for high-nutrient products such as juices, apple chips or purees.

1. Introduction

Apple (Malus domestica Borkh.) is the most widely cultivated temperate fruit crop, with a global yearly production of over 86 million tons in 2021 [1]. Due to its long storage potential fresh apples are available on the market year-round. Additionally, apple concentrates, juices, purees, ciders, and other apple products labeled as functional food are offered to consumers [2]. Consequently, apple is regarded as one of the most important fresh and processed fruit sources of beneficial compounds in the Western diet [3].

Numerous studies report the favorable composition of apples as they contain balanced levels of different primary and secondary metabolites such as sugars, organic acids, vitamins and phenolics [4]. These compounds are very important for a plant’s growth and development. Their synthesis is genetically predefined and cultivar-specific or boosted as a response to the plant’s environment [5]. Sugars, organic acids and phenolics define inner fruit quality traits, and several groups of secondary metabolites (for example, anthocyanins) considerably affect apple fruit appearance. The intake of phenolics and dietary fibers has been associated with improved human health [6], lower incidence of various diseases [3,7] and even enhanced cognitive functions [8]. When compared to other fruits, apples contain high amounts of readily available phenolic compounds, which are easily absorbed into human cells [9].

A shift towards a healthier diet increased consumer’s interest in fresh apples, and several hundred cultivars competed for their share on the market. Apples with red peel are preferential to green- or yellow-colored cultivars because they are considered tastier and healthier [10]. White-fleshed apple cultivars are dominating, but recently, red-fleshed cultivars have gained interest as they are novel, attractive and a potentially promising source of bioactive compounds [7]. From the first discovery of an astringent and small wild, red-fleshed apple in the Tian Shan mountains of Central Asia [4] to modern, red-fleshed apple cultivars with improved size and taste, only a decade has passed. In the last years, four cultivars of red-fleshed apples have become commercially available: ‘Redlove’ from Switzerland, ‘Rosette’ from England, and ‘Weirouge’ and ‘Baya Marisa’ from Germany and Italy. The latter is characterized by completely red pulp, crispy flesh texture and a complex taste of red currants, strawberries, and raspberries. These traits make ‘Baya Marisa’ apples appealing for fresh consumption, culinary highlights in restaurants and hotels, and dried apple slices, crisps and juices [4]. The color of ‘Baya Marisa’ apples is retained during processing, and this is potentially related to their unique phenolic composition. Interest in the cultivation of this red-fleshed cultivar is growing in Europe, and biochemical descriptions of its special visual characteristics are of interest to producers and consumers.

Therefore, the objective of the present study was to present the composition of primary and secondary metabolites of ‘Baya Marisa’ apple fruit (peel and pulp), leaves, and bark on young shoots. A special emphasis was on studying the group of anthocyanins as they are responsible for the attractive color of apple peel and flesh. As the pigmentation of apple pulp gradually decreases inwards to the core, a significant aim of the study was to monitor the distribution of phenolic compounds, particularly anthocyanins, in different parts of apple pulp. It has been proposed that the gradation in the content of major metabolites is not uniform and different classes of phenolics are predominant in different sections of ‘Baya Marisa’ pulp as well as other tissues analyzed.

2. Materials and Methods

2.1. Plant Material

Red-fleshed apple cultivar ‘Baya Marisa’ was selected for the study. Samples of fruit, fully developed leaves, and young shoots (approximately 60 cm in length) were collected from a three-year-old production orchard located in Zdole in the South-Eastern part of Slovenia (45°98′02″ N; 15°52′07″ E; 307 m a.s.l). Trees were grafted on an M9 rootstock, spaced 1 m × 3.2 m with a North-South row orientation. The orchard was managed according to standard phytosanitary recommendations and integrated pest control practices. Technologically mature apples (n = 30, 6 apples for each repetition), fully developed leaves (n = 30, 6 leaves for each repetition) and one-year shoots (n = 30, 6 shoots for each repetition) were collected on 17 September 2020 (Figure 1) and immediately transported to the laboratory of the Department of Agronomy at Biotechnical Faculty of the University of Ljubljana (Slovenia) where they were subjected to detailed analyses.

Leaves were washed in double-distilled water and tapped with tissue paper. Bark was carefully removed from the shoots and cut into sections. Bark and leaf samples were shock-frozen in liquid nitrogen and immediately stored in a freezer at −20 °C for up to 5 days until further analyses. Apples were cut into 1 cm thick rings at the equatorial plane, and pulp samples were taken with a 7.9 mm tip at three different sampling positions: (P1) outer pulp adjacent to the apple peel (0.1 cm depth), (P2) middle pulp section (1–2 cm depth), (P3) pulp adjacent to the fruit core (2–3 cm depth) and (S) peel (Figure 2). Pulp and peel samples were immediately shock-frozen in liquid nitrogen and immediately stored in a freezer at −20 °C until further analyses of sugars, organic acids, and phenolic content.

