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Article

Microclimate Condition Influence on the Physicochemical Properties and Antioxidant Activity of Pomegranate (Punica granatum L.): A Case Study of the East Adriatic Coast

by
Mira Radunić
1,2,*,
Maja Jukić Špika
2,3,*,
Jelena Gadže
4,
Smiljana Goreta Ban
2,5,
Juan Carlos Díaz-Pérez
6 and
Dan MacLean
7
1
Department of Plant Sciences, Institute for Adriatic Crops and Karst Reclamation, Put Duilova 11, 21000 Split, Croatia
2
Centre of Excellence for Biodiversity and Molecular Plant Breeding, Svetošimunska 25, 10000 Zagreb, Croatia
3
Department of Applied Science, Institute for Adriatic Crops and Karst Reclamation, Put Duilova 11, 21000 Split, Croatia
4
Department for Pomology, Division of Horticulture and Landscape Architecture, Faculty of Agriculture, University of Zagreb, Svetošimunska 25, 10000 Zagreb, Croatia
5
Institute of Agriculture and Tourism Poreč, Karla Huguesa 8, 52440 Poreč, Croatia
6
Department of Horticulture, University of Giorgia, 2360 Rainwater Road, Tifton, GA 31793, USA
7
AgroFresh, 510-530 Walnut Street, Suite 1350, Philadelphia, PA 19106, USA
*
Authors to whom correspondence should be addressed.
Agriculture 2025, 15(21), 2210; https://doi.org/10.3390/agriculture15212210
Submission received: 29 September 2025 / Revised: 20 October 2025 / Accepted: 22 October 2025 / Published: 24 October 2025
(This article belongs to the Special Issue Advanced Cultivation Technologies for Horticultural Crops Production)
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Abstract

The pomegranate cultivar Barski slatki, the most widely cultivated on the Eastern Adriatic coast, was evaluated over one growing season across four growing areas to assess its pomological and chemical properties and antioxidant activity. Results showed that location significantly influenced fruit weight, volume, number of arils per fruit, and both total and individual aril weight, with the Kaštela (CRO) site producing the largest fruits and highest aril yields. Climatic factors, such as precipitation during bud differentiation, flowering, and early fruit development, were found to impact fruit set, aril number, and fruit size. Aril and juice yields, however, remained relatively stable across sites. Notable differences were observed in total soluble solids, titratable acidity, pH, total phenolic content, and anthocyanin profiles. Location with higher rainfall occurring during fruit growth favored enhanced phenolic accumulation. Although total anthocyanin content remained consistent among locations, significant variation occurred in aril coloration and composition of individual anthocyanins. In conclusion, microclimatic factors, particularly rainfall distribution, temperature, and altitude, play a decisive role in shaping the physical, chemical, and visual attributes of ‘Barski slatki’. Despite being cultivated under similar Mediterranean conditions, the observed differences across sites highlight the strong adaptability of this cultivar to diverse agroecological environments, while maintaining stable quality.

1. Introduction

The pomegranate (Punica granatum L.) has been cultivated throughout the Mediterranean region since ancient times, particularly in South Europe and North Africa, as well as in parts of Asia, especially Persia. Countries such as Turkey, Spain, Italy, Greece, and Morocco, which are characterized by a favorable Mediterranean climate and long-standing agricultural traditions, play a central role in global pomegranate production [1]. In recent decades, pomegranate cultivation has expanded to new subtropical and tropical regions in the Americas, South Africa, and Australia, driven by the increasing recognition of the pomegranate as a functional food and a rich source of nutraceuticals, including bioactive compounds such as anthocyanins, phenolic acids, flavonoids, tannins, and vitamins [2].
Pomegranates thrive in climatic conditions characterized by hot, dry summers and mild, rainy winters [3,4]. The Mediterranean region, with its varied topography and microclimates, ranging from coastal plains to inland valleys, offers optimal conditions for cultivating different pomegranate cultivars. Despite their tolerance to low temperatures (as low as −10 °C) and moderate water requirements (rainfall or equivalent irrigation of around 500 mm annually) [4], pomegranate trees are prone to physiological fruit disorders caused by weather and microclimate fluctuations [5]. Optimal fruit development is achieved under a regime of summer drought, followed by winter rains that replenish soil moisture. This climatic combination plays a crucial role in achieving the desired balance of sweetness, acidity, and juiciness, while fruit quality can also be objectively evaluated using modern non-destructive methods [6].
Fruit quality, both internal and external, is determined by a combination of factors, including genotype, growing region, climate, and cultivation practices [7,8]. While genotype plays a fundamental role in determining physicochemical traits of fruit peel and arils (weight and red color), environmental variables such as temperature, micro location, and altitude have a decisive influence on the expression of traits such as fruit size, skin coloration, and biochemical composition [2,9,10]. In particular, temperature has a pronounced influence on the physiological and biochemical development of different fruit species [11]. Although warmer temperatures can enhance sugar metabolism in the fruit, high temperatures are generally associated with a reduction in fruit size and sugar content, as well as changes in skin pigmentation and firmness [12,13]. During key phenological stages, elevated temperatures can alter fruit morphology, sugar accumulation, firmness, and coloration [11]. In addition to temperature, other microclimatic elements such as humidity, UV light intensity, PAR (Photosynthetically Active Radiation), day–night temperature differences, precipitation, and soil properties also exert a cumulative influence, albeit to a lesser extent [14,15].
On the eastern Adriatic coast, pomegranate cultivation remains rooted in tradition and is mostly limited to small orchards and home gardens. Numerous cultivars are successfully grown in these areas, but the predominant local cultivar is ‘Barski slatki’, which is highly valued for its sensory and nutritional properties. Nevertheless, there is a notable gap in the scientific literature regarding the influence of specific environmental parameters on the physicochemical and antioxidant properties of this cultivar. This study aims to fill this gap by investigating how different microclimatic conditions on the eastern Adriatic coast affect the physical, chemical, and antioxidant properties of ‘Barski slatki’ pomegranates. Understanding these microclimatic interactions is essential not only for improving cultivation practices and postharvest handling, but also for advancing varietal selection and increasing the market potential of fruit destined for both fresh consumption and value-added processing.

