*Article* **Assessment of Morphological Traits, Nutritional and Nutraceutical Composition in Fruits of 18 Apricot cv. Sekerpare Clones**

**Neva Karatas <sup>1</sup> , Sezai Ercisli 2,\* and Mehmet Ramazan Bozhuyuk <sup>3</sup>**


**Abstract:** Apricot (*Prunus armeniaca* L.) is one of the most important members of *Prunus* and its trees bears delicious and nutritious fruits during summer months in the temperate zones in the world. Apricot cultivars are propagated asexually which consists of clones. Information on interclonal variations in apricot cultivars can assist us in the selection of better clones from commercial cultivars. We aimed to determine morphological traits (fruit weight, seed weight, kernel weight, flesh/seed ratio, shape index, fruit firmness, color index), nutritional (sugars and organic acids) and nutraceutical (total phenolic, total flavonoids, total carotenoid and antioxidant activity) composition of 18 clones of Sekerpare apricot cultivar grown together in Kagizman district in eastern Turkey. Results showed significant differences among clones concerning most of the morphological traits, nutritional and nutraceutical compositions. Fruit weight, flesh/seed ratio and fruit firmness of clones were in range of 23.14–27.11 g, 11.21–13.14 and 3.88–5.11 kg/cm<sup>2</sup> , respectively. Fruit shape index was slightly similar among all clones which was between 0.95 and 1.03. Citric acid and sucrose were found to be the predominant organic acid and sugar among clones which varied from 728 to 915 mg/100 g and 7.11 to 9.94 g/100 g, respectively. The clone 'KS2' exhibited the highest level of total phenol (67.1 mg gallic acid equivalent per 100 g) and antioxidant activity (2.16 µmol trolox equivalent per g). The study confirmed the diversity among Sekerpare clones and effectiveness of combining morphological, nutritional and nutraceutical analyses in assessment of ¸Sekerpare clones and its use for future pre-breeding programs.

**Keywords:** apricot; Sekerpare; nutraceuticals

#### **1. Introduction**

Due to suitable climate and soil conditions, Turkey is among the most important fruit producer countries in the world in terms of both the number of fruit species and the amount of production. Turkey ranks first in the world's apricot, fig, hazelnut, sweet cherry and quince production [1]. Nine apricot species and subspecies are known in the world. Among these species, *Prunus armeniaca* L. is cultivated in main apricot growing countries and spreads over the widest geographical area in the world [2,3]. The origin of apricot, which has been cultivated since ancient times, covers a wide area from Turkistan to Western China. Although apricots are grown geographically almost everywhere in the world, commercial production mostly occurs in countries of southern Europe, north America and north Africa [4].

Apricot can be consumed fresh, dried and canned throughout the year. The fruits of apricots are important for human nutrition, being rich in sugars, organic acids, fiber, vitamin A, vitamin E and potassium [5–9].

**Citation:** Karatas, N.; Ercisli, S.; Bozhuyuk, M.R. Assessment of Morphological Traits, Nutritional and Nutraceutical Composition in Fruits of 18 Apricot cv. Sekerpare Clones. *Sustainability* **2021**, *13*, 11385. https:// doi.org/10.3390/su132011385

Academic Editor: Boris Duralija

Received: 5 August 2021 Accepted: 12 October 2021 Published: 15 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

According to the data of the Food and Agriculture Organization (FAO) based on the year 2018, the amount of apricot production increased 19.5% compared to the previous year, while the area shows a decrease of 2% in apricot growing countries. When the data for the last five years (2014–2018) are examined, apricot production increased from 3.3 million tons to 3.8 million tons worldwide, but the production area stayed stable [1]. Turkey is leading world apricot production with a yearly average 750 thousand tons production. The country shares 20% of the world apricot production and is followed by Uzbekistan (13%), Iran (9%), Algeria (6%) and Italy (6%) [1].

In Turkey, apricot trees are grown mainly in the Aegean region, the Mediterranean region, and in particular the Central and Eastern Anatolia regions. Within the regions, Malatya, Elazig, Erzincan, Kahramanmaras, Kars, Mersin and I ˘gdır provinces are well known for commercial apricot cultivation and significant portions of the apricots are dried traditionally in these areas. Except for drying, apricots are generally used in the fruit juice industry in Turkey as well [10,11]. In recent years, depending on the technological developments, apricot fruits are frozen and become widespread in the market outside of the production period [12].

Each apricot growing region in Turkey has their own apricot cultivars and interregional cultivar transfer generally results in negative results. This is because apricots show low environmental adaptability, and the introduction of foreign germplasm may also result in fluctuating or limited yield. This is associated with differences in fertilization, chilling requirements, late-frost resistance, cold-hardiness, etc. [13]. In Turkey, the cultivar–region relationship is very strong as well in apricots. However, the ¸Sekerpare cultivar can be grown in every region and shows great environmental plasticity. The cultivars are mostly grown in Malatya, Erzincan provinces and Aras valley in Turkey and show variable fruit weight ranging from 20 to 40 g [2,4,10,14]. The cultivar called Shakarpara in Pakistan and India and Shekarpareh in Iran shows great phenotypic variability as well. Phenotypic variation within Sekerpare grown in similar ecological conditions arises from an accumulation of somatic mutations due to vegetative propagation during centuries in different Sekerpare growing countries [2,15,16]. The concept of sustainable apricot production can be described as a "three-legged stool", with legs of economic viability, environmental soundness, and social acceptability. Communicating the health benefits of apricot fruit to consumers is an essential ingredient in sustaining apricot product demand, which is a prerequisite for sustainable apricot production. Thus, the cultivar Sekerpare grown in different parts of the world could be adding value for economic viability, environmental soundness, and social acceptability.

Sekerpare is found in most of the apricot growing regions in Turkey and still retains importance and provides interesting economic results in local markets, remaining a popular option for most of the apricot growing regions. This locally adapted cultivar is appreciated for its superior flavor and suitability for both fresh consumption and as a dried product [2].

The Aras valley (Kars-Igdir region) is one of the important apricot growing areas of Turkey. Kagizman district provides almost all of the apricot production in Kars province. In the district, Aprikoz, Sekerpare and wild apricots are grown [17–19]. There are numerous clones of Sekerpare available in the Kagizman district that exhibit differences in key horticultural traits.

Identifying plant varieties is an age-old human endeavor. Historically, morphological traits and later nutritional and nutraceutical characteristics were used to categorize specimens into families, genera, species, cultivars, genotypes, landraces (for perennials: a plant selected from seedlings and asexually re-propagated for its desired characteristics). Thus, varietal characterization based on morphological, nutritional and nutraceutical traits is an important component of fruit tree improvement and breeding [20,21].

Apricot has gained great value in human consumption and commercial importance in recent years, attracting researchers to study its morphological, nutritional and in particular nutraceutical traits.

Advances in fruit species improvement programmes is only possible when intense and more defined genetic variability exists. The phonological expression of any fruit tree species is mostly governed by two factors viz. heredity and environment. Given the fact that environmental variations can be reduced by growing the identical genotypes under uniform site and climatic conditions, studying genetic parameters is of immense use to obtain superior genotypes of any species.

The present study intended to capture variability across morphological, nutritional and nutraceutical parameters of 18 clones of Sekerpare apricot from a particular similar environmental condition.

#### **2. Materials and Methods**

#### *2.1. Plant Samples*

Twenty fruits were harvested from different parts of trees of 18 Sekerpare clones grown together in Kagizman district during July in 2018. Kagizman is located at 40. 1406◦ N and 43.1191◦ E and 1406 m above mean sea-level. All trees of the 18 Sekerpare clones were found at nearly the same altitude in Kagizman district. All examined trees were pre-selected clones according to higher yield, pest and disease free status and more attractive bigger fruit characteristics. Special attention was given on harvest and fruits were harvested in the same period with the same degree of maturity. A total of 80 fruits per clone were collected and then sorted and cleaned. Mature and healthy fruits were transported to the laboratory and divided into two equal parts for morphological measurements and nutritional and nutraceutical analysis.

#### *2.2. Morphological Parameters*

A total 40 fruits per clone were used for morphological measurements which included fruit weight, seed weight, kernel weight, flesh/seed ratio, shape index and fruit color coordinates (L, a and b values). Fruit weight (g) was measured with a digital scale sensitive to 0.01 g (Scaltec SPB31). Fruit firmness was determined with a non-destructive Acoustic Firmness Sensor (Aweta B.V., Pijnacker, The Netherlands) expressed as kg/cm<sup>2</sup> . Fruit shape index (SI) was calculated with the following equation [22].

$$\text{SI}\frac{\text{W} + \text{T}}{\text{2L}} \quad \text{where } \text{W}: \text{Width, T}: \text{Thickness and L}: \text{Length} \tag{1}$$

Color coordinates (*L*\*, *a*\*, and *b*\*) of fruit skin were determined by a Konica Minolta, CR-400 Plus fruit colorimeter (Konica Minolta, Inc., Chiyoda City, Tokyo, Japan) at four different positions around the equator of the fruits [23].

#### *2.3. Nutritional and Nutraceutical Composition*

#### 2.3.1. Sample Preparation and Extraction

A representative of 40 fruits/clone were randomly selected from the harvested lot at commercial maturity stage. The fruits were then introduced to a High-Speed Pulp Ejection Juicer (Omega Products International, Corona, CA, USA), allowing the separation of pomace and juice. The juice was stored at −80 ◦C until use for nutritional and nutraceutical content. During the analysis, the frozen fruits were taken and thawed to 24–25 ◦C. A laboratory blender was used to homogenize the fruit samples (100 g lots of fruits per clone) and a single extraction procedure (taking 3 g aliquots transferred inside tubes and extracted for 1 h with 20 mL buffer including acetone, water (deionized), and acetic acid (70:29.5:0.5 *v*/*v*)) was used [24].