2.2. Extraction of Individual Sugars and Organic Acids

For the determination of sugar and organic acid content in different pulp samples, the extractions were carried out as described by Mikulic-Petkovsek et al. [11] with minor modifications. Pulp plugs were taken at five points from three designated pulp positions (P1, P2 and P3) on each fruit, and 30 plugs (5 plugs from 6 different fruit) represented one combined sample (the number of repetitions per treatment, n = 5). Samples were compressed into a mixture for better homogeneity, and 1 g of pulp samples were extracted in 5 mL of double distilled water. Samples were placed on a shaker for 30 min (UNI MAX 1010; Heidolph, Schwabach, Germany), centrifuged (5810 R; Eppendorf, Hamburg, Germany) at 10,000× g for 10 min at 4 °C and filtered through 0.2-μm polyamide filters (Chromafil XTRA 20/25; Macherey-Nagel, Düren, Germany) into vials. Extracts were stored at −20 °C until further analyses.

2.3. HPLC Analysis of Individual Sugars and Organic Acids

The analysis of individual sugars and organic acids was carried out using a Vanquish HPLC system (Thermo Scientific, San Jose, CA, USA). The separation of individual sugars was carried out on a Rezex RCM monosaccharide column from Phenomenex (Torrance, CA, USA) operated at 85 °C. The mobile phase was double distilled water, and the flow rate was maintained at 0.6 mL/min. The run time was 15 min, and a refractive index (RI) detector was used to monitor the eluted carbohydrates as described by Mikulic-Petkovsek et al. [12]. Organic acids were analyzed with the same HPLC system, equipped with a UV detector set at 210 nm with a Rezex ROA column from Phenomenex, as described by Mikulic-Petkovsek et al. [12]. The column temperature was 65 °C. The elution solvent was 4 mM (H₂SO₄) with a flow rate of 0.6 mL/min. The content of sugars and organic acids was expressed in g/kg FW (fresh weight).

2.4. Extraction of Individual Phenolic Compounds

Phenolic compounds were extracted as previously described by Mikulic-Petkovsek et al. [13] with minor modifications. Briefly, 1 g of peel, bark, pulp, and leaves were extracted with 80% methanol + 3% formic acid in water (MeOH; Sigma-Aldrich, Steinheim, Germany). The extraction ratio for pulp was 1:2 (w/v) and for peel, leaves, and bark, 1:3 (w/v). The samples were sonicated in iced water (Sonis 4 ultrasonic bath; Iskra pio, Sentjernej, Slovenia) for 60 min, centrifuged (5810 R; Eppendorf, Hamburg, Germany) at 10,000× g for 10 min at 4 °C and filtered through 0.2-μm polyamide filters (Chromafil AO-20/25; Macherey-Nagel, Düren, Germany). Extracts were stored in vials at −20 °C until further analysis.

2.5. HPLC–MS Analysis of Individual Phenolic Compounds

Quantification of phenolic compounds was performed on a UHPLC system (Thermo Finnigan Surveyor Dionex UltiMate 3000 series; San Jose, CA, USA) using a diode array detector at 530 nm (for anthocyanins), 350 nm (for flavonoids) and 280 nm (for other phenolic groups). The protocol previously described by Mikulic-Petkovsek et al. [13] was used. The spectra were recorded in a range between 200 nm and 600 nm. Separation of compounds was achieved using a C18 column (Gemini; 150 × 4.60 mm, 3 μm; Phenomenex, Torrance, CA, USA) operated at 25 °C. Elution was made with a discontinuous gradient, as described by Medic et al. [14]. The volume of extract injection was 20 μL, and the flow rate was maintained at 0.6 mL/min. Identification of phenolic compounds was performed using tandem mass spectrometry (MS/MS; LTQ XL; Thermo Scientific, Waltham, MA, USA) with heated electrospray ionization that operates in negative ion mode. The conditions were as previously described by Mikulic-Petkovsek et al. [13]. External standards were used for further identification and quantification of compounds. Unknown compounds were identified based on MS fragmentation data. Individual phenolic compounds were expressed in mg/kg fresh weight (FW) with standard compounds or similar compounds. The content of the total analyzed phenolic compounds (TAPC) represents the sum of all identified phenolic compounds.

2.6. Chemicals

The following standards were used for quantification of compounds: cyanidin-3-O-galactoside, neochlorogenic acid, chlorogenic acid, procyanidin B1, myricetin-3-O-rhamnoside, kaempferol-3-O-galactoside, (-)epicatechin, quercetin-3-O-rutinoside, quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin-3-O-xyloside, quercetin-3-O-arabinopyranoside, quercetin-3-O-rhamnoside, quercetin-3-O-arabinofuranoside (Sigma–Aldrich Chemie GmbH, Steinheim, Germany); p-coumaric acid and phloridzin (Fluka Chemie GmbH, Buchs, Switzerland); and cyanidin-3-O-arabinoside (Apin Chemicals, Abingdon, UK). The chemicals for the mobile phases (acetonitrile, formic acid, methanol) were HPLC–MS grade (Fluka Chemie GmbH, Buchs, Switzerland). The water used for sample preparation, solutions and analyses was double distilled and purified using a Milli-Q water purification system (Millipore, Bedford, MA, USA).