2. Materials and Methods

2.1. Plant Material

Fruit samples of pomegranate ‘Barski slatki’ were collected from four orchards along the Eastern Adriatic coast (Kaštela and Metković in Croatia; Mostar, Bosnia and Herzegovina, and Bar, Montenegro). The 12-year-old trees grown on their own roots were trained into a shrub with 3–5 main trunks and planted at a spacing of 3 × 4.5 m. The trees were in good physiological condition, exhibiting optimal vigor and productivity. The orchards were drip-irrigated, and other cultivation practices, such as plant protection, pruning, and fertilization, were applied in accordance with accepted commercial practices [16]. The main soil types at the study sites were as follows: Kaštela—anthrosol developed from flysch, karst synclines, and colluvial deposition; Metković—alluvial soil; Mostar—eutric cambisol; and Bar—eutric brown soil. Flowering at the study sites occurred as follows: Kaštela—12 May; Metković—14 May; Mostar—10 May; and Bar—17 May. Fruits were harvested at the time of harvest maturity (6–10 October), which was determined according to standard local practices, when the fruit skin began to show superficial cracking. At each site, fruits were collected from three trees and pooled to form one composite sample representing a single biological replicate. Three such composite samples were obtained per site. From each tree, three fruits of uniform size were randomly collected around the canopy.

2.2. Climatic Condition

The sampling locations (Figure 1) are situated in the Mediterranean climate, which is defined as a Csa climate type according to the Köppen–Geiger climate classification [17].
The climatic conditions shown in Table 1 are as follows:
  • Kaštela is situated on the coastal part of Croatia (CRO), Middle Dalmatia (43°33′05″ N, 16°20′23″ E, 9 altitude). The main climatic characteristics were an average annual temperature of 16.4 °C, an absolute minimum of −3.7 °C (December), and an absolute maximum of 37.2 °C (July). Annual rainfall in this location was 1428 mm, but during April to October, it was 712 mm (50% of the total). Average sunny hours totaled 2700.
  • Metković is situated in the lower part of the Neretva river valley, South Dalmatia, Croatia (CRO) (43°00′29″ N, 17°38′47″ E, 5 m altitude). The main climatic characteristics were a mean daily temperature of 15.6 °C, an absolute minimum of −6.4 °C (December), and an absolute maximum of 36.5 °C (August). Annual rainfall in this location was 1555.4 mm, with 646 mm (42% of total) recorded from April to October. Average sunny hours totaled 2700.
  • Mostar is situated in the upper part of the Neretva Valley, in the southern part of Bosnia and Herzegovina (BIH) (43°25′13″ N, 17°52′26″ E, 100 m altitude). Mostar has a temperate Mediterranean climate with milder but colder winters (characterized by little to no snow) and hot summers. Average annual temperature was 15.7 °C, with an absolute minimum of −7.8 °C (December) and an absolute maximum of 38.9 °C (July). Annual rainfall was 1848.9 mm, with 741.6 mm (40% of total) recorded from April to September. Annual sunny hours totaled 2285.
  • Bar is situated on the southeast coastal part of Montenegro (MNE) (42°02′22″ N, 19°09′09″ E, 143 m altitude). The climate of Bar is determined by the proximity of the Adriatic Sea and Lake Skadar and the mountain massif of Rumia. The average annual temperature of 16.9 °C characterizes it, with an absolute minimum of 0 °C (February) and an absolute maximum of 35.4 °C (August). Annual rainfall was 1826.4 mm, with 641.8 mm (35% of total) recorded during April to October. Annual sunny hours totaled 2555.

2.3. Physical Properties

The weight of the fruit (FW; g) was measured using a digital balance (Mettler Toledo AB 204, Greifensee, Switzerland) with a sensitivity of ±0.01 g. Fruit volume was determined using the liquid displacement method. The measurements of fruit length without calyx (FL; mm), equatorial fruit diameter (FD; mm), calyx length (CL; mm), calyx diameter (CD; mm), and peel thickness (PT; mm) were carried out using digital micrometers (Powerfix Profi, Neckarsüm, Germany). For fruit diameter and calyx diameter, two perpendicular measurements were taken around the horizontal (equatorial) plane, while peel thickness was measured on two opposite sides of the fruit. Fruit shape was determined by calculating the ratio of fruit length to fruit diameter. The calyx index (CI, %) was defined as the ratio of the calyx length to the total length of the fruit. Arils were manually separated from the pericarp and membrane, and the total aril weight (TAW) per fruit was recorded. The total number of arils per fruit (NoA/F) was estimated by counting the number of arils in a 100 g sample and extrapolating this number based on the total aril weight per fruit. Aril yield (AY; %) was calculated as the ratio of aril to fruit weight multiplied by 100. Juice was extracted from a 50 g aril sample by pressing it through four layers of cheesecloth. The juice yield (JY; %) was determined by calculating the volume of juice extracted (in milliliters) per 50 g sample and expressing the result as a percentage.
Pomegranate aril color was measured using a Croma meter CR-400 (Konica Minolta, Osaka, Japan). Color was estimated according to the CIE L*a*b* method and expressed as L*, a*, b*, C, and hue angle (h°) values. Thirty arils were randomly taken from each location for the color measurements. L* indicates the lightness and ranges from 0 to 100. Values a* and b* define red-greenness and blue-yellowness, respectively. Croma (C) and hue angle denote the visual color appearances. The C value defines color intensity. The hue angle represents the visual experience according to which the color is evaluated, with the following values: 0°–90° = red-purple; 90°–180° = yellow; 180°–270° = bluish-green; and 270°–360° = blue.