#### 2.3.2. Organic Acids

Organic acid composition in fruits of Sekerpare apricot clones was determined by Bevilacqua and Califano [25]. Fruit extracts were obtained by crushing the fruits in cheesecloth. Then, 0.009 N H2SO<sup>4</sup> was added and shaken for 1 h. The mixture was then centrifuged at 15,000 rpm for 15 min and the supernatants were filtered twice through a

0.45 µm membrane filter with a coarse filter (Millipore Millex-HV Hydrophilic PVDF, Millipore, MA, USA) and passed through a SEP-PAK C18 cartridge. Organic acid readings were performed by HPLC using the Aminex column (HPX-87 H, 300 × 7.8 mm, Bio-Rad Laboratories, Richmond, CA, USA) at 214 and 280 nm wavelengths in the Agilent package program (Agilent Technologies, Santa Clara, CA, USA). Results are expressed as mg/100 g.

#### 2.3.3. Determination of Individual Sugars

For individual sugar (fructose, glucose, and saccharose) analyses, the method of Melgarejo et al. [26] was used. First, homogenized fruits (5 g) were diluted with purified water and homogenate was centrifuged at 6000 rpm for 5 min. Supernatants were filtered through a 0.45 µm membrane filter (Iwaki Glass, Jawa Barat, Indonesia) before the analysis. The HPLC analysis was conducted using a PerkinElmer HPLC system with Amino NH<sup>2</sup> column, and 85% acetonitrile/15% H2O (*v*/*v*) as a mobile phase. Refractive index detector (RID) was used. Samples were identified and quantified by standards. Results were expressed as g/100 g fw. To specify the sweetness perception of 40 fruit per clone, their sweetness indices (SI) were calculated according to Roussos et al. [27]. The SI index considers the relative sweetness as a factor of each of the three sugars measured. It is described in the following equation (1): where *Glu* stands for glucose concentration, *Fru* for fructose concentration, and *Sacch* stands for saccharose concentration.

$$\text{SI} = 1.00 \cdot Glu + 2.3 \cdot Fru + 1.35 \cdot Scalh \tag{2}$$

#### 2.3.4. Total Phenol Content

The total phenolic content (TPC) of the samples was evaluated using the Folin– Ciocalteu method according to Singleton and Rossi [24]. In this procedure, each extract (1 mL) was mixed with Folin–Ciocalteu's reagent and water 1:1:20 (*v*/*v*). The samples were incubated for 8 min. Then, sodium carbonate (10 mL) with a concentration of 7% (*w*/*v*) was added. After incubation for 2 h, the absorbance at 750 nm was measured. The total phenolic content was calculated against the reference standard calibration curve of gallic acid. The TPC was expressed as mg of gallic acid equivalents (GAE) per 100 g of fresh sample.

#### 2.3.5. Total Carotenoid Content

The total carotenoid content was determined by Lichtenthaler [28]. For total carotenoid content, 1 g of fruit sample was homogenized with 5 mL of acetone in a cold porcelain mortar in an ice bath. Then, 1 g of anhydrous sodium sulfate (Na2·SO4) was added to the homogenate, which was elutriated using a paper filter. The filtered solution was made up to 10 mL with acetone and centrifuged at 2600× *g* for 10 min. The upper phase was collected and the absorbance of the solution at 662, 645 and 470 nm was measured. Acetone was used as control. Total carotenoid content is expressed as mg per 100 g fresh fruit sample.

#### 2.3.6. Antioxidant Capacity

The TEAC value of each sample was detected according to the method described by Rice-Evans et al. [29]. The 7 mM ABTS reagent solutions were prepared and diluted with sodium acetate (C2H3NaO2) until 0.700 ± 0.01 spectrophotometrical absorbance level at 734 nm. Following this, 2.97 mL buffered solution was mixed with 30 µL fruit extract and kept in dark at room temperature for 10 min and their absorbance levels were measured at 734 nm using a spectrophotometer. Obtained results were calculated according to TEAC standard calibration curve and expressed as µmol of trolox equivalent/g fresh fruit weight (µmol TE/g FW).

#### *2.4. Statistical Analysis*

All data were analyzed using SPSS software and procedures. Analysis of variance tables were constructed using the least significant difference (LSD) method at *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Morphological Traits*

As presented in Table 1, statistically significant differences (*p* < 0.05) were recorded in fruits of among 18 clones of cv. Sekerpare for most of the morphological traits. Fruit weight and skin color are the most important and distinct external fruit traits in apricots for consumer acceptance. Skin color is a practical and simple indicator with which to instruct harvesters on what to harvest. In addition, consumers in general prefer attractive medium-sized apricot fruits. The flesh/seed ratio is also an important fruit characteristic for apricots [2].

**Table 1.** Fruit weight, seed weight, kernel weight, flesh/seed ratio, shape index and fruit firmness of 18 Sekerpare clones.


Different letters in the same column indicate significant differences (*p* < 0.05) among clones.

Fruit weight, seed weight and kernel weight of Sekerpare clones differed from each other statistically (*p* < 0.05) which ranged from 23.02 to 27.11, 1.80 to 2.09 and 0.68 to 0.84 g, respectively (Table 1). The variation in fruit and seed weight of the clones was also reflected in the fruit flesh/seed ratio which were in the range of 10.46–13.14.

Shape index and fruit firmness of clones were found between 0.96–1.03 and 3.88–5.11 kg/cm<sup>2</sup> (Table 1). KS3 and KS9 were notable among clones due to relatively high fruit weight and flesh/seed ratio.

Studies on apricots in different parts of Turkey identified variations in apricot fruit weight [18,30–32]. Turkish national apricot cultivars have relatively small fruit size and previous studies indicated this fact. For example, Akin et al. [33] studied main apricot cultivars grown in Malatya and determined fruit weight between 21.16 and 38.24 g. Asma and Ozturk [31] reported that 128 Turkish apricot cultivars that belong to the Iran-Caucasian ecogeographical group generally had low fruit weight (lower than 50 g). The authors reported that the fruit weight of only seven apricot cultivars was over 50 g, and the rest of cultivars had lower fruit weight. Karaat and Serce [10] reported fruit weight as 25.12 g in Cagataybey cultivar and 25.65 g in Sekerpare cultivar in Malatya which supports our findings. They also found seed weight to be 1.97 g, flesh/seed ratio as 12.02 and fruit firmness as 2.58 kg/cm<sup>2</sup> in cv. Sekerpare. Akca and Asma [34] conducted a clonal selection study on cv. Kabasi that aimed to find better Kabaasi clones with promising horticultural characteristics. They determined 13 promising clones among 450 Kabaasi trees. Fruit weight, seed weight, kernel weight and flesh/seed ratio were found between 31.81 and

60.91 g, 2.35–3.01 g, 0.52–0.98 g and 12.38–16.64, respectively, indicating similarities with our study. Previously, the flesh/seed ratios of the foreign apricot cultivars grown in Turkey varied between 8.9 and 21.8 [35,36]. In the literature, the shape index of apricots was reported between 0.91 and 1.09 [37,38]. In apricots, if fruit shape index values are found around 1, fruit tend to have a round shape. The Sekerpare cultivar in general gave round shaped fruits while if these values are higher than 1, fruits correspond to ovoid shape.

*L\**, *a\** and *b\** color coordinates of clones are given in Table 2 and it was found that the *L\**, *a\** and *b\** values of the Sekerpare clones significantly differed from each other at *p* < 0.05 (Table 2).


**Table 2.** Fruit skin color parameters of 18 Sekerpare clones.

Different letters in the same column indicate significant differences (*p* < 0.05) among clones.

Color is of primary importance for consumers in the judgment of different fruit groups and accepted as one of the important quality attributes. Lightness (*L\**), red/greenness (*a\**), and yellow/blueness (*b\**) values of the 18 clones of cv. Sekerpare are shown in Table 2. The highest *L\** values were observed in clones KS14 as 66.32 and followed by KS10 (65.41) while KS18 had the lowest *L\** values (58.00) compared to the other clones. The lightness (*L\**) was dependent on exposure to sun [39]. Higher *a\** and *b\** values were observed in KS12 (17.12) and 44.13 (KS3). The chromaticity coordinate *a\** is the most important factor of maturity appearance describing color of the fruit. The intensity of red color normally indicates full maturity and ripeness [40]. Karaat [41] indicated *L\**, *a\** and *b\** values in Sekerpare fruit as 64.17, 14.07 and 42.27, respectively, which is in agreement with our results. Karatas and Sengul [4] reported *L\**, *a\** and *b\** values as 48.66, 19.41 and 19.72 in Sekerpare fruits which indicated differences with our study. In India, *L\**, *a\** and *b\** values of cv. Shakarpara (Sekerpare) were 71.51, 1.03 and 40.56, respectively [16]. These results also reveal that Sekerpare is probably the name of a group of apricots because quite diverse results were obtained from different countries even in the same countries and also strong clonal variation is evident because different clones of the cultivar show significant variation in color values as well.

Among 18 clones, seven had yellow ground color, five clones had dark yellow ground color, four clones had light orange ground color and two clones had light yellow ground color (Table 2).