2.7. Statistical Analysis

Statistical analysis was performed with the R commander i386 4.0.4. software using one-way analysis of variance (ANOVA). Duncan’s test was used to determine the differences in primary and secondary metabolites among different pulp sections (p < 0.05).

3. Results and Discussion

3.1. Sugars and Organic Acids

In addition to phenolic compounds, sugars and organic acids are the most important factors determining inner apple fruit quality [15]. Ten primary metabolites were identified in apple pulp: four sugars and six organic acids. The most abundant sugar was fructose, followed by sucrose, glucose, and sorbitol (

Table 1. Content of sugars and organic acids in different pulp sections of ‘Baya Marissa’ apples (means ± standard errors in g/kg FW).

	Compound	P1	P2	P3
sugars	fructose	55 ± 5 ab *	62 ± 3 b	52 ± 4 a
	glucose	21 ± 2 a	25 ± 2 b	24 ± 2 b
	sucrose	14 ± 2 a	18 ± 2 ab	21 ± 4 b
	sorbitol	4 ± 0.5 a	4 ± 0.3 a	5 ± 0.8 b
	sum of sugars	96 ± 6 a	108 ± 5 b	105 ± 3 b
organic acids	citric acid	1.9 ± 0.03 b	1.2 ± 0.10 a	1.1 ± 0.1 a
	malic acid	12 ± 0.4 a	13.1 ± 0.1 a	14 ± 0.4 a
	sum of acids	15 ± 1 a	15 ± 1 a	16 ± 2 a

* Different letters (a,b) for each parameter denote statistically significant differences among treatments by LSD multiple range test at p < 0.05. P1, pulp adjacent to the apple peel; P2 middle pulp section; P3 pulp adjacent to the fruit core.

), which is consistent with numerous studies on different apple cultivars [16,17,18,19]. When comparing the content of individual and total sugars in ‘Baya Marisa’ apples with other commonly cultivated apple cultivars, the levels are somewhat lower or comparable to more sour-tasting cultivars such as ‘Granny Smith’ [17]. Hecke et al. [20] measured the total sugar content of ‘Gala’, ‘Elstar’, ‘Idared’, ‘Golden Delicious’, ‘Braeburn’, ‘Fuji’ and ‘Jonagold’ in the range from 115 to 183 g/kg FW but the variation in primary metabolites is expected as these also depend on technological measures, ripeness stage and environmental factors [4]. The content of individual and total sugars differed among the analyzed pulp sections, but significant differences were only detected in the level of sorbitol and the sum of sugars. The innermost sections of the fruit (P2 and P3) were generally characterized by a higher content of sugars compared to the outer section P1 (Table 1). This could tentatively be explained by declining air content from just beneath the peel to the center of the fruit, which correlates with cell density and potentially leads to higher sugar levels nearer to the core [21]. Differences in sugar content dependent on radial distance were also reported by Cebulj et al. [17], who studied the composition of metabolites in ‘Jonagold’ and ‘Granny Smith’ apples.

Malic acid was the most abundant organic acid in ‘Baya Marisa’ apples, followed by citric acid, which corresponds with studies on other apple cultivars [12,20] (Table 1). Shikimic, oxalic, quinic and fumaric acids were also identified, but their levels combined represented less than 5% of the sum of organic acids determined in ‘Baya Marisa’ apples. The sum of organic acids in ‘Baya Marisa’ apples (≈15.0 g/kg) was higher than reported for sweet tasting ‘Golden Delicious’ (≈6 g/kg) and ‘Idared’ (≈11 g/kg), and similar as in sweet-sour ‘Elstar’ (≈14 g/kg) and sour tasting ‘Granny Smith’ (≈14 g/kg) apples of integrated production [17,20]. Differences among pulp sections were only determined for citric acid and not for the prevalent malic acid or the sum of organic acids (Table 1). Etienne et al. [22] speculated that the variation in acidity in various parts of fruit pulp is linked to the metabolism of malate and citrate. This was partly confirmed by our results on ‘Baya Marisa’ apples.

3.2. Identification of Individual Phenolic Compounds

In our study, 34 different individual phenolic compounds from the groups of hydroxycinnamic acids, flavanols, flavonols, flavanones, anthocyanins and dihydrochalcones were identified in different parts of ‘Baya Marisa’ cultivar. Characteristic fragmentation and molecular ions mass-to-charge ratios (m/z) were the basis for the identification of phenolic compounds. Compounds lacking standards were tentatively identified using their pseudomolecular ions (i.e., [M − H]⁻) and their specific fragmentation patterns (i.e., MS² and MS³) and were compared to previously published data. All 34 phenolic compounds and their fragmentation as well as the standards used for their expression, are presented in

Table 2. Tentative identification by mass spectrometry fragmentation of 34 phenolic compounds in different parts of ‘Baya Marisa’ and the standard compounds used for their quantification.