2.4. Chemical Analysis

2.4.1. Total Soluble Solids, Titratable Acidity, TSS/TA Ratio, pH, and Total Sugar Content

The measurements were performed on fresh aril juice extracted with cheesecloth as previously described. Each replicate included three pomegranate fruits. The total soluble solids (TSS) content was determined using a digital refractometer (Mettler Toledo, Columbus, OH, USA), with results expressed as percentage TSS at 20 °C. Titratable acidity (TA) was determined using the AOAC method [18] and expressed as a percentage of citric acid equivalents. The TSS/TA ratio was then calculated based on these values. The pH of the juice was measured at 20 °C using a Mettler Toledo MP 230 digital pH meter. The total sugar content (TSC) was quantified using the Luff–Schoorl method [18] and expressed in g/100 g.

2.4.2. Total Phenolic Content, Anthocyanin Content, and Antioxidant Activity

Extraction: Approximately 30 g of pomegranate arils was frozen at −80 °C prior to extraction. The frozen material was then freeze-dried using a FreeZone 2.5 L benchtop freeze dryer (LabConco, Kansas City, MO, USA). The lyophilized arils were then ground to a coarse powder under liquid nitrogen using a pre-cooled mortar and pestle. For extraction, 0.5 g of the powdered sample was mixed with 5 mL of an extraction solvent consisting of methanol, water, and formic acid (60:37:3, v/v/v), and extracted for 1 h. The mixture was homogenized for 10 min and then centrifuged at 4000 rpm for 10 min. To increase the efficiency of the extraction, the procedure was repeated under the same conditions, and the resulting supernatants were pooled. The combined extracts were concentrated to a volume of less than 1 mL using a rotary vacuum evaporator (Jouan RC10.22, Thermo Fisher Scientific, Waltham, MA, USA). The concentrate was further centrifuged in a microcentrifuge at 14,000 rpm for 15 min, and the extract was transferred to a vial. The extract was brought to a final volume of 1 mL, capped, and then stored at −20 °C until analysis.
Determination of Total Phenolic Content
Total phenolic content (TPC) was quantified using a modified version of the Folin–Ciocalteu colorimetric assay described by Singleton and Rossi [19]. Briefly, 300 μL of the extract (previously diluted to 1:10 with an extraction solvent of methanol, water, and formic acid in a 60:37:3 ratio) was combined with 1.5 mL of the 10-fold-diluted Folin–Ciocalteu reagent, followed by the addition of 1.2 mL of sodium carbonate solution (7.5% w/v). The reaction mixture was incubated at room temperature for 90 min. The absorbance was then measured at 760 nm using a Beckman Coulter DU730 UV–Vis spectrophotometer (Allegra™ 25R Centrifuge, Beckman Coulter, Brea, CA, USA) in comparison to a reagent blank. Calibration was performed using six standard solutions of gallic acid ranging from 50 to 500 mg/100 mL. The results were expressed as gallic acid equivalents (GAE).
Identification and Quantification of Anthocyanin Compounds
A 20 µL sample of the pomegranate extract was analyzed on an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a photodiode array detector (DAD, G1315 D series) and an autosampler (G1329A series) and controlled by ChemStation software (Rev. B.03.01, Agilent Technologies, Santa Clara, CA, USA). The separation was performed on an Agilent Zorbax Eclipse XDB reversed-phase C18 column (150 mm × 3.5 mm; particle size 5 µm) maintained at 30 °C. The analytes were eluted using a two-solvent gradient system consisting of solvent A [formic acid and water (5:95, v/v)] and solvent B [formic acid, water, and acetonitrile (5:90:5, v/v/v)]. The gradient started with 5% B (0–2 min) and increased to 15% B at 19 min and then to 20% B at 20 min. From 20 to 21 min, the gradient was increased to 100% B and held until 24 min. The column was re-equilibrated to the initial conditions (5% B) from 24 to 28 min. The flow rate was 1 mL/min, and the chromatograms were recorded at 520 nm to detect the anthocyanins. Peaks were quantified using a standard curve generated by injecting analytical-grade cyanidin-3-glucoside (Indofine Chemical Company, Hillsborough, NJ, USA) at concentrations ranging from 0.2 to 500 µg/g in triplicate (R2 = 0.9999). The results were expressed as cyanidin-3-glucoside equivalents. To identify the individual peaks, the collected fractions were concentrated to near dryness using a rotary evaporator. The concentrated samples were analyzed by the core facility using MALDI-TOF mass spectrometry on a Bruker Autoflex instrument (Bruker Daltonics, Billerica, MA, USA). The resulting mass spectra (m/z values) were compared with data from the literature to identify the compounds [20,21]. The total anthocyanin content (TAC) was determined by summing the concentrations of all individually identified anthocyanin compounds.
Determination of Antioxidant Activities
DPPH assay: The radical scavenging activity was evaluated according to the method of Brand-Williams and collaborators [22] with minor modifications. The extract (diluted to 1:10 with a mixture of extraction buffer, methanol, and water/formic acid, 60:37:3) was mixed with 0.5 mM DPPH in methanol (400 μL of extract and 200 μL of DPPH). After 30 min of incubation in the dark at room temperature, the absorbance was measured at 517 nm (Beckman Coulter DU730 UV–Vis spectrophotometer). A blank without DPPH was used for correction. The results were expressed as percent inhibition.
TOSC determination: Total antioxidant capacity was determined using the TOSC assay [23]. The extracts were dried, reconstituted in a 100 mM phosphate buffer (pH 7.4), and diluted to 1:20. An aliquot of 100 μL was added to reaction vials containing α-keto-γ-(methylthio)-butyric acid (KMBA) and a buffer; one vial also contained Trolox as a standard. Reactions were initiated by injection of 200 mM 2,2′-azobis(2-amidinopropane) dihydrochloride (ABAP), followed by incubation at 39 °C for 90 min. Ethylene production was measured using a gas chromatograph (Agilent 7890A, Agilent Technologies, CA, USA). The system was equipped with a capillary injection port with electronic pressure control (EPC), which was set to 50 °C and operated in split mode (20:1). Separation was performed using an HP-Plot Q column (15 m × 0.32 mm × 20 µm) maintained at 40 °C, while detection was performed with a flame ionization detector (FID), also equipped with EPC and set at 225 °C. The carrier gas was high-purity helium at a flow rate of 2.66 mL/min. Data acquisition was performed at a rate of 50 Hz and processed using the ChemStation software (Rev. B.03.01). Ethylene concentrations were quantified using a second-degree polynomial calibration curve (R2 = 0.981), and antioxidant capacity was expressed as millimole Trolox equivalents (mM TE).