The majority of clones had red blushed skin and eight clones lacked red blushed skin development (Table 2). The majority of apricot (*Prunus armeniaca* L.) cultivars display orange or yellow background skin, whereas some cultivars are particularly preferred by consumers because of their red blushed skin on the background. Anthocyanins are

responsible for the blushed skin of apricots and the PaMYB10 gene was found as a positive regulator of anthocyanin biosynthesis in apricots and demonstrates that blush formation depends on light [42]. Apricots with a blush on orange or yellow skin are becoming more and more popular in the market due to their colorful appearance and excellent nutritional value [43].

### *3.2. Nutritional Traits*

#### 3.2.1. Organic Acids

The results on organic acid content in fruits of 18 clones of cv. Sekerpare apricots are reported in Table 3. The order of organic acid depending on their content was in descending order citric > malic > ascorbic > tartaric for all clones. Citric acid was the predominant organic acid for all studied clones that ranged from 728 to 915 mg/100 g. Malic acid and tartaric acid content of 18 Sekerpare clones were in range of 261–452 and 2.8–5.7 mg/100 g, respectively (Table 3). Ascorbic acid was identified in all clones from 13.9 mg to 18.6 mg/100 g and this indicates that apricot fruits contain a moderate level of ascorbic acid among fruit species. Organic acid results also indicated that citric, malic, tartaric and ascorbic acid concentrations are greatly varied among clones (*p* < 0.05). Organic acids are of increasing interest because of their role in plant physiology as cofactors, buffering agents, etc. [44]. The organic acid content and profile of fruit species differs depending on species, cultivars, accessions, etc. Alajil et al. [16] showed that citric acid comprised approximately 55% of the organic acids in apricot fruits and ranged from 550 to 1170 mg/100 g, followed by malic acid, which comprised approximately 25% of the organic acids and ranged from 400 to 1430 mg/100 g. Some organic acids have an antioxidant role (tartaric, malic and citric acids). Fruit acids that allow nutrient digestion and stimulate blood circulation are considered among the quality parameters of apricot fruits. Numerous studies have quantified and detailed the organic acid content of apricot fruits and there have been differences between them due to the species, location, used methods, sampling periods, etc. [16,44–52]. Fan et al. [49] showed that malic acid was mainly responsible for sourness of apricots, although malic acid was not the prominent organic acid in all apricot cultivars. It has also been reported that malic acid has an apparent acidic taste compared to citric acid or other organic acids in fruit [53].


**Table 3.** Organic acids in fruits of 18 Sekerpare clones (mg/100 g).

Different letters in the same column indicate significant differences (*p* < 0.05) among clones.

#### 3.2.2. Individual Sugars and Sweetness Indices

Sugar content in different Sekerpare clones is shown in Table 4. The dominant sugar was sucrose in 18 clones that ranged from 7.11 to 9.94 mg/100 g, followed by glucose in the range of 2.03–3.31 g/100 g, respectively. Fructose content of fruits was relatively lower and found between 0.78 and 1.05 g/100 g (Table 4). Overall, the highest sucrose, glucose and fructose contents were found in KS17, KS1, KS3 and KS4 clones, respectively. Sweetness index (SI) was obtained between 13.35 (KS13) and 18.46 (KS17). Alajil et al. [16] used a number of apricots including Sekerpare in nutritional analysis and reported that sucrose was the dominant sugar, accounting for more than 63% of total sugars and ranged from 4.15 to 10.13 g/100 g, glucose contributed about 22% of total sugars and ranged from 2.28 to 4.31 g/100 g, and fructose contributed about 15% of total sugars and ranged from 1.22 to 4.19 g/100 g which shows parallel values with our study. Saridas and Agcam [44] examined individual sugars and organic acids in Agerik and Teberze apricot cultivars and reported that both contents change greatly according to cultivars. They reported sucrose, glucose and fructose content between 5.33 and 8.57, 1.90 and 2.95 and 0.60 and 0.88 g/100 g, respectively. Furthermore, the composition of individual sugars in the current study agrees with that documented by Akin et al. [33] for different Turkish apricot cultivars. ˙Imrak et al. [45] found that the dominant sugar in apricot fruits was sucrose. Karata¸s and Sengul [4] reported sucrose content between 1.83 and 3.97 g/100 g in main apricot cultivars sampled in Malatya province in Turkey. Su et al. [46] used local apricots in sugar analysis and found that sucrose was the main sugar. Individual sugars differ in sweetness, with fructose perceived as sweeter than sucrose and sucrose perceived as sweeter than glucose [27]. The sweetness is important to apricot consumers and breeders, and it also leads to market acceptance of the fruit [47].


**Table 4.** Individual sugars (g/100 g) and sweetness indices (SI) in fruits of 18 Sekerpare clones.

Different letters in the same column indicate significant differences (*p* < 0.05) among clones.

The sweetness index (SI) in fruits of Sekerpare clones ranged from 13.35 to 18.46 (Table 3). Previously, the sweetness index (SI) ranged from 13.58 to 22.30 in apricot fruits grown in India and Shakarpara reported a SI of about 13.58 [16], indicating lower values than our study. In Greece, SIs were found between 8.16 and 11.25 among apricot cultivars [27]. Our findings are consistent with those published SIs for Spanish apricot genotypes ranging from 8.5 to 15.9 [48]. Despite the fact that SI determines taste, the final perception of fruit sweetness is influenced by the presence of other compounds such as phenolics and other aroma compounds [49].

#### *3.3. Nutraceutical Traits*

Total Phenolic Content, Total Flavonoids, Total Carotenoids and Antioxidant Activity

Table 5 shows total phenolic, total flavonoid, total carotenoid content and antioxidant activity in fruits of 18 clones of cv. Sekerpare. We found statistically significant differences among clones in terms all nutraceutical traits at 0.05 level (Table 5).

**Table 5.** Nutraceuticals in fruits of 18 Sekerpare clones (fresh weight basis).


Different letters in the same column indicate significant differences (*p* < 0.05) among clones.

As mentioned in Table 5, total phenolic content was found between 49.5 and 67.1 mg GAE/100 g fresh weight basis. Saeed et al. [54] used fruits of eight apricot cultivars sampled from Pakistan and reported total phenolic content between 50 and 220 mg GAE/100 g FW indicating higher values than our results. Gecer et al. [18] used a number of wild apricots and cv. Aprikoz and found total phenolic content between 34.2 and 52.8 mg GAE/100 g which is in accordance with our study. In Hungary, a large number of apricot cultivars were used in nutraceutical analysis and total phenolic content greatly varied among cultivars from 12.0 to 89.0 mg GAE/100 g [7]. In Turkey, Karaat and Serce [10] used main apricot cultivars in Malatya and reported total phenolic content between 35.1 and 90.7 mg GAE/100 g. In the Mediterranean region in Turkey, apricots show total phenolic content between 14.4 and 177.1 mg GAE/100 g, with a mean value of 64.4 mg GAE/100 g indicating similarities with our samples [55]. Alajil et al. [16] reported total phenolic content among apricots, ranging from 25.31 (Shakarpara) to 89.95 mg GAE/100 g (Roxana). Wani et al. [56] and Leccese et al. [57] previously reported similar findings in apricots grown in India and Italy, respectively.

Total flavonoids were in range of 9.2–14.1 mg CE/100 g (Table 5). Saeed et al. [54] reported total flavonoids between 48 and 382 mg QE/100 g on fresh weight basis in apricots indicating higher values than our results. Alajil et al. [16] found that total flavonoid amounts in apricot genotypes ranged from 5.00 to 15.46 mg CE/100 g which indicated good agreement with our study. Our results are also consistent with those reported by Carbone et al. [58], who reported total flavonoid content (TFC) ranging from 1.9 to 12.0 mg CE/100 g for different apricot genotypes. Kafkaletou et al. [59] and Wani et al. [56] found TFC values ranging from 16.87 to 41.42 and 12.2 to 36.2 mg/100 g in apricot genotypes grown in Greece and India, respectively. Phenolics and flavonoids are essential measures of nutraceutical quality and have been linked to the treatment of a variety of chronic diseases, including cancer, cardiovascular disease, and neurodegeneration [60–64].

Total carotenoid content of 18 apricot clones of cv. Sekerpare were found between 7.72 and 10.13 mg/100. Gecer et al. [18] reported total carotenoid content ranged from 1.1 to 12.5 mg/100 g of edible portion in wild apricots and cv. Aprikoz. Ruiz et al. [65] found total carotenoid content between 1.5 and 16.5 mg/100 g among a large number of apricot cultivars in Spain. Shemesh et al. [66] found total carotenoid content between 0.5 and 9.5 mg/100 g among 113 apricot cultivars in Israel. These studies are in harmony with our results. The content and composition of carotenoids in apricots determine their fruit color. Apricots are high in carotenoids, which influence the color and visual appearance of the fruit; the color of the fruit can vary from yellow to orange depending on the carotenoids content [67]. Carotenoids are also essential dietary sources of vitamin A.

The antioxidant activity determined by TEAC assay in fruits of the 18 Sekerpare clones were evaluated and the results are presented in Table 5. Amongst the 18 clones of Sekerpare cultivar, the KS15 clone showed the lowest antioxidant activity (1.88 µmol TE/g), whereas the KS2 clone showed the highest antioxidant activity (2.16 µmol TE/g) (Table 5). The KS6 clone showed the second highest antioxidant activity (2.12 µmol TE/g) and the clones KS7 and KS12 showed the third highest antioxidant activity (2.09 µmol TE/g). In Italy, among the apricot cultivars analyzed, the variability of the antioxidant capacity was obtained showing a range from 1.36 to 4.55 µmol TE/g. They found that the latest cultivars had two-fold higher TEAC values with respect to the earliest ones [68].