Phenolic Compounds	Rt	[M − H]−	MS2	MS3	Expressed as
	(Min)	[M − H]+ (m/z)	(m/z)	(m/z)		Pulp	Peel	Leaves	Bark
cyanidin-3-O-galactoside	8.93	449	287		cyanidin-3-O-galactoside	X *	X	X	X
p-coumaric acid hexoside derivative	11.2	371	325, 163		p-coumaric acid		X
p-coumaric acid derivative 1	11.91	325	163, 145, 187		p-coumaric acid	X
cyanidin-3-O-arabinoside	11.93	419	287		cyanidin-arabinoside	X	X	X	X
neochlorogenic acid	12.43	353	191, 179		neochlorogenic acid			X
p-coumaric acid hexoside	13.10	325	265, 235, 163		p-coumaric acid				X
chlorogenic acid	13.12	353	191, 179		chlorogenic acid	X		X
p-coumaric acid derivative 2	13.12	435	389	163, 179, 119	p-coumaric acid	X		X	X
p-coumaric acid derivative 3	13.12	435	389	163, 179, 119	p-coumaric acid			X
procyanidin dimer	13.80	577	451, 425, 407	289, 245	procyanidin B1		X		X
(-)epicatechin	14.65	289	245, 205, 179		epicatechin	X	X		X
myricetin hexoside	14.90	479	317, 316		myricetin-rhamnoside				X
procyanidin trimer	15.84	865	739, 695, 577		procyanidin B1		X		X
4-p-coumaroylquinic acid	15.97	337	191, 173, 163		p-coumaric acid	X			X
myricetin pentoside	17.38	463	317, 316		myricetin-rhamnoside				X
eriodictyol hexoside	19.41	449	287		kaempferol-3-O-galactoside				X
(epi)catechin derivative	19.70	583	289, 271	245, 205, 179	epicatechin		X
quercetin-3-O-rutinoside	19.72	609	301		quercetin-3-O-rutinoside		X	X
quercetin-3-O-galactoside	20.52	463	301		quercetin-3-O-galactoside		X	X	X
quercetin-3-O-glucoside	20.80	463	301		quercetin-3-O-glucoside		X	X	X
(epi)catechin isomer	20.93	289	245, 205, 179		(-)epicatechin	X
p-coumaric acid derivative 4	21.29	497	453, 325, 235, 163		p-coumaric acid			X
phloretin-2-O-xyloside	21.54	567	273, 167		phloridzin	X
quercetin-3-O-xyloside	21.59	433	301		quercetin-3-O-xyloside		X	X	X
quercetin-3-O-arabinopyranoside	21.80	433	301		quercetin-3-O-arabinopyranoside		X		X
kaempferol derivative	21.89	477	285, 284		kaempferol-3-O-galactoside				X
quercetin-3-O-arabinofuranoside	22.40	433	301		quercetin-3-O-arabinofuranoside		X	X	X
quercetin-3-O-rhamnoside	22.50	447	301		quercetin-3-O-rhamnoside			X	X
phloridzin	23.33	481	435, 273		phloridzin	X	X	X	X
kaempferol-3-O-rhamnoside	24.20	447	285		kaempferol-3-O-galactoside			X	X
phloretin diglucoside	29.33	597	273		phloridzin				X
phloretin	30.12	273	167,179		phloridzin			X	X
phloretin derivative 1	29.79	571	409	273	phloridzin			X	X
phloretin derivative 2	30.63	581	419	273	phloridzin			X	X

* Letter “X” denotes the presence of a specific compound in tissue.

.

Phloridzin, cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, quercetin-3-O-glucoside, quercetin-3-O-rutinoside, quercetin-3-O-galactoside, quercetin-3-O-xyloside, quercetin-3-O-arabinopyranoside, quercetin-3-O-arabinofuranoside, quercetin-3-O-rhamnoside, (-)epicatechin, procyanidin dimer, neochlorogenic acid and chlorogenic acid were identified using specific standards.

(Epi)catechin isomer was identified according to its [M − H]⁻ at (m/z 289) and MS² fragments of (m/z 245, 205, 179) as described by de Souza et al. [23] in Maytenus ilicifolia. p-Coumaric acid hexoside, which produced [M − H]⁻ at (m/z 325) and MS² fragments of (m/z 163), was identified according to Bystrom et al. [24], who reported it in Melicoccus bijugatus. p-Coumaric acid derivative 1 produced [M − H]⁻ at (m/z 325) and MS² fragments of (m/z 163, 145, 187) as described by Jiménez et al. [25] in Annona muricata. p-Coumaric acid derivative 2 and 3 with [M − H]⁻ at (m/z 435) and MS² fragments of (m/z 389) with MS³ fragments of (m/z 163, 179, 119) as well as p-coumaric acid derivative 4 with [M − H]⁻ at (m/z 497) and MS² fragments of (m/z 453, 325, 235, 163) were identified based on a study on apple pomace by Sanchez-Rabaneda et al. [26].