2.5. Statistical Analysis

The data obtained from this study were analyzed using Statistica 14.0.0.15 [24]. Analysis of variance, specifically a one-way ANOVA, was performed to determine the differences between locations in the physical and chemical characteristics of the fruit, including the % radical scavenging activity (DPPH) and Trolox equivalents (TOSC), as well as to determine differences within the individual locations in the proportion of individual anthocyanidins. Differences between means were considered significant at p ≤ 0.5 using the LSD Test.

3. Results

3.1. Physical Properties

Properties of the pomegranate fruit ‘Barski slatki’ cultivar grown at four different locations (Kaštela and Metković, CRO; Mostar, BIH; and Bar, MNE) were analyzed. The location had a significant influence on some of the physical properties of the fruit and aril (Table 2). This indicates that local climatic and environmental conditions can influence fruit morphology, which is important for understanding the impact of the growing site on cultivar performance.
Fruit weight and fruit volume ranged from 386.9 g to 506.8 g and from 407.2 mL to 531.1 mL, respectively, and showed significant differences among different growing areas (Table 2). In general, fruit weight and fruit volume were higher in the northernmost-fruit-grown location (Kaštela, CRO) than in the southernmost location in Bar (MNE). While no differences were found in fruit length between growing locations, the location affected fruit diameter. At Mostar (BIH) (96.1 mm), FD was significantly larger than at Kaštela (91.5 mm) and Metković (86.4 mm) (CRO). The FL/FD ratio defines the fruit shape index. At both locations in Croatia (Kaštela and Metković), the fruits were round (0.92), whereas at the Mostar (BIH) and Bar (Montenegro) locations, they had a more flattened shape. Also, at both Croatian locations, the length of the calyx was significantly lower than in Mostar (BIH) and Bar (MNE).
Significant influence of growing area on the number of arils per fruit was recorded, varying from 561 to 749, and at Kaštela (CRO), it was higher than at the other investigated location. The total aril’s weight and the weight of individual arils also depended on the growing region (Table 2). Total arils’ weight varied between 235 and 309.2 g. The highest TAW was recorded at Kaštela (CRO) and was higher than at Bar (MNE) and Metković (CRO). Aril weight varied between 0.39 g and 0.47 g and was higher in Mostar (BIH) than in Kaštela (CRO) and Bar (MNE). Although fruit size varied between locations, no differences were found in terms of peel thickness, aril yield, and juice yield. This highlights that some fruit traits may be less sensitive to environmental variation than others.

3.2. Chemical Properties

We found a significant influence of location on TSS, TA, TSC, and pH in juice (Table 3).
The total soluble solid content varied between 12.8 and 15 °Brix (Table 3). The TSS content was the highest at the Metković site (CRO) and the lowest at the Mostar (BIH) and Bar (MNE) sites. Location also had a significant influence on total acidity, with higher acidity (0.59%) found at the Metković location compared to Kaštela (CRO) (0.53%) and Mostar (BIH) (0.48%). Despite the differences in TSS and TA, the TSS/TA ratio was not found to vary by location, suggesting that overall taste balance remains relatively stable across sites despite variation in individual components.
The total sugar content at Bar (MNE) (13.9%) was higher than that at other locations. The difference in pH between the locations was also significant: at Kaštela (CRO) and Mostar (BIH), the pH was higher than at Bar (MNE).
Table 4 shows that the location has an influence on the total phenol content, which varied between 2603 and 3101.9 mg L−1 GAE. At the Kaštela (CRO) location, the TPC was significantly higher than at the other locations. Although no differences were found in total anthocyanidin content or antioxidant activity across locations (Table 4), the concentration of specific individual anthocyanins varied depending on location (Figure 2).
Anthocyanins identified in this study included delphinidin-3,5-diglucoside, cyanidin-3,5-diglucoside, cyanidin-3-glucoside, pelargonidin-3-glucoside, and delphinidin-3-glucoside. The profile was consistent across all locations studied. The most prevalent anthocyanin pigment at all locations was cyanidin-3-glucoside, the content of which did not differ significantly between the investigated locations. The second most abundant anthocyanin, delphinidin-3-glucoside, also showed no significant differences between locations. The remaining three anthocyanins showed variability depending on the cultivation site. The content of cyanidin-3,5-diglucoside was highest at the Bar location (10.34 mg/100 g f.w) and lowest in Kaštela, with no significant differences between Metković and Mostar. The highest content of delphinidin-3,5-diglucoside (10.20 mg/100 g f.w) was found in Bar, while the lowest was in Kaštela (3.88 mg/100 g f.w), with no significant differences between Kaštela and Mostar. The content of pelargonidin-3-glucoside differed only in Mostar compared to Metković.
Aril color defined by the CIELAB color space parameters L* (lightness), a* (green to red), b* (blue to yellow), C (chroma, representing color intensity), and h° (hue angle) varied significantly across different growing locations (Table 5).
Lightness (L*) of arils from both locations in Croatia and Mostar (BIH) was higher than in Bar (MNE). L* = 0 denotes black, and L* = 100 indicates diffuse white. The results showed that the arils from fruits from Bar (MNE) were more colorful than those from other locations studied. In Bar (MNE), arils had significantly higher redness (a*; more magenta color components) than the arils from both locations in Croatia, where the content of yellow color components (b*) is higher. The (C) value represents the ‘purity’ of the color. A lower value, i.e., lower color purity, was recorded at the location Mostar (BIH) compared to Metković (CRO). Although the h° value was higher at both locations in CRO than in BIH and MNE, the arils had a red-purple color at all locations, suggesting that while hue remains relatively consistent, subtle differences in color parameters reflect the impact of growing site on fruit visual quality.