Previous studies conducted in different horticultural plants indicated that horticultural crops rich for antioxidant components and antioxidant activity were found to be cultivar/genotype/clone dependent [69–80].

#### **4. Conclusions**

A detailed morphological, nutritional and nutraceutical traits analysis was reported here for the first time in a large number of clones of cv. Sekerpare. The results indicated that even in a small single growing location, Sekerpare clones showed rich diversity on most of the morphological traits and nutritional and nutraceutical compositions. The KS3 clone showed the highest fruit weight as 27.11 g. KS2, KS7 and KS12 had the highest antioxidant activity. The promising clones could be used as breeding material. The results could have practical implications for orchard management to select better Sekerpare clones and bring them into production.

**Author Contributions:** Conceptualization, N.K., S.E. and M.R.B.; data curation, N.K., S.E. and M.R.B.; formal analysis, N.K., S.E. and M.R.B.; methodology, N.K., S.E. and M.R.B.; visualization, S.E.; writing—original draft, N.K., S.E. and M.R.B.; writing—review and editing, N.K., S.E. and M.R.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** No external funding for this research.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All new research data were presented in this contribution.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

#### **References**


### *Article* **Determination of Selected Beneficial Substances in Peach Fruits**

**Martina Mrázová 1 , Eliška Rampáˇcková 1 , Petr Šnurkoviˇc <sup>2</sup> , Ivo Ondrášek <sup>1</sup> , Tomáš Neˇcas 1,\* and Sezai Ercisli <sup>3</sup>**


**Abstract:** Peaches (*Prunus persica* L.) are a popular and sought-after dessert fruit. This is mainly due to their flavour, aroma, attractive appearance, and high content of substances that play an important role in human nutrition. The present study was carried out to determine some important analytical properties (sugars/sucrose, glucose, fructose and sorbitol), total acid, total phenolics, flavonoids, antioxidant capacity, carotenoids and anthocyanins of 34 selected peach varieties. The analyses are also complemented by colorimetric measurements of peach skin colour using CIELAB and other chromatic parameters. The results show, for example, that all peach varieties are good sources of phenolic compounds (9.43–577 mg gallic acid equivalent (GAE).100 g−<sup>1</sup> ), flavonoids (1.12–95.1 mg catechin equivalent (CAE).100 g−<sup>1</sup> ), and antioxidant capacity (136–462 mg Trolox equivalent (TE).100 g−<sup>1</sup> ).

**Keywords:** *Prunus persica* L.; colour; chemical contents; antioxidant capacity; sugar

#### **1. Introduction**

There are a great variety of peach trees (*Prunus persica* L.), not only in terms of the length of ripening period, but also in terms of the pomological characteristics of the fruit, where we can distinguish yellow-fleshed, white-fleshed, red-fleshed, fully separable from the stone or clings, flat-shaped varieties, referred to as Peento, that are very popular in southern Italy and Asia. There are also well-known selections of varieties without any anthocyanin content, originating in Italy (the 'ice peach'), and the Californian 'Royal' series of varieties, which are characterised by their very hard flesh and very low acid content, giving the fruit a sweet taste.

From a nutritional point of view, peaches contain a number of beneficial substances, making them an interesting addition to the human diet. Peaches are a rich source of dietary fibre (1.5 g.100 g−<sup>1</sup> ) and provitamin A [1]. This fruit is considerably rich in antioxidants and is an important source of vitamins A, B, and C, carotenoids and phenolic compounds. Among the most important phenolic acids are chlorogenic and neochlorogenic acids, catechin, epicatechin, 3-glucoside of cyanidin (chrysanthemin), and quercetin derivatives [2–5]. Polyphenols represent the majority of antioxidants present in the diet and their daily intake should exceed 1 g/day, which is much higher than that of all other classes of phytochemicals and known dietary antioxidants [6]. They are low in fat and contain a lot of water, approximately 89 g per 100 g of fruit [7,8]. Peaches are very low in sugars (9–20 ◦Rf), with the main sugars present being sucrose, fructose, sorbitol, and glucose. The proportions of these sugars undergo changes during fruit ripening, with glucose and fructose being present in greater amounts in immature fruit and increasing as ripening progresses. At full maturity, sucrose content dominates [9–11]. Carbohydrates are an important source

**Citation:** Mrázová, M.; Rampáˇcková, E.; Šnurkoviˇc, P.; Ondrášek, I.; Neˇcas, T.; Ercisli, S. Determination of Selected Beneficial Substances in Peach Fruits. *Sustainability* **2021**, *13*, 14028. https://doi.org/10.3390/ su132414028

Academic Editor: Rajeev Bhat

Received: 3 November 2021 Accepted: 13 December 2021 Published: 20 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of energy in the human diet and also play an important role in the regulation of the gut microbiota [12]. They also have low levels of organic acids (0.13–1.16%) such as malic, citric, and folic acids. The content of L-ascorbic acid (vitamin C) in peaches is relatively low compared to other fruits such as kiwifruit or oranges, in which it is the most important antioxidant. Quinic, fumaric, and shikimic acids are present in smaller concentrations [13,14]. Amino acids (arginine, asparagine, isoleucine, lysine, serine, threonine, valine, leucine, phenylalanine, tryptophan, tyrosine, proline, and alanine) also contribute to the flavour of fruit and are found in peaches in different concentrations depending on the cultivar [15,16]. Among the mineral elements, they contain nitrogen, phosphorus, potassium, calcium, magnesium, iron, manganese, zinc, cooper, chromium, nickel, cobalt, lead, selenium, and fluoride [17,18]. Similar to apricots, the glycoside amygdalin (26%), protein amandine (3.8%), enzymes, lactase, and oleic acids are present in peach kernels. The leaves contain about 1% prunasin and are used against rheumatism, gastritis, headaches, and as a diuretic; when used externally, they are effective against eczema, ulcers, and other dermatoses [19].

The potential of peaches, especially those rich in phenolics, lies in delaying or even preventing the onset of neurogenerative diseases such as Alzheimer's and Parkinson's. They also help in the prevention of inflammation, atherosclerosis, diabetes, obesity, and cardiovascular disease. Due to their low sugar content, they can easily be included in nutritional therapy. They are easily digestible, have a strong alkaline effect on the body, and stimulate the secretion of digestive juices. They have both a laxative and a diuretic effect. Peach phenolics have been shown to display several biological activities such as antioxidant activity [20,21], anti-allergic and anti-inflammatory activities [22], antibacterial activity [23], hepatoprotective activity [24], nephroprotective activity [25], antiproliferative [26], chemopreventive, and anticancer activities [27,28].

The aim of this study was to compare varieties from different pomological groups as well as different geographical origins and thus get an overview of the differences in content composition from the point of view of titratable acidity, soluble solid content, sugars, phenolic compounds, flavonoids, antioxidant activity, carotenoids, and total anthocyanin content.

#### **2. Materials and Methods**

#### *2.1. Site of Planting and Plant Material*

In total, 34 peach cultivars of different origin were analysed in this study (Table 1). 20 cultivars from USA, 6 from Yalta, 5 from Italy, 1 from Czech Republic, 1 from France, and 1 from Slovakia. Trees of these cultivars were grown in the experimental orchard at the Faculty of Horticulture in Lednice, Mendel University in Brno (localisation 48.80◦N/16.80◦E, at an altitude of 172 m), with an average annual temperature of 9.7 ◦C.

**Table 1.** The cultivars obtained in this study and their flesh colour, fruit type, and origin.


Five fruits from each variety were harvested at their harvest maturity and transported to the laboratory for chemical analyses.

#### *2.2. Determination of Titratable Acidity*

The determination of titratable acidity was performed by potentiometric titration, with a solution of 0.1 mol.L−<sup>1</sup> NaOH of a known factor up to pH 8.1 measured by a combined SenTix™ 81 pH electrode (WTW™, Prague, Czech Republic) coupled with inoLab 7110 pH meter (WTW™, Prague, Czech Republic). Titratable acidity was expressed as % malic acid equivalent [29]. Mixed fruits were used as a sample for titration.

#### *2.3. Preparation of the Plant Samples for Analysis of Total Phenolic Content, Total Flavonoids, and Total Antioxidant Capacity*

Prior to determination of content of secondary metabolites (phenolic compounds, flavonoids, and antioxidant capacity), methanol extract from fresh fruit material was performed. Five grams of the sample was homogenized with a hand blender in 25 mL 75% methanol. The extract was left to stand for 24 h and then filtered through a filter paper into a 50 mL measuring flask. The filtrate was then adjusted to the line with 75% methanol. Samples were transferred into a 20 mL plastic bottles and kept at −20 ◦C until the analysis [30].

#### *2.4. Determination of Total Phenolic Content, Total Flavonoids, and Total Antioxidant Capacity*

Analyses of all parameters were carried out according to the protocols of Zloch et al. (2004) [31] by using a SPECORD® 50 PLUS spectrophotometer (Analytik, Jena, DE). Total phenolic content was measured after reaction of sample extracts with Folin–Ciocalteu reagent at a wavelength of 765 nm and expressed in milligrams GAE per 100 g FW. Total flavonoid content was determined by using chloride and sodium nitrite and the results were expressed in milligrams CAE per 100 g FW. For determination of total antioxidant activity, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method was used. This method is based on the decolorizing property of the hydrogen radical of DPPH with hydrogen donors, which are included in phenolic compounds as well. Trolox (6-hydroxy-2.5.7.8-tetramethylchroman-2 carboxylic acid) was used as a standard, and the measurement was performed at 515 nm and then expressed in milligrams TE per 100 g FW.