Quercetin-3-O-rhamnoside produced [M − H]⁻ at (m/z 477) and MS² fragment of (m/z 301). Quercetin-3-O-arabinofuranoside, quercetin-3-O-arabinopyranoside and quercetin-3-O-xyloside with [M − H]⁻ at (m/z 433) also yielded MS² fragment of (m/z 301). Quercetin-3-O-glucoside and quercetin-3-O-galactoside with [M − H]⁻ at (m/z 463) yielded their main fragmentation ions of (m/z 301) and kaempferol derivative produced [M − H]⁻ at (m/z 477) while yielding fragmentation ions of (m/z 285 and 284) as described by Schieber et al. [27]. Myricetin hexoside produced [M − H]⁻ at (m/z 479) and MS² fragments at (m/z 317 and 316) as reported by Michodjehoun-Mestres et al. [28]. Myricetin pentoside produced MS (m/z 463) and MS² fragments of (m/z 317), as described by Vvedenskaya et al. [29]. Kaempferol-3-O-rhamnoside, [M − H]⁻ at (m/z 447) yielded MS² ions at (m/z 285) [30]. Eriodictyol hexoside, which produced [M − H]⁻ at (m/z 449) and MS² fragments of (m/z 287), was identified according to Zhao et al. [31].

Two anthocyanins were identified in various parts of the ‘Baya Marisa’ cultivar. Cyanidin-3-O-galactoside produced [M − H]⁻ at (m/z 449) and yielded MS² fragmentation ions of (m/z 287), and cyanidin-3-O-arabinoside with [M − H]⁻ at (m/z 419) yielded MS² ions at (m/z 287) as reported by Bizjak et al. [9] and Lin et al. [32].

Phloretin and its derivatives were also identified in samples. Phloretin produced [M − H]⁻ at (m/z 273) and yielded fragmentation ions at (m/z 167 and 179); meanwhile, phloretin derivative 1 had MS at (m/z 571), MS² at (m/z 409) and MS³ at (m/z 273) and phloretin derivative 2 produced [M − H]⁻ at (m/z 581), MS² at (m/z 419) and MS³ at (m/z 273) as reported by Sanoner et al. [33]. Phloretin diglucoside that produced [M − H]⁻ at (m/z 597) yielded MS² ions at (m/z 273) [34], while compound that produced [M − H]⁻ at (m/z 567) and MS² fragments of (m/z 273 and 167) was tentatively identified as phloretin-2-O-xyloside as reported by De La Luz Cádiz-Gurrea et al. [35].

In the group of hydroxycinnamates, chlorogenic, neochlorogenic and 4-p-coumaroylquinic acid were detected. Chlorogenic and neochlorogenic acids produced [M − H]⁻ at (m/z 353) and MS² fragments at (m/z 191 and 179) as described by Sanoner et al. [33] and Wojdyło et al. [36]. The compound with [M − H]⁻ of (m/z 337), which yielded ions at (m/z 173, 191 and 163), was identified as 4-p-coumaroylquinic acid as reported by Wojdyło et al. [36].

3.3. Distribution of Phenolic Compounds in Different Tissues

The composition of individual phenolics differed among ‘Baya Marisa’ tissues, and therefore, the results are presented separately for fruit (peel and pulp) and other samples (bark of young shoots and leaves). The content of phenolic groups varied in the following order: pulp (hydroxycinnamic acids > dihydrochalcones = anthocyanins = flavanols > flavonols), peel (flavanols > anthocyanins > dihydrochalcones > flavonols > hydroxycinnamic acids), leaves (flavonols > dihydrochalcones > hydroxycinnamic acids > anthocyanins) and bark (dihydrochalcones > flavonols > flavanols > anthocyanins > hydroxycinnamic acids (Figure 3).

The greatest phenolic diversity was detected in the bark (24), followed by leaves (17), peel (16) and finally, pulp (10) (

Table 3. Individual and total phenolic compounds in three pulp sections and peel of ‘Baya Marisa’ apples (mean ± SE, mg/kg FW).