4. Discussion

Extensive scientific knowledge about the health-promoting properties of pomegranates, along with public interest in functional foods [25,26,27,28,29], has led to a significant increase in the demand for pomegranate fruit and its byproducts in recent years, particularly in Western countries [30].
The pomegranate is a Mediterranean fruit species that is best adapted to the Mediterranean climate, which is characterized by intense sunlight, mild winters with minimum temperatures not below −12 °C, and warm, dry summers without rainfall during the final stage of fruit development [31,32]. Under these conditions, the fruit can reach its optimal size, color, and sugar accumulation without the risk of skin cracking. The area under pomegranate cultivation worldwide has expanded considerably, and it is now grown in various subtropical and tropical regions with different microclimatic conditions [33].
The ‘Barski slatki’ cultivar is grown extensively on the eastern Adriatic coast, and this study has established the influence of four different locations on select physical and chemical fruit properties.

4.1. Microclimate Condition Influence on Physical Properties

A significant influence of location was found on physical properties (Table 2), including fruit weight, fruit diameter, fruit volume, fruit shape index, calyx length, number of arils per fruit, and total and individual aril weight.
Mditshwa et al. [34] also indicated a significant influence of growing location on fruit and aril weight, fruit length and diameter, and total aril weight. Borochov–Neori et al. [35], Schwartz et al. [32], and Ghasemi Soloklui et al. [36] confirm the influence of growing area on the fruit and aril weight, while Boussaa et al. [2] reported that location had no influence on the fruit weight, length, and diameter, but influenced the peel weight and thickness, as well as aril and juice yield. Feng et al. [37] reported that the adaptability of the cultivar to environmental conditions significantly affects the physical fruit characteristics. The quality and proper flower bud initiation, flowering, and pollination determine fruit set, as these are particularly sensitive stages of development influenced by various physiological and environmental factors, such as soil moisture, temperature, rainfall, wind, and humidity [32]. Flower bud initiation begins about 4–5 weeks before flowering [38,39] which typically occurs in April under the climatic conditions of the study area. In our study area, flowering began in May and lasted about two months. Early-flowering flowers usually have larger floral structures and ovaries, resulting in higher fruit set (up to 90%) and better fruit quality [40,41], probably due to more favorable environmental conditions during their development [42]. However, unfavorable weather conditions during the flowering period can negatively affect pollination and fruit set. At the Kaštela site, about 50% of the annual rainfall occurred during the vegetation period (April to October), which is 8–15% more than at the other sites during the same period (Table 1). This rainfall distribution was particularly favorable during the critical period of flower bud differentiation and flowering. Higher precipitation at Kaštela in April (Table 1) improved soil moisture availability and created favorable conditions for early initiation and differentiation of hermaphrodite flowers, which are known to produce larger fruits. In contrast, at the same location, lower rainfall in May probably favored better pollination and fertilization, contributing to higher fruit set and a greater number of arils per fruit. During the first two weeks after fruit set, fruit growth progresses evenly, followed by a rapid increase in fruit size (weight, length, diameter, and volume) until the end of June, after which growth gradually slows down [43]. Sufficient soil moisture combined with adequate heat is crucial for optimal fruit development and growth during this phase, which ultimately affects yield and fruit quality. Although pomegranate is considered a drought-tolerant fruit species, deficit irrigation during fruit development and ripening has an adverse effect on fruit size and total yield [44]. At the Kaštela site, microclimatic conditions, especially higher rainfall in June and July (Table 1), provided higher soil moisture and more favorable conditions for fruit growth, resulting in larger fruits at harvest. In contrast, the lowest rainfall in June was recorded at the Bar site (MNE), where the smallest fruits were also harvested.
In our study, aril yield and juice yield showed no significant differences between locations for either parameter (Table 2), which contrasts with the results of Mditshwa et al. [34] and Schwartz et al. [32]. Interestingly, despite the variation in fruit weight across locations, aril yield remained constant. This response can be attributed to climatic factors that may favor the development of internal membrane structures over aril formation [34]. According to Borochov-Neori et al. [35], the number of membrane walls influences both the number of arils and the peel thickness. Environmental conditions typical of Mediterranean and subtropical climates promote the development of these structures. This finding could also explain the relatively high juice content observed in fruits from these regions.