#### *2.5. Determination of Total Carotenoids*

Prior to determination of the carotenoid content, sliced thin fruit sections were dried in a heat chamber FED 400 (Binder, Tuttlingen DE) at 50 ◦C for 24 h and pulverised in a mill Pulverisette 11 (Fritsch, Weimar, DE). Next, acetone was used to extract the pigments from the samples. Determination of photosynthetically active pigments (carotenoids) was performed with a SPECORD® 50 PLUS spectrophotometer (Analytic Jena AG, Germany) at 440 nm according to Holm (1954) [32]. Total carotenoids were expressed in milligrams per 100 g dry weight (DW).

#### *2.6. Determination of Total Anthocyanin Content (TAC)*

The determination of TAC was based on a pH differential method using changes in the colour of samples containing anthocyanins in various pH value environments. Five grams of homogenized whole fruit of peach was mixed with 25 mL of 0.1 M HCl. After 1 h of extraction, the solution was filtered and 0.5 mL of the filtrate was pipetted into 6 test tubes. A 2.5 mL (0.025 mol.L−<sup>1</sup> ) of KCl solution of pH 1 was added into the first 3 test tubes and 2.5 mL (0.4 mol.L−<sup>1</sup> ) solution of C2H3NaO<sup>2</sup> of pH 4.5 was added into the remaining 3 test tubes. Prepared rest tubes were measured at wavelengths of 510 nm and 700 nm with a spectrophotometer SPECORD® 50 PLUS (Analytic Jena AG, Germany). The results were expressed in mg.100 g−<sup>1</sup> fresh weight (FW).

#### *2.7. Determination of Sugar Content*

The soluble solids content was determined using the Abbé refractometer and expressed in weight percentage.

The determination of sugar content was performed by high performance liquid chromatography (HPLC). Juice was squeezed from the fruit and diluted with distilled water at a 1:4 ratio (2 mL juice + 8 mL H2O). The diluted sample was filtered through a microfilter and analysed. A Clarity chromatography station (Watrex, Prague, Czech Republic) with a Polymer IEX Ca\_SN8422 column (250 × 8 mm; Watrex, Prague, Czech Republic) was used for making the analysis. The flow rate of the mobile phase (deionized water) was 0.5 mL.min−<sup>1</sup> , pressure 1.9 MPa, temperature 80 ◦C. A refractometric detector was used for making the evaluation. Fructose, glucose, sucrose, and sorbitol contents were converted into the fresh weight of plant material and expressed as g sugar per 100 g fruit.

#### *2.8. Colour Analysis*

Colour of cleaned skin of 5 fruits was analysed using colorimeter CR-400 (Konica Minolta®, Tokyo, Japan), equipped with D65 illuminant. The over colour and ground colour were distinguished where possible within the analysis. The data were processed by software SpectraMagic NX Lite (Konica Minolta®, Tokyo, Japan). The analysis is based on CIELAB scale. The colour parameters *L\**, *a\**, *b\** are directly measured in terms of standard observed and standard illuminant [33], where parameter *L\** represents the lightness of the fruit, parameter *a\** represents the axis in the direction from green to red and parameter *b\** represents the axis in the direction from blue to yellow. Values were displayed with the mean ± standard deviation. Cylindrical coordinates *C\*ab* and *h* ◦ *ab* were calculated from coordinates *a\** and *b\** by Equations (1) and (2) [34]:

$$\mathbf{C}^\*\_{ab} = (a^{\*2} + b^{\*2})^{1/2} \tag{1}$$

$$h^{\diamond}\_{\;\;\;ab} = \tan^{-1} \left( b^\* / a^\* \right) \tag{2}$$

*C\*ab* denotes the purity of saturation of the colour [35], which means the higher is the chroma (*C\*ab*) the colour is more intense. Hue angle (*h* ◦ *ab*) refers to the colour wheel and is measured in angles [36]. The colour difference ∆*E\*ab* was accomplished for cultivars with measurable ground and over colour. Values were displayed with the mean ± standard deviation of ten replications. Given two colours in the CIELAB colour space, (*L\**1, *a\**1, *b\**1) and (*L\**2, *a\**2, *b\**2), the CIE76 colour difference formula is defined as (3):

$$
\Delta E^\*\_{ab} = (\Delta L^{\*2} + \Delta a^{\*2} + \Delta b^{\*2})^{1/2} \tag{3}
$$

∆*E\*ab* ≈ 2.3 corresponds to a JND (just noticeable difference) [37].

#### *2.9. Statistical Analysis*

Statistical analysis was performed in Statistica 12 (TIBCO, USA) and Microsoft Excel software. Single-factor ANOVA analysis (level of significance α = 0.05) was used for statistical processing and the Tukey HSD test was subsequently used to evaluate the statistical significance of differences between the individually measured values (TAC and chromatic parameters *L\**, *a\**, *b\**). Between colour parameters and TAC, the Spearman's correlation coefficient *ρ* was determined using Statistica 12 (TIBCO, USA) and regression function with coefficient of determination R<sup>2</sup> were determined using Microsoft Excel.

#### **3. Results**

The highest acid content was recorded in the fruit of the varieties 'Benedicte' (1.32% malic acid), 'Helene' (0.91% malic acid), and 'Royal Majestic' (0.85% malic acid). The varieties with the lowest acid content were 'UFO 3' (0.25% malic acid), 'Fidelia' (0.26% malic acid) and 'Royal Glory' (0.26% malic acid, Figure 1). The average value of the test set

highly significant (Table 2).

was 0.59% malic acid. The differences between the varieties were confirmed as statistically

Sustainability 2021, 13, x FOR PEER REVIEW 6 of 18

Table 2. The total content of titratable acids in peach cultivars. The data are displayed as the mean ± standard deviation of three replications; a–p refer to the grouping based on the Tukey HSD test.

Admiral de Wey 0.44 ± 0.01 b,c Iris Rosso 0.676 ± 0.009 j,k,l,m

Aurelia 0.643 ± 0.004 i,j Narjadnyj Nikitskij 0.513 ± 0.002 <sup>d</sup>

Figure 1. The photos of some cultivars obtained in this study. **Figure 1.** The photos of some cultivars obtained in this study.

#### Cultivars Titratable Acidity [%] Cultivars Titratable Acidity [%]


**Table 2.** The total content of titratable acids in peach cultivars. The data are displayed as the mean ± standard deviation of three replications; a–p refer to the grouping based on the Tukey HSD test.

Significantly, the highest representation of total phenolic compounds was found in fruits of the variety 'Carolina Belle' (577.72 mg GAE.100 g−<sup>1</sup> FW), then in the variety 'Krasava' (334.02 mg GAE.100 g−<sup>1</sup> FW, Figure 1), 'Dixigem' (285.24 mg GAE.100 g−<sup>1</sup> FW, Figure 1), and in the variety 'Benedicte' (238.09 mg GAE.100 g−<sup>1</sup> FW). On the other hand, the lowest values of phenolic compounds content were observed in fruits of 'Favorita Morettini' (9.43 mg GAE.100 g−<sup>1</sup> FW), 'Early Redhaven' (12.90 mg GAE.100 g−<sup>1</sup> FW), and 'Strelec' (17.39 mg GAE.100 g−<sup>1</sup> FW). In the studied set of cultivars, the total phenolic content in fruits ranged from 9.43 to 577 mg GAE.100 g−<sup>1</sup> FW. The differences between the values were highly statistically significant (Table 3).

**Table 3.** Total phenolic content in peach cultivars. The data are displayed as the mean ± standard deviation of three replications; a–w refer to the grouping based on the Tukey HSD test.


The highest concentration of flavonoids was measured in the fruits of 'Carolina Belle' (95.1 mg CAE.100 g−<sup>1</sup> FW), 'Benedicte' (53.2 mg CAE.100 g−<sup>1</sup> FW), and 'Admiral de Wey' (50.8 mg CAE.100 g−<sup>1</sup> FW). The lowest values were observed in 'UFO 3', 'Favorita Morettini', 'Alexandra' and 'Candor' (1.12; 3.37; 4.09 and 5.16 mg CAE.100 g−<sup>1</sup> FW). The average flavonoid value in the test set was 22.3 mg CAE.100 g−<sup>1</sup> FW. The differences between the varieties were confirmed as statistically highly significant (Table 4).