Phenolic Group	Compound	P1	P2	P3	Peel
hydroxycinnamic acids	chlorogenic acid	160 ± 1 a *	189 ± 8 b	169 ± 4 ab	ND
	p-coumaric acid derivative	0.82 ± 0.04 a	0.87 ± 0.1 a	1 ± 0.1 a	ND
	p-coumaric acid derivative	1.4 ± 0.03 a	1.3 ± 0.1 a	1.1 ± 0.1 a	ND
	p-coumaric acid hexoside derivative	ND	ND	ND	12 ± 1
	4-p-coumaroylquinic acid	1.5 ± 0.1 a	1.7 ± 0.1 a	1.8 ± 0.1 a	ND
flavanols	epicatechin isozyme	17 ± 0.6 b	12 ± 0.4 a	10 ± 1.3 a	ND
	Epicatechin	29 ± 2 a	16 ± 1 a	6± 0.4 a	309 ± 10 b
	epicatechin derivative	ND	ND	ND	24 ± 2
	procyanidin dimer	ND	ND	ND	320 ± 8
	procyanidin trimer	ND	ND	ND	147 ± 6
flavonols	quercetin-3-O-arabinofuranoside	ND	ND	ND	40 ± 3
	quercetin-3-O-glucoside	ND	ND	ND	31 ± 3
	quercetin-3-O-rhamnoside	1.4 ± 0.03 a	0.9 ± 0.03 a	0.5 ± 0.02 a	34 ± 2 b
	quercetin-3-O-arabinopyranoside	ND	ND	ND	9 ± 1
	quercetin-3-O-galactoside	ND	ND	ND	82 ± 9
	quercetin-3-O-rutinoside	ND	ND	ND	23 ± 2
	quercetin-3-O-xyloside	ND	ND	ND	26 ± 3
anthocyanins	cyanidin-3-O-arabinoside	11 ± 0.4 a	3.2 ± 0.5 a	2.2 ± 0.2 a	74 ± 7 b
	cyanidin-3-O-galactoside	64 ± 0.7 a	23 ± 3 a	8 ± 0.7 a	492 ± 45 b
dihydrochalcones	phloretin	ND	ND	ND	1.3 ± 0.09
	phloretin glucoside	9 ± 0.2 a	10 ± 0.3 a	20 ± 2 b	ND
	phloridzin	11 ± 0.2 a	14 ± 1 a	15 ± 0.6 a	197 ± 20 b
	total dihydrochalcones	21 ± 0.8 a	28 ± 2 a	46 ± 3 a	339 ± 17 b
	total hydroxycinnamic acids	164 ± 0.7 b	182 ± 9 b	172 ± 4 b	11 ± 0.1 a
	total flavanols	41 ± 2 a	28 ± 2 a	15 ± 0.7 a	640 ± 11 b
	total flavonols	2.7 ± 0.1 a	1.8 ± 0.1 a	1.7 ± 0.1 a	337 ± 19 b
	total anthocyanins	82 ± 1 a	31 ± 2 a	10 ± 0.7 a	608 ± 60 b
	TAPC	302 ± 3 a	271 ± 3 a	245 ± 3 a	1934 ± 43 b

* Data represent mean ± standard error. Means followed by different letters within the row are significantly different (p < 0.05); ND not detected. P1, pulp adjacent to the apple peel; P2 middle pulp section; P3 pulp adjacent to the fruit core.

and

Table 4. Individual and total phenolic compounds in leaves and bark of ‘Baya Marisa’ cultivar (mean ± SE, mg/kg FW).

Phenolic Group	Compound	Leaves	Bark
hydroxycinnamic acids	chlorogenic acid	67 ± 6	ND *
	p-coumaric acid derivative	1076 ± 108	50 ± 5
	p-coumaric acid derivative	20.2 ± 0.9	ND
	p-coumaric acid hexoside	ND	36 ± 3
	p-coumaric acid derivative	ND	50± 5
	neochlorogenic acid	178.84 ± 11.91	ND
	4-p-coumaroylquinic acid	ND	108 ± 9
flavanols	Epicatechin	ND	320 ± 12
	procyanidin dimer	ND	572 ± 27
	procyanidin trimer	ND	196 ± 14
flavanones	eriodictyol hexoside	ND	226 ± 18
flavonols	kaempferol derivative	ND	1292 ± 53
	kaempferol-3-O-rhamnoside	119.31 ± 7.10	65 ± 2
	myricetin hexoside	ND	245 ± 15
	myricetin pentoside	ND	261 ± 15
	quercetin-3-O-arabinofuranoside	841 ± 79	930 ± 163
	quercetin-3-O-glucoside	1289 ± 90	234 ± 16
	quercetin-3-O-rhamnoside	2310 ± 212	978 ± 16
	quercetin-3-O-arabinopyranoside	ND	499 ± 17
	quercetin-3-O-galactoside	2238 ± 149	1130 ± 240
	quercetin-3-O-rutinoside	1075 ± 122	ND
	quercetin-3-O-xyloside	175 ± 17	449 ± 24
anthocyanins	cyanidin-3-O-arabinoside	2.7 ± 0.3	34 ± 3
	cyanidin-3-O-galactoside	19 ± 3	344 ± 22
	peonidin-3-O-galactoside	0.64 ± 0.12	24 ± 2
dihydrochalcones	phloretin	181 ± 15	179 ± 17
	phloretin derivative	389 ± 18	76 ± 13
	phloretin derivative	63 ± 1	ND
	phloretin diglucoside	ND	240 ± 25
	phloridzin	7468 ± 88	8318 ± 61
	total dihydrochalcones	8039 ± 97	9031 ± 129
	total hydroxycinnamic acids	1421 ± 47	220 ± 6
	total flavanols	ND	959 ± 7
	total flavonols	8345 ± 285	6278 ± 192
	total anthocyanins	20.1 ± 0.6	339 ± 26
	TAPC	17,825 ± 178	16,828 ± 154

* Data represent mean ± standard error; ND not detected.

). Phenolic compounds act as antioxidants and contribute to the natural defense against various pests and diseases [37]. In this context, the distribution of phenolic compounds was expected as other studies also reported different compositions of various apple tissues [36,38]. For instance, Mikulic Petkovsek et al. [39] compared the diversity and levels of phenolics in apple leaves and fruit of integrated and organic production. They reported that the latter accumulated higher levels of hydroxycinnamic acids, flavanols, dihydrochalcones, quercetins and total phenolics. Many of these compounds have been linked to stress caused by environmental factors (high insulation) and infection with apple scab.