4.2. Microclimate Condition Influence on Chemical Properties

Previous studies have reported that genotype, agroclimatic conditions, cultivation practices, fruit maturity, harvest time, storage, and postharvest handling affect the content of bioactive compounds in pomegranate fruits [45,46,47]. With some experience, harvest maturity can be assessed by tapping the fruit and listening for a metallic sound, a traditional method mentioned by Mir et al. [48]. In the region where our study was conducted, harvest time is typically determined based on skin color and the growers’ experience when the fruit skin begins to show superficial cracking. Our results showed that the growing location influences specific chemical characteristics (Table 3). The total soluble solids (TSS) content significantly varied. As previously described by Fawole and Opara [45], TSS content increases faster in the early stages of fruit development, which is probably due to the active hydrolysis of starch into sugars during fruit ripening [49]. At the time of harvest, climatic conditions, especially the amount and distribution of rainfall, have a significant impact on TSS content [34,36,50,51]. The relatively low TSS content observed in fruits from Bar (MNE) (Table 3) could reflect the adverse effects of higher rainfall during ripening. Fruits grown under drier conditions are often sweeter than those from humid or irrigated areas [50].
Along with total soluble solids (TSS) content, titratable acidity (TA) is a crucial quality characteristic of pomegranate juice that significantly contributes to its taste and consumer acceptance [32]. In our study, the highest TA value was measured at the Metković (CRO) site, situated in the lower reaches of the Neretva River at an altitude of 5 m in a typical Mediterranean climate zone. Conversely, the lowest TA was found in fruits from Mostar (BIH), which is located in the upper reaches of the same river at an altitude of 100 m in the inland region where the Mediterranean influence persists. This pattern is consistent with earlier findings by Shulman et al. [52], who found higher acidity in pomegranates grown in coastal regions of Israel compared to those in warmer inland valleys.
The pH value, which is directly related to the perception of the acidity of pomegranate juice, is also an important parameter [53]. Immature fruits usually have lower pH values due to the higher acidity, while the pH increases as the fruits ripen. In our study, lower pH values were observed in fruits from Bar (MNE), whereas the fruits from Kaštela (CRO) and Mostar (BIH) had a higher pH value (Table 3). These differences may reflect developmental stages as well as site-specific growing conditions. Previous research has attributed variations in TSS, TA, and pH to both genotype and regional environmental factors [45,54]. Similarly, Mditshwa et al. [34] confirmed the significant influence of location on TSS and TA, although they found no significant differences in pH.
The phenolic content of pomegranate fruit is influenced by a complex interplay of factors, including genetic background, climatic conditions, and cultivation practices [55,56,57]. Among the environmental variables, altitude has been identified as a factor that can significantly affect the phenolic biosynthetic pathway [2,46] by altering temperature, UV radiation, and water availability. In our study, total phenolic content (TPC) varied significantly between locations (Table 4). Fruits harvested from the lower altitude (Kaštela, at 5 m above sea level) had a higher TPC than fruit from higher altitude sites such as Mostar and Bar (100 and 143 m above sea level, respectively). This result aligns with the results reported for pomegranates [34], and similar trends were observed for olives [58,59], sweet cherries [60], and grapes [61]. Schwartz et al. [32] also reported a higher TPC in the juice of pomegranates from the Mediterranean compared to the arid desert region, underlining the importance of mesoclimatic suitability. Attanayake et al. [30] observed lower phenolic content in pomegranates grown in the colder and rainier regions of Sri Lanka, suggesting that excessive rainfall may inhibit phenolic accumulation. Some studies, however, indicated that higher altitudes can lead to higher phenolic content and antioxidant capacity, probably due to environmental stress and radiation exposure [36]. In our study, lower altitudes seem to favor higher phenolic accumulation, possibly due to better hydration, favorable temperature, and radiation exposure, although this was not consistent across all sites; Kaštela and Metković (5 and 9 m a.s.l., respectively) did not follow the same trend as the two other locations at significantly higher altitudes. This suggests that rainfall distribution may also play an important role, as a more favorable distribution of precipitation during fruit growth and development (e.g., in Kaštela) resulted in fruits with higher moisture content at the developmental stage, which in turn promoted phenolic accumulation. Overall, these results emphasize the need to consider not only altitude but also the broader climatic context, including latitude, rainfall patterns, and microclimatic conditions, when interpreting the effects of growing environment on phenolic composition.
Color is a key visual quality parameter for pomegranate fruit, playing a decisive role in its attractiveness and marketability for the consumer. The distinctive red color of pomegranate juice is primarily attributed to anthocyanins, a class of flavonoids responsible for the pigmentation of many fruits [34,52]. In our study, although the total anthocyanin content was not significantly affected by location, the growing location had a significant effect on the color of the arils (Table 5) and the concentration of individual anthocyanin compounds (Figure 2). Fruits grown in Bar (MNE) at 147 m altitude had the lightest arils and the most pronounced red hue, as reflected in the significantly higher a* values indicating a greater proportion of magenta-red color components. In contrast, the arils from Croatian areas (Kaštela and Metković) had a higher yellow hue, indicated by higher b* values. These results are in line with previous reports by Ubi [62] and Mditshwa et al. [34], which highlight an environmental modulation of pomegranate coloration.
At all locations, cyanidin-3-glucoside was the predominant anthocyanin, followed by delphinidin-3-glucoside (Figure 2), which is consistent with previous anthocyanin profiles in pomegranate. Interestingly, despite apparent differences in color intensity (a*) and lightness (L*), the concentrations of these two main anthocyanins remained relatively stable across the different locations. On the other hand, the remaining three anthocyanins showed site-specific variation. The content of cyanidin-3,5-diglucoside and delphinidin-3,5-diglucoside was highest at Bar (147 m altitude) and lowest in Kaštela (9 m altitude). Pelargonidin-3-glucoside was present in significantly different concentrations only in Mostar compared to Metković (Figure 2).
Environmental temperature is a crucial factor regulating the biosynthesis and degradation of anthocyanins. These pigments are known to accumulate in response to cooler temperatures, which enhances gene expression related to the anthocyanin biosynthetic pathway [30,63,64]. Anthocyanin accumulation is inversely correlated with harvest season temperatures, and phenolic compounds are often more abundant under cooler growing conditions. While low temperatures can stimulate anthocyanin production, excessively high temperatures can inhibit synthesis and promote pigment degradation [63,65]. Temperature not only affects the anthocyanin concentration but also influences the chemical stability of anthocyanins. Under warmer conditions, anthocyanins are predominantly glucosylated, which may enhance their structural stability [45,66]. According to our findings, the fruits from Bar (MNE) with milder temperatures showed higher levels of diglucoside forms, supporting the hypothesis that temperate climatic conditions favor both anthocyanin biosynthesis and stabilization (Figure 2). However, it is essential to note that genotype–environment interactions can result in varied responses. For example, Attanayake et al. (2018) [30] observed increased expression of anthocyanin synthase genes and higher anthocyanin accumulation in drier, warmer environments, highlighting the role of crop-specific sensitivity. Despite these nuances, a consensus in several studies confirms that cooler temperatures during ripening tend to increase anthocyanin accumulation, resulting in deeper red pigmentation of the skin and arils [45,63]. In addition, cyanidins appear to have more temperature stability, while delphinidins prefer to accumulate under cooler conditions. In summary, although the total anthocyanin content remained stable across all locations, variations in local growing conditions, including temperature, were associated with changes in the color parameters and profile of individual anthocyanins, highlighting the contribution of the environment to the visual and nutritional quality of pomegranate fruit.