**Cultivars Flavonoids [mg CAE.100 g**−**<sup>1</sup> ] Cultivars Flavonoids [mg CAE.100 g**−**<sup>1</sup> ]** Admiral de Wey 50.8 ± 0.3 <sup>t</sup> Iris Rosso 12 ± 1 h,i Alexandra 4.1 ± 0.5 b,c Krasava 45.1 ± 0.5 <sup>s</sup> Anita 27.7 ± 0.2 <sup>n</sup> Lakomyj 10.6± 0.1 g,h Aurelia 18.0 ± 0.2 <sup>k</sup> Narjadnyj Nikitskij 31.8 ± 0.1 <sup>p</sup> Avalon Pride 24.2 ± 0.4 <sup>m</sup> Nerine 15.0 ± 0.1 <sup>j</sup> Benedicte 53.2 ± 0.5 <sup>u</sup> Otliˇcnik 9.60 ± 0.09 f,g Candor 5.16 ± 0.06 <sup>c</sup> Queen Lady 20.1 ± 0.1 <sup>l</sup> Carolina Belle 95.1 ± 0.8 <sup>v</sup> Red Robin 34.1 ± 0.2 <sup>q</sup> Dixigem 35.6 ± 0.2 <sup>r</sup> Redhaven 24.5 ± 0.2 <sup>m</sup> Dostojnyj 12.7 ± 0.1 <sup>i</sup> Romea 8.2 ± 0.1 d,e Early Glo 27.4 ± 0.8 <sup>n</sup> Royal Glory 9.5 ± 0.7 e,f,g Early Redhaven 8.68 ± 0.09 d,e,f Royal Majestic 10.0 ± 0.7 <sup>g</sup> Favorita Morettini 3.4 ± 0.3 <sup>b</sup> Sonet 14.8 ± 0.2 <sup>j</sup> Fénix 7.55 ± 0.08 <sup>d</sup> Strelec 11.8 ± 0.2 h,i Fidelia 10.0 ± 0.3 <sup>g</sup> Suncrest 46.1 ± 0.3 <sup>s</sup> Harvester 23.3 ± 0.1 <sup>m</sup> Sunshine 30.20 ± 0.09 <sup>o</sup> Helene 18.4 ± 0.4 <sup>k</sup> UFO 3 1.12 ± 0.02 <sup>a</sup>

**Table 4.** Total flavonoid content in peach cultivars. The data are displayed as the mean ± standard deviation of three replications; a–v refer to the grouping based on the Tukey HSD test.

Using the DPPH (2,2-diphenyl-1-picrylhydrazyl) method, values of antioxidant activity in peach fruits ranging from 136 to 462 mg TE.100 g−<sup>1</sup> FW were determined. Specifically, the cultivar 'Carolina Belle' (249.08 mg TE.100 g−<sup>1</sup> FW) had the highest value. All other varieties analysed showed relatively high values. The results varied within a few units. High values were also found in the fruit of the variety 'Admiral de Wey' (280.46 mg TE.100 g−<sup>1</sup> FW) and in the variety 'Dixigem' (255.61 mg TE.100 g−<sup>1</sup> FW). The Czech variety 'Krasava' also had high antioxidant capacity (250.07 mg TE.100 g−<sup>1</sup> FW). The lowest total antioxidant capacity was measured in the fruits of 'Favorita Morettini' (136.15 mg TE.100 g−<sup>1</sup> FW) and 'Candor' (150.72 mg TE.100 g−<sup>1</sup> FW). The differences in the values were highly statistically significant (Table 5).

**Table 5.** Antioxidant activity in peach cultivars. The data are displayed as the mean ± standard deviation of three replications; a–z refer to the grouping based on Tukey HSD test.



**Table 5.** *Cont.*

The average carotenoids content in the fruits of the studied varieties reached 1.67g.100 g−<sup>1</sup> DW. The varieties with the highest carotenoids (4.77 mg.100 g−<sup>1</sup> DW) include fruits of the variety 'Romea' (3.50 mg.100 g−<sup>1</sup> DW, Figure 1), followed by fruits of the variety 'Royal Majestic' (3.14 mg.100 g−<sup>1</sup> DW), 'Favorita Morettini' (3.12 mg.100 g−<sup>1</sup> DW), and 'Early Redhaven' (3.12 mg.100 g−<sup>1</sup> DW). On the other hand, the lowest total carotenoids content was determined in the fruits of 'Krasava', 'Fidelia', and 'Anita' (0.05; 0.24 and 0.24 mg.100 g−<sup>1</sup> DW). Total carotenoids content was not detected in the cultivars 'Benedicte' and 'Royal Glory'. The differences between the varieties were confirmed as statistically highly significant (Table 6).

**Table 6.** Total carotenoids in peach cultivars. The data are displayed as the mean ± standard deviation of three replications; a–j refer to the grouping based on the Tukey HSD test.


\* Not measured.

High levels of anthocyanins were measured in the fruits of 'Helene' (3.74 mg.100 g−<sup>1</sup> FW), 'Royal Majestic' (2.64 mg.100 g−<sup>1</sup> FW), and 'Favorita Morettini' (2.13 mg.100 g−<sup>1</sup> FW). On the other hand, low values were recorded in fruits of 'Early Redhaven', 'UFO 3', 'Dostojnyj', 'Strelec' and 'Admiral de Wey' (0.05; 0.05 0.14; 0.18 mg.100 g−<sup>1</sup> FW). The average value of total anthocyanins of the tested set of varieties reached 0.70 mg.100 g−<sup>1</sup> FW. The differences between the varieties were confirmed as statistically highly significant (Table 7).


**Table 7.** Total anthocyanin content (TAC) in peach cultivars. The data are displayed as the mean ± standard deviation of three replications; a–p refer to the grouping based on the Tukey HSD test.

\* Not measured.

In the set of varieties studied, the total soluble solids content of the fruit ranged from 8.3 to 14.7 ◦Rf. The varieties with the highest content were 'Royal Majestic' (14.7 ◦Rf), followed by 'Helene' (13.8 ◦Rf) and 'Nerine' (13.7 ◦Rf). The lowest values of the evaluated set of varieties were measured for the fruits of the 'Fénix' variety (8.3 ◦Rf, Figure 1), 'Krasava' and 'Romea', which had the same soluble solids value for both varieties (9.2 ◦Rf). The differences in the values found were highly statistically significant (Table 8).

**Table 8.** Soluble solid content (SSC) in peach cultivars. The data are displayed as the mean ± standard deviation of three replications; a–k refer to the grouping based on Tukey HSD test.


The average sucrose, glucose, fructose, and sorbitol contents of the fruit were determined for each variety. The average sucrose content was 9.62 g.100 g−<sup>1</sup> FW. The highest sucrose content was measured in the varieties 'Narjadnyj Nikitskiy' (16.57 g.100 g−<sup>1</sup> ) and

'Sonet' (16.44 g.100 g−<sup>1</sup> ). The lowest contents were observed in the cultivars 'Alexandra', 'Suncrest'(Figure 1), and 'Iris Rosso' (4.89, 4.69 and 4.66 g.100 g−<sup>1</sup> , respectively). The glucose content ranged from 0.74 to 3.67 g.100 g−<sup>1</sup> . The highest contents were determined in the varieties 'Sunshine' (3.67 g.100 g−<sup>1</sup> ) and 'Admiral de Wey' (3.50 g.100 g−<sup>1</sup> ). The lowest content was measured in the varieties 'UFO 3' (0.82 g.100 g−<sup>1</sup> ) and 'Nerine' (0.74 g.100 g−<sup>1</sup> ). The average value of glucose content was 1.94 g.100 g−<sup>1</sup> . In the studied set of varieties, the total fructose content ranged from 0.48 to 2.39 g.100 g−<sup>1</sup> , with an average value of 1.37 g.100 g−<sup>1</sup> . The highest content was measured in the cultivars 'Sunshine' and 'Dixigem' (2.39 and 2.36 g.100 g−<sup>1</sup> ). The lowest fructose content was observed in the variety 'UFO 3' (0.48 g.100 g−<sup>1</sup> ). The average value of alcoholic sugar sorbitol in our study was 0.23 g.100 g−<sup>1</sup> . The variety 'Benedicte' greatly exceeded all other varieties in sorbitol content, with its content being determined at 1.57 g.100 g−<sup>1</sup> . Very low amounts were measured in the cultivars 'Lakomyj', 'Nerine', 'Iris Rosso', and 'Alexandra' (0.09; 0.09; 0.08 and 0.06 g.100 g−<sup>1</sup> ). The differences in the values found were highly statistically significant (Table 9).

**Table 9.** Sugars in peach cultivars. The data are displayed as the mean ± standard deviation of three replications; a–p refer to the grouping based on the Tukey HSD test.


Colorimetric parameters *L\**, *a\**, *b\** for the basic skin colour of the fruit were measured for all varieties. In the varieties 'Alexandra', 'Anita', 'Helene', 'Iris Rosso', 'Royal Glory'

and 'Royal Majestic', the skin was completely covered by the blush. The average values of *L\**, *a\**, *b\** are summarised in Table 10. The highest values of *L\** were found for the basic colour in the varieties 'Krasava', 'Aurelia', 'Sunshine' and for the cheek in the varieties 'Romea', 'Dostojnyj', 'Carolina Belle'. In our study the highest value of *a\** were found for 'Nerine', 'Admiral de Wey', 'Avalon Pride', the lowest value were found for 'Krasava', 'Otliˇcnik', 'Carolina Belle' and 'Queen Lady'. For chromatic parameter *b\** the highest values were measured for 'Romea', 'Otliˇcnik', 'Lakomyj' and the lowest values were found for 'Fidelia', 'UFO 3' and 'Red Robin'. Colour intensity is represented by the chromatic parameter *C\*ab*, which was determined using the chromatic parameters *a\** and *b\**, and its highest values were found for the basic colour of 'Romea', 'Otliˇcnik', 'Lakomyj' and for the cheek colour of 'Romea', 'Sunshine', 'Admiral de Wey'. From the measured values for base colour and cheek colour, the greatest colour difference ∆*E\*ab* (Table 11) was found for the cultivars 'Otliˇcnik', 'Lakomyj', 'Queen Lady'. These varieties had the richest cheeks when compared to the base colour. On the other hand, the lowest ∆*E\*ab* were found for the varieties 'Red Robin', 'Romea', 'UFO 3', where the cheek almost merged with the base colour. Figure 2 captures the exact colour found in the *L\**, *a\**, *b\** coordinates.