Phloridzin was the major dihydrochalcone present in pulp, peel, leaves, and bark, and other dihydrochalcones were mostly present in leaves and bark, which correlates with the study of Pontais et al. [40], El-Hawary et al. [41] and Adamcová et al. [42]. The latter conducted research on 13 apple cultivars and concluded that bark, buds and leaves accumulate high levels of these compounds as they play a major physiological role in plants’ defense mechanisms. Few other species belonging mainly to the Rosaceae and Ericaceae families contain phloridzin in much lower amounts compared to Malus × domestica [43]. Gosch et al. [44] and Ehrenkranz et al. [45] described phloridzin as the main polyphenol in apple leaves, which accumulate exceptionally high levels of this compound. Liaudanskas et al. [46] measured approximately 110 mg phloridzin per g of leaf tissue which is higher than the levels measured in ‘Baya Marisa’ leaves. Phloridzin has been proposed to be involved in pathogen resistance, particularly to apple scabs, as it inhibits the growth of Venturia inaequalis in vitro [44]. The fungitoxic effect of phloridzin has been studied in leaves of resistant cultivars, which contained higher amounts of phloridzin compared to susceptible cultivars [39,47]. Moreover, Slatnar et al. [48] reported that differences in phenolic compounds between scab-susceptible and resistant apple cultivars are especially evident in the content of flavanols, phloridzin and hydroxycinnamic acids accumulated in the scab spot and areas around the infection. It can be speculated that high levels of phloridzin in ‘Baya Marisa’ leaves contribute to moderate resistance of this cultivar to apple scab as Mikulic-Petkovsek et al. [49] reported that scab resistant cultivars content of phloridzin in leaves range from 100 to 180 mg/g DW and in scab susceptible cultivars from 100 to 40 mg/g DW, but further studies are needed to confirm this. The levels of dihydrochalcones (mainly phloridzin) differed among pulp sections, and significantly higher levels were detected in the ‘Baya Marisa’ apple peel (Table 3). Surprisingly, when comparing pulp sections, the highest levels of phloridzin were measured in the P3 section (closest to the core). Its accumulation may be ascribed to intense metabolite transport around vascular cells, which are mainly located in the P3 section. This has been proposed in a study by Buer et al. [50] and Gaucher et al. [51] and further confirmed in research by Cebulj et al. [17], who also detected higher levels of dihydrochalcones in the inner part of ‘Granny Smith’ and ‘Jonagold’ apples. Studies report high variability in phloridzin content in apple fruit of different cultivars ranging from 60 mg/kg to 113 mg/kg [46]. Gosch et al. [43] determined that higher content of phloridzin in apple peel and pulp lowers oxidative stress. The content of phloridzin in the ‘Baya Marisa’ peel was approx. 196.63 mg/kg FW (Table 3). In comparison, the ‘Golden Delicious’ cultivar, which is considered highly susceptible to scab [52], contained only 56 mg/kg of phloridzin in its peel [53]. High levels of phloridzin in ‘Baya Marisa’ apples make them a potent source of dihydrochalcones, which possess a wide spectrum of biological effects [46]. For example, a direct anti-hyperglycaemic effect of phlorizin has been measured in a study by Makarova et al. [54], who subjected healthy volunteers to phloridzin-enriched powder from unripe apples. The results indicated inhibition of the re-uptake of glucose in the kidneys and lower plasma glucose concentration compared to control.

Two anthocyanins (cyanidin-3-O-galactoside > cyanidin-3-O-arabinoside) were determined in all ‘Baya Marisa’ parts analyzed, and the content of total anthocyanins was significantly highest in the peel. These natural pigments have been associated with the typical red coloration of plant tissues [53,55,56], and therefore, high levels in the peel are expected. Other studies [57,58] on red apple cultivars have reported similar compositions and content of anthocyanins in their peel. The most distinctive trait of the ‘Baya Marisa’ apple is the red color of its pulp. Visual perception of apple tissues has been linked to the content of anthocyanins in many studies [53,58]. Correspondingly, a decreased content of total anthocyanins from the outer pulp tissue (P1 contained 13.5% of total anthocyanins measured in the peel) towards the fruit core (P3 contained 1.6% of total anthocyanins measured in the peel) has been determined in our study (Table 3) which is expressed in the fading color of the pulp. Interestingly, the relative content of individual anthocyanins differed among pulp sections (Figure 4), with cyanidin-3-O-galactoside as the prevalent red pigment. The levels of anthocyanins in P1 were comparable to the levels reported in other red-fleshed apple cultivars such as ‘Weirouge’ [59], which is one of the parents in the ‘Baya Marissa’ lineage. Anthocyanins contribute to protection against UVB rays as cyanidins have the ability to successfully prevent the UVB-induced apoptosis of plant cells [60].