5. Conclusions

Microclimatic conditions, including rainfall distribution, altitude, and temperature, significantly influenced the physical, chemical, and visual attributes of ‘Barski slatki’ pomegranate. The results indicated that precipitation at the location during critical phenological stages, such as bud differentiation, flowering, and early fruit development, was associated with variation in fruit set, aril number, and fruit size, although these effects are part of a complex interplay. While aril and juice yields were relatively stable across sites, notable differences were observed in total soluble solids, titratable acidity, pH, total phenolic content, and anthocyanin profiles. Locations with higher spring rainfall (Kaštela, CRO) favored enhanced fruit growth and higher phenolic accumulation. Although the total anthocyanin content remained consistent across locations, significant variation occurred in aril coloration and the relative abundance of individual anthocyanins. Fruits from Bar (MNE), the highest-altitude site, significantly exhibited the highest accumulation and concentration of diglucoside forms.
These findings highlight the importance of incorporating environmental variables into cultivar selection and orchard management strategies to maximize fruit quality for both fresh consumption and processing. Overall, pomegranate ‘Barski slatki’ demonstrates a notable adaptability to diverse agroecological conditions within the Mediterranean climate zone, maintaining stable fruit quality.

Author Contributions

M.R., conceptualization, methodology, sample collection, investigation, formal analyses, writing—original draft preparation, editing, data curation, and review; M.J.Š., formal analyses, software and data analyses, writing—original draft preparation, review, and editing; J.G., sample collection, formal analyses, writing—original draft preparation, and editing; S.G.B., conceptualization, review, and editing; J.C.D.-P., review and editing; D.M., formal analyses, methodology, review, and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Croatian National Program for Conservation and Sustainable Use of Plant Genetic Resources for Food and Agriculture 2021–2027 and by the project INOMED-2I (09-207/1-23) granted by the European Union—“NextGenerationEU”.

Data Availability Statement

All data are included within the article.

Acknowledgments

We especially note our appreciation of the support from Dennis Phillips of the University of Giorgia Proteomic and Mass Spectrometry (OAMS) Core Facility in the identification of pomegranate fruit chemical compounds, as well as from technical assistant Radojka Plećaš for help in the laboratory, grower Jozo Obšivač for providing the plant material, and Mate Čarija for assistance with the visualization of the sampling site.