**Table 10.** The average values of individual chromatic parameters for peach skin ground colour and over colour of peach.


**Table 11.** Values of chromatic parameters for ground colour skin and over colour skin of the peach cultivars.



Figure 2. Values of ΔE\*ab of individual peach cultivars. The ground colour is shown in the lower part of the column, and over colour is shown in the upper part according to the measured coordinates L\*, a\*, b\*. **Figure 2.** Values of ∆*E\*ab* of individual peach cultivars. The ground colour is shown in the lower part of the column, and over colour is shown in the upper part according to the measured coordinates *L\**, *a\**, *b\**.

#### Table 11. Values of chromatic parameters for ground colour skin and over colour skin of the peach cultivars. **4. Discussion**

Ground Colour Over Colour <sup>Δ</sup><sup>E</sup>\*ab Cultivar <sup>L</sup>\* a\* b\* C\* h L\* a\* b\* C\* <sup>h</sup> Admiral de Wey 65.5 19.3 39.1 43.6 1.1 35.8 37.6 18.0 41.7 0.45 40.8 Alexandra - - - - - 29.1 21.9 10.2 24.2 0.43 - Anita - - - - - 36.0 33.5 15.5 37.0 0.43 - Aurelia 73.6 1.96 50.8 50.8 1.53 39.6 30.0 19.1 35.6 0.57 54.3 Avalon Pride 67.5 16.8 40.8 44.1 1.18 37.8 33.6 18.2 38.2 0.50 41.0 Benedicte 72.5 0.36 29.8 29.8 1.56 33.1 32.1 16.6 36.1 0.48 52.3 Candor 68.7 10.8 44.3 45.7 1.33 35.3 33.7 17.3 37.9 0.48 48.6 Carolina Belle 71.9 −2.44 32.3 32.4 −1.50 42.5 29.6 18.8 35.1 0.57 45.5 Dixigem 73.2 9.21 47.6 48.5 1.38 37.4 29.3 14.5 32.7 0.46 52.7 The acid content of fruit is a key quality parameter and is an important factor in determining the taste of the fruit. Titratable acidity indicates the concentration of organic acids present in the fruit. Peaches have a very low level of organic acids. The total titratable acid content found in our set of varieties ranged from 0.26 to 1.32% malic acid on fresh weight. These values are similar to the results found in many other publications. Scordino et al. (2012) [38] reported TA contents ranging from 0.52–0.86% malic acid in Sicilian yellow flesh peaches on fresh weight. Similar values were also found in the work by Tomás-Barberán et al. (2010) [39], where the contents ranged from 0.53–0.97% malic acid in yellow flesh peaches on fresh weight, and 0.15–0.34% malic acid in white flesh peaches on fresh weight. Gil et al. (2002) [40] investigated the differences between white- and yellow-fleshed peach cultivars grown in California. The average TAC content found in the white-fleshed varieties was 0.22%, and in the yellow-fleshed varieties it was 0.69%.

Dostojnyj 69.9 0.78 52.4 52.4 1.56 48.4 25.7 32.5 41.4 0.90 38.4 Early Glo 72.6 9.38 45.9 46.9 1.37 39.4 31.8 18.8 37.0 0.53 48.4 Early Redhaven 72.4 8.69 48.9 49.7 1.40 36.8 33.1 19.0 38.2 0.52 52.5 Favorita Morettini 64.1 10.3 43.6 44.8 1.34 39.0 25.3 13.4 28.6 0.49 42.1 Fénix 65.9 9.11 43.2 44.1 1.36 35.5 27.9 17.1 32.7 0.55 44.2 Fidelia 66.7 16.2 28.5 32.8 1.05 33.0 33.0 14.9 36.2 0.42 40.0 In a publication by Cantin et al. (2009) [41], the total phenolic content ranged from 12.7 to 71.3 mg GAE.100 g−<sup>1</sup> FW, with an average of 36.4 mg GAE.100 g−<sup>1</sup> FW. In our selected set of cultivars, the average content reached 122.4 mg GAE.100 g−<sup>1</sup> FW. Marinova et al. (2005) [42] investigated the determination of all phenolic compounds in fruit grown in Bulgaria. The total phenolic content in peach fruits was 50.9 mg GAE.100 g−<sup>1</sup> FW, and similar values were reached by figs—*Ficus carica* (59.0 mg GAE.100 g−<sup>1</sup> FW). Another

Iris Rosso - - - - - 33.2 16.0 7.01 17.5 0.41 - Krasava 74.5 −6.95 33.8 34.5 −1.37 40.1 34.1 19.9 39.5 0.53 55.4 Lakomyj 73.1 0.71 52.7 52.7 1.56 37.7 35.3 20.6 40.9 0.53 58.9 Narjadnyj Nikitskij 62.3 13.1 43.1 45.0 1.28 35.0 29.7 16.9 34.2 0.51 41.4 Nerine 64.9 20.3 45.8 50.1 1.15 37.8 34.8 20.3 40.3 0.53 39.9 Otličnik 71.6 −6.38 53.5 53.9 −1.45 37.5 26.8 16.5 31.4 0.55 60.2 Queen Lady 69.9 −2.00 47.2 47.2 −1.53 34.3 29.3 16.0 33.4 0.50 56.8 Red Robin 59.8 11.4 29.2 31.3 1.20 36.2 30.1 16.2 34.2 0.49 32.7 Redhaven 71.3 9.62 49.3 50.3 1.38 37.1 30.6 18.8 35.9 0.55 50.4 Romea 71.9 13.7 54.5 56.2 1.32 50.4 32.1 33.1 46.1 0.80 35.5 Royal Glory - - - - - 25.6 30.0 10.0 31.6 0.32 -

publication by Saidani et al. (2017) [43] dealt with the determination of phenolic compounds separately in the peel and in the pulp. In the peel, contents ranging from 88.9 to 277 mg GAE.100 g−<sup>1</sup> FW were determined, while in the pulp, contents ranging from 25.1 to 139 mg GAE.100 g−<sup>1</sup> FW were determined. Previously, Zhao et al. (2015) [44] monitored the content of total phenolics in selected Chinese peach cultivars, ranging from 4.58 to 12.68 mg gallic acid equivalent (GAE).100 g−<sup>1</sup> DW in the peel and from 0.82 to 6.52 mg GAE.100 g−<sup>1</sup> DW in the pulp.

The obtained results of total flavonoids content in the tested set of varieties ranged from 1.1 to 95.1 mg CAE.100 g−<sup>1</sup> FW. Di Vaio et al. (2015) [45] determined the total flavonoid content, and it ranged from 35.05-58.85 g CAE.kg−<sup>1</sup> FW within the test set. In another publication by Cantin et al. (2009) [40], total flavonoid content ranged from 1.8 to 30.9 mg CAE.100 g−<sup>1</sup> FW, with an average of 8.8 mg CAE.100 g−<sup>1</sup> FW. Marinova et al. (2005) [42] investigated the determination of all phenolic compounds, as well as flavonoids in crops grown in Bulgaria. The total flavonoid content in peach fruits was 15.0 mg CAE.100 g−<sup>1</sup> FW; similar values are seen in figs—*Ficus carica* (20.2 mg CAE.100 g−<sup>1</sup> FW) and sweet cherries (19.6 mg CAE.100 g−<sup>1</sup> FW). The highest representation of flavonoids was found in this work in blueberries (190.3 mg CAE.100 g−<sup>1</sup> FW). Saidani et al. (2017) [43] determined the flavonoid content in the skin of peach fruits to be between 39 and 245 mg CAE.100 g−<sup>1</sup> FW, and in the flesh between 8.18 and 112 mg CAE.100 g−<sup>1</sup> FW.

Analyses of antioxidant components in products are fast becoming a recognized profile, primarily emphasizing antioxidant capacity as a quality index for many fruits and vegetables. The high phenolic content showed an increased antioxidant capacity in the studied varieties. The average value of antioxidant capacity determined by the DPPH (1-diphenyl-2,2-picrylhydrazyl) method showed values of 205.7 mg TE.100 g−<sup>1</sup> FW. The authors of Di Vaio et al. (2015) [45] determined average antioxidant capacity values of 111.1 mg TE.100 g−<sup>1</sup> FW in four peach cultivars. Saidani et al. (2017) [43], in a tested set of peach cultivars, determined the antioxidant capacity value in the skin of the fruit ranging from 133 to 401 mg TE.100 g−<sup>1</sup> FW, and in the flesh ranging from 22.7 to 194 mg TE.100 g−<sup>1</sup> FW. Zhao et al. (2015) [44] found antioxidant capacity contents in Chinese peach cultivars from 6.35 to 19.84 mg trolox equivalent antioxidant capacity (TE).100 g−<sup>1</sup> DW in the peel and from 1.05 to 15.01 mg TE.100 g−<sup>1</sup> DW in the pulp.