The greatest diversity of flavonols was determined in ‘Baya Marisa’ bark (10 compounds), followed by leaves and peel (7 compounds each), and finally, pulp (1 compound). Adamcová et al. [42] similarly concluded that bark and leaves were abundant in flavanols (rutin and quercitrin), which is potentially linked to the protective role of these compounds. Quercetin-3-O-rhamnoside was the only flavonol detected in all investigated tissues. Quercetin-3-O-rhamnoside and quercetin-3-O-galactoside dominated in the flavonol group, with the highest contents measured in leaves. Flavonols have a role in plant protection against UV rays, and quercetins specifically act as reactive oxygen species (ROS) quenchers [61]. Therefore, the accumulation of these compounds is common in the green tissues of many plants [62] and is linked to their resistance. Correspondingly, Juhart et al. [53] reported that the scab-susceptible cultivar ‘Golden Delicious’ contained significantly less total quercetins in peel and pulp compared to ‘Baya Marisa’ tissues. The content of quercetin-3-O-rhamnoside in ‘Baya Marisa’ pulp gradually decreased from P1 (4.1% of quercetin-3-O-rhamnoside measured in the peel) to P3 (1.6% of quercetin-3-O-rhamnoside measured in the peel) but was significantly lower to those measured in the peel. Interestingly, common apple cultivars such as ‘Elstar’, ‘Fuji’ and ‘Idared’ do not accumulate flavonols in their pulp [38]. ‘Baya Marisa’ apples can therefore be promoted as a diet source rich in flavonols.

The content of flavanols was much lower compared to that of flavonols. The levels of procyanidin dimer and epicatechin were highest in bark and peel. Contrary, no flavanols were detected in leaves, and the level of these compounds was very low in ‘Baya Marisa’ pulp in which a decrease from P1 (6.4% of total flavanols measured in the peel) to P3 sections (2.4% of total flavanols measured in the peel) was detected. Epicatechin level in the peel was approximately 182 mg/kg FW which is lower than the content reported for ‘Granny Smith’ (246–312 mg/kg FW) and ‘Gala’ (438 mg/kg FW) [37]. The effects of the environment, cultivar and climatic conditions significantly affect the accumulation of flavanols, as reported by Bizjak Bat et al. [63].

In the group of hydroxycinnamic acids, four different compounds were identified in the leaves, bark, and pulp of the ‘Baya Marissa’ cultivar and only one compound in its peel (Table 3 and Table 4). p-Coumaric acid derivative 1 and chlorogenic acid were the major hydroxycinnamic acid determined in ‘Baya Marisa’ leaves (1076 mg/kg FW), which accumulated the highest levels of hydroxycinnamic acids of all tissues analyzed. Similarly, Adamcová et al. [42] detected chlorogenic acid in the leaves of 13 apple cultivars but not in their bark. The most abundant hydroxycinnamic acid in pulp was also chlorogenic acid, with the highest levels detected in the P2 section. This compound has a protective role against diseases in plants [64]. In humans, it decreases blood glucose levels and may reduce cardiovascular risk status [65]. Chlorogenic acid was not detected in ‘Baya Marisa’ peel, although it was previously determined in the peel of ‘Idared’, ‘Elstar’, ‘Fuji’, ‘Gala’, ‘Golden Delicious’, ‘Granny Smith’, ‘Jonagold’, ‘Pinova’, ‘Red Delicious’ and other cultivars [38]. The level of total hydroxycinnamic acids was significantly lowest in the ‘Baya Marisa’ peel, which only contained 5.8% of levels detected in the P2 section.

The TPC values differed significantly among analyzed tissues and were measured in the following order: leaves (17,825 mg/kg), bark (16,827), peel (1934) and pulp (from 245 to 310). Wang et al. [7] reported an equivalent content of total analyzed phenolics in the peel and pulp of some red-fleshed apple cultivars. Correspondingly, Sadilova et al. [59] measured 1684 mg/kg TPC in peel and 379 mg/kg in the pulp of the red-fleshed ‘Weirouge’ cultivar. The highest TPC levels were detected in the P1 section (Table 3) and decreased towards the apple core. Adyanthaya et al. [66] reported that consumption of apples rich in phenolic compounds can reduce the risk of type 2 diabetes by the modulation of post-meal glucose increase and via inhibition of ά-glucosidase. Based on our findings, ‘Baya Marisa’ apples can be promoted as a good food source, rich in phenolic compounds, that can improve our health status.

4. Conclusions

‘Baya Marisa’ apples are abundant in phenolic compounds, organic acids, and sugars in all segments of the fruit, which characterize this cultivar as unique in taste and appearance. It can represent an interesting novelty cultivar with attractive fruit containing above-average levels of specific phenolic compounds, particularly anthocyanins and dihydrochalcones. The most distinctive feature of ‘Baya Marisa’ apples is the accumulation of anthocyanins in all plant tissues analyzed. They are most abundant in peel and bark and, contrary to other common apple cultivars, are also present in all segments of the apple pulp. Differences in anthocyanin levels can be observed along the equatorial fruit plane: pulp adjacent to the skin contains the highest levels of total anthocyanins, and pulp nearest to the core is less rich in red pigments. The distribution of specific groups of metabolites in different depths of apple fruit has been reported for the first time in any red-fleshed cultivar and is of special interest to the processing industry.

Further studies on phenolic/color persistence in processed products are currently in progress to endorse this cultivar and its utilization for juice, preserves, dried apple slices and other goods.