The content of carotenoids (especially β-carotene, zeaxanthin, lutein, neoxanthin) and anthocyanins increases with fruit maturity, largely due to the colouring of the fruit (formation of the cheek). The results obtained for total anthocyanin content in the tested set of cultivars ranged from 0.04 to 3.74 mg.100 g−<sup>1</sup> FW. Cantín et al. (2009) [41] monitored the content of total anthocyanins in selected cultivars and found contents ranging between 0.1 and 26.7 mg of C3GE.kg−<sup>1</sup> FW (0.1-26.7 mg of cyanidin-3-glucoside equiv. (C3GE) per kg of FW). In another publication by Saidani et al. (2017) [43], they discussed the determination of total anthocyanins separately in the peel and in the pulp. The average anthocyanin content in the peel was 5.53 mg C3GE.100 g−<sup>1</sup> FW, while in the pulp the average content was 0.37 mg C3GE.100 g−<sup>1</sup> FW. In other research on total anthocyanin content in apricot fruits, Rababah et al. (2011) [46] reported an average anthocyanin content of 2.54 mg.100 g−<sup>1</sup> FW, whereas Contessa et al. (2013) [47] reported an anthocyanin content of 0.99 mg C3GE.100 g−<sup>1</sup> FW.

The carotenoids content found in the set of cultivars ranged from 0.00 to 4.77 mg.100 g−<sup>1</sup> DW. Gil et al. (2002) [40] observed differences in carotenoids content between white- and yellow-skinned peach cultivars. The average carotenoids content found in white-fleshed cultivars was 11.6 µg.100 g−<sup>1</sup> , while in yellow-fleshed cultivars it was 131.6 µg.100 g−<sup>1</sup> . Vizzotto et al. (2007) [48] also found higher carotenoids content in genotypes with yellow flesh (0.8 to 3.7 milligrams β-carotene per 100 g tissue) than in peaches with white flesh (0.0 to 0.1 milligrams β-carotene per 100 g tissue).

Soluble solid content (SSC) is an important characteristic of fruit, as it is closely related to consumer satisfaction and how well the fruit is liked. Zhao et al. (2015) [44] evaluated the soluble solid content of different Chinese peach cultivars; their findings ranged from 8.34

to 15.48 ◦Rf. These results are similar to ours, with values ranging from 8.30 to 14.70 ◦Rf in our set of cultivars. In another work, Gil et al. (2002) [40] investigated the differences between white- and yellow-fleshed peach cultivars grown in California. The average SSC content found in the white-fleshed varieties was 11.22 ◦Rf, while in the yellow-fleshed varieties it was 11.90 ◦Rf. For the Spanish varieties, Legua et al. (2011) [49] found SSC contents between 9.98 and 18.36 ◦Rf. Tavarini et al. (2008) [50] determined an average SSC value of 12.42 ◦Rf for Italian varieties.

In this study, sucrose, glucose, fructose, and sorbitol were determined as the basic sugars of peaches and there were differences found among the cultivars (Table 10). The mean values of sucrose, glucose, fructose, and sorbitol were 9.62 g.100 g−<sup>1</sup> , 1.94 g.100 g−<sup>1</sup> , 1.37 g.100 g−<sup>1</sup> and 0.23 g.100 g−<sup>1</sup> , respectively. These values are very similar to those determined by Forcada et al. (2014) [9]. The values found ranged from 3.5–9.8 g.100 g−<sup>1</sup> sucrose, 0.4–1.5 g.100 g−<sup>1</sup> glucose, 0.2–1.4 g.100 g−<sup>1</sup> fructose, and 0.2–3.5 g.100 g−<sup>1</sup> sorbitol. Nowicka et al. (2019) [51] investigated the sugar content of 20 peach cultivars. They determined sucrose content ranging from 3.4–5.4 g.100 g−<sup>1</sup> , glucose 0.27–0.84 g.100 g−<sup>1</sup> , fructose 0.41–1.03 g.100 g−<sup>1</sup> , and sorbitol content ranging from 0.15–0.74 g.100 g−<sup>1</sup> . Colaric et al. (2005) [13] determined sucrose levels between 46.14-66.92 g.kg−<sup>1</sup> in some nectarine and peach cultivars. Cantin et al. (2009) [41] determined a similar sucrose content (47.10–64.00 g.kg−<sup>1</sup> ), and further investigated the determination of glucose (5.60–8.00 g.kg−<sup>1</sup> ) and fructose (6.9–10.3 g.kg−<sup>1</sup> ) in peach and nectarine fruits. Gecer (2020) [52] measured sucrose (5216.3–9122.4 mg.100 g−<sup>1</sup> ), glucose (721.7–1902.1 mg.100 g−<sup>1</sup> ), and fructose (325.7–1048.1 mg.100 g−<sup>1</sup> ) in some peach and nectarine cultivars. Robertson et al. (1990) [53] determined the average sorbitol content in yellow-fleshed cultivars, 0.46% and in white-fleshed cultivars, 0.37%. The colour of the fruit is an important parameter that influences the attractiveness of the fruit to consumers. A colorimetric analysis can also provide information on the degree of ripeness of the fruit. The colour of peaches using CIELAB was measured in studies before [54–57]. The value of *a\** has been suggested as colour index maturity [54]. The study was associated with changes of *a\** with chlorophyl degradation and an increase of anthocyanin content. Because of low values of anthocyanin in most cultivars of peaches, Ferrer et al. (2010) [55] found that changes of chromatic parameter *b\** can be a good indicator of ripeness of peach fruit. These changes correlated to an increase of carotenoids pigments. In our study the correlation relationship between carotenoids and chromatic parameter *b\** with a correlation coefficient R = 0.7951 and *C\** with R = 0.8051 were found (Figure 3). The cultivars 'Otliˇcnik' and 'Aurelia' were accomplished as outliers and they are not included in the correlations; this can be attributed to insufficient maturity of these two cultivars. Sustainability 2021, 13, x FOR PEER REVIEW 16 of 18 associated with changes of a\* with chlorophyl degradation and an increase of anthocyanin content. Because of low values of anthocyanin in most cultivars of peaches, Ferrer et al. (2010) [55] found that changes of chromatic parameter b\* can be a good indicator of ripeness of peach fruit. These changes correlated to an increase of carotenoids pigments. In our study the correlation relationship between carotenoids and chromatic parameter b\* with a correlation coefficient R = 0.7951 and C\* with R = 0.8051 were found (Figure 3). The cultivars 'Otličnik' and 'Aurelia' were accomplished as outliers and they are not included in the correlations; this can be attributed to insufficient maturity of these two cultivars.

Figure 3. Relationship between b\*, C\*ab, and total carotenoids content (TCC) in peach cultivars. Blue points represent correlation between b\* and TCC, orange points represent correlation between C\*ab and TCC. **Figure 3.** Relationship between *b\**, *C\*ab*, and total carotenoids content (TCC) in peach cultivars. Blue points represent correlation between *b\** and TCC, orange points represent correlation between *C\*ab* and TCC.

carotenoids, and anthocyanins, and can also provide valuable antioxidants. The Czech 'Krasava' variety was found to be a variety that has a very high content of titratable acids, phenolics, flavonoids, and antioxidant capacity. It can be said that this variety is very interesting from a biochemical point of view and offers a certain potential. Peach consumption represents one of the main fruit incomes during the summer months and is subject to seasonal demand, i.e., the short period of availability in the year. While pome fruits may form the bulk of typical dietary intake during longer periods of the year, peaches are only

Author Contributions: Conceptualization, M.M. and E.R.; methodology, M.M. and P.Š.; formal analysis, M.M. and P.Š.; investigation, M.M.; resources, I.O.; data curation, M.M and E.R.; writing original draft preparation, M.M. and E.R.; writing—review and editing, M.M. and S.E.; supervision, T.N.; funding acquisition, T.N. All authors have read and agreed to the published version of the

Funding: This research received external funding from project by activity no. 6.2.10 ref. 51834/2017- MZE-17253, subprogram "National Program of Conservation and Utilization of Plant Genetic Resources and Agrobiodiversity," which is funded by the Ministry of Agriculture of the Czech Repub-

Acknowledgments: This research used the infrastructure acquired by the project CZ.02.1.01/0.0/0.0/16\_017/0002334 Research Infrastructure for Young Scientists, which is co-fi-

nanced by the Operational Program of Research, Development and Education.

5. Conclusions

a seasonal concern.

Data Availability Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable.

manuscript.

lic.

#### **5. Conclusions**

Peach fruits have an important specific nutritional status among stone fruits. This means that peaches can serve as a source of sugars, mainly sucrose, as well as phenolics, carotenoids, and anthocyanins, and can also provide valuable antioxidants. The Czech 'Krasava' variety was found to be a variety that has a very high content of titratable acids, phenolics, flavonoids, and antioxidant capacity. It can be said that this variety is very interesting from a biochemical point of view and offers a certain potential. Peach consumption represents one of the main fruit incomes during the summer months and is subject to seasonal demand, i.e., the short period of availability in the year. While pome fruits may form the bulk of typical dietary intake during longer periods of the year, peaches are only a seasonal concern.

**Author Contributions:** Conceptualization, M.M. and E.R.; methodology, M.M. and P.Š.; formal analysis, M.M. and P.Š.; investigation, M.M.; resources, I.O.; data curation, M.M and E.R.; writing—original draft preparation, M.M. and E.R.; writing—review and editing, M.M. and S.E.; supervision, T.N.; funding acquisition, T.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received external funding from project by activity no. 6.2.10 ref. 51834/2017- MZE-17253, subprogram "National Program of Conservation and Utilization of Plant Genetic Resources and Agrobiodiversity," which is funded by the Ministry of Agriculture of the Czech Republic.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This research used the infrastructure acquired by the project CZ.02.1.01/0.0/0.0/ 16\_017/0002334 Research Infrastructure for Young Scientists, which is co-financed by the Operational Program of Research, Development and Education.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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