Conflicts of Interest

Author Dan Maclean was employed by the company AgroFresh. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Agriculture 15 02210 g001
Figure 1. Sampling locations: Kaštela and Metković (Croatia), Mostar (Bosnia and Herzegovina), and Bar (Montenegro). The inset in red in the top right corner shows the position of the study area within Europe.
Figure 1. Sampling locations: Kaštela and Metković (Croatia), Mostar (Bosnia and Herzegovina), and Bar (Montenegro). The inset in red in the top right corner shows the position of the study area within Europe.
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Agriculture 15 02210 g002
Figure 2. Anthocyanin concentration (mg cyanidin-3-glucoside equivalents per 100 g fresh weight) in the ‘Barski slatki’ at four locations. Values are presented as mean ± SE (n = 3). Different lowercase letters indicate significant differences among locations for each individual anthocyanidin at p ≤ 0.05 according to the LSD test. Different uppercase letters indicate significant differences within locations in the relative proportions of individual anthocyanidins. Abbreviations: D-3,5-dGc, delphinidin-3,5-diglucoside; Cy-3-dGc, cyanidin-3-diglucoside; D-3-Gc, delphinidin-3-glucoside; Cy-3-Gc, cyanidin-3-glucoside; P-3-Gc, pelargonidin-3-glucoside.
Figure 2. Anthocyanin concentration (mg cyanidin-3-glucoside equivalents per 100 g fresh weight) in the ‘Barski slatki’ at four locations. Values are presented as mean ± SE (n = 3). Different lowercase letters indicate significant differences among locations for each individual anthocyanidin at p ≤ 0.05 according to the LSD test. Different uppercase letters indicate significant differences within locations in the relative proportions of individual anthocyanidins. Abbreviations: D-3,5-dGc, delphinidin-3,5-diglucoside; Cy-3-dGc, cyanidin-3-diglucoside; D-3-Gc, delphinidin-3-glucoside; Cy-3-Gc, cyanidin-3-glucoside; P-3-Gc, pelargonidin-3-glucoside.
Agriculture 15 02210 g002
Table 1. Microclimatic variables in four pomegranate growing areas: Kaštela and Metković (Croatia), Mostar (Bosnia and Herzegovina), and Bar (Montenegro).
Table 1. Microclimatic variables in four pomegranate growing areas: Kaštela and Metković (Croatia), Mostar (Bosnia and Herzegovina), and Bar (Montenegro).
LocationKaštela, Croatia (CRO)Metković, Croatia (CRO)
MonthTemperature (°C)Rainfall (mm)Humidity (%)Temperature (°C)Rainfall (mm)Humidity (%)
MeanMaxMinMeanMaxMin
January8.115.9−3.0228.4657.116.7−4.0243.168
February7.115.1−3.081.8576.017.5−3.971.063
March10.219.02.088.2589.820.7−0.2132.060
April15.224.58.5111.27015.127.16.954.468
May20.934.79.717.45420.834.08.233.067
June22.231.913.7242.46121.532.812.6203.169
July26.337.217.434.05225.636.414.725.869
August26.836.118.014.65025.936.515.534.769
September22.531.014.690.45821.832.012.852.967
October15.726.25.5202.05914.027.71.7242.266
November12.019.73.1119.27610.819.60.3130.472
December9.418.6−3.7198.6668.321.0−6.4332.873
Average16.4 15.6
Summa 1428.2 1555.4
LocationMostar, Bosnia and Herzegovina (BIH)Bar, Monte Negro (MNE)
MonthTemperature (°C)Rainfall (mm)Humidity (%)Temperature (°C)Rainfall (mm)Humidity (%)
MeanMaxMinMeanMaxMin
January6.316.3−3.6336.9639.617.21.0351.169
February5.918.0−3.0117.4558.120.10.0141.954
March9.520.01.6173.66010.822.22.7188.761
April15.626.47.368.16515.525.28.252.572
May21.335.07.635.75520.530.810.739.766
June22.036.112.0190.06222.332.014.7175.868
July26.338.915.015.85525.533.817.727.066
August26.837.918.157.85125.835.419.911.866
September22.334.814.2127.85622.830.815.6103.162
October14.828.63.6246.66216.926.56.7231.965
November10.419.42.9164.27513.121.15.9236.779
December7.518.2−7.8315.06811.919.91.1265.970
Average15.7 16.9
Summary 1848.9 1826.4
Climate parameters registered for the studied locations were obtained from the Croatian Meteorological and Hydrological Service, Federal Hydrometeorological Institute, Federation of BIH, and Institute of Hydrometeorology and Seismology, Montenegro, respectively.
Table 2. Physical properties of the fruit and aril of the pomegranate ‘Barski slatki’ evaluated at four different locations.
Table 2. Physical properties of the fruit and aril of the pomegranate ‘Barski slatki’ evaluated at four different locations.
Location
Fruit PropertiesKaštela, CROMetković, CROMostar, BIHBar, MNE
Fruit weight (FW; g)506.8 * ± 18.5 a **421.6 ± 20.09 ab453.9 ± 26.4 ab386.9 ± 31.9 b
Fruit volume (FV; mL)531.1 ± 12.9 a448.9 ± 20.9 ab477.2 ± 27.6 ab407.2 ± 33.8 b
Fruit length (FL; mm)84.6 ± 0.879.6 ± 1.283.5 ± 2.279.7 ± 2.6
Fruit diameter (FD; mm)91.5 ± 1.2 b86.4 ± 0.8 c96.1 ± 0.7 a92.6 ± 2.5 ab
Fruit shape index (FL/FD)0.92 ± 0.02 a0.92 ± 0.01 a0.87 ± 0.02 b0.86 ± 0.02 b
Calix length (CL; mm)17.0 ± 0.5 b17.2 ± 0.6 b18.9 ± 0.3 a19.2 ± 0.3 a
Calix diameter (CD; mm)23.9 ± 1.223.6 ± 0.926.3 ± 0.423.6 ± 1.1
Peel thickness (PT; mm)4.2 ± 0.44.1 ± 0.43.5 ± 0.24.4 ± 0.4
Number of arils per fruit (NoA/F)749 ± 41 a607 ± 27 b611 ± 46 b561 ± 23 b
Total aril weight (TAW; g)309.2 ± 14.8 a261.1 ± 17.1 bc294.3 ± 10.9 ab235.0 ± 17.9 c
Aril weight (AW; g)0.41 ± 0.02 b0.42 ± 0.02 ab0.47 ± 0.02 a0.39 ± 0.03 b
Aril yield (AY; %)60.8 ± 0.960.9 ± 1.758.6 ± 1.961.1 ± 1.4
Juice yield (JY; %)71.8 ± 1.774.2 ± 0.473.3 ± 1.671.0 ± 2.6
* Values are given as mean ± SE. ** Different lowercase letters in each row indicate a significant difference between locations at p ≤ 0.05 by LSD test.
Table 3. Total soluble solids (TSS), titratable acidity (TA), TSS/TA ratio, total sugar content (TSC), and pH of juice from pomegranate ‘Barski slatki’ grown at four locations.
Table 3. Total soluble solids (TSS), titratable acidity (TA), TSS/TA ratio, total sugar content (TSC), and pH of juice from pomegranate ‘Barski slatki’ grown at four locations.
Location
Juice PropertiesKaštela, CROMetković, CROMostar, BIHBar, MNE
Total soluble solids (TSS; °Brix)14.1 * ± 0.1 b **15.0 ± 0.2 a13.1 ± 0.0 c12.8 ± 0.4 c
Total acidity (TA; %)0.53 ± 0.01 bc0.59 ± 0.03 a0.48 ± 0.01 c0.55 ± 0.01 ab
TSS/TA ratio26.4 ± 0.525.7 ± 0.927.0 ± 0.323.2 ± 1.1
Total sugar content (TSC; %)11.8 ± 0.4 b12.1 ± 0.2 b12.6 ± 0.1 b13.9 ± 0.5 a
pH3.57 ± 0.02 a3.52 ± 0.05 ab3.60 ± 0.03 a3.42 ± 0.03 b
* Values are given as mean ± SE. ** Different lowercase letters in each row indicate a significant difference between locations at p ≤ 0.05 by LSD test.
Table 4. Total phenol content (TPC), total anthocyanin content (TAC), and antioxidant activity (DPPH and TOSC) of arils of pomegranate ‘Barski slatki’ grown at four locations.
Table 4. Total phenol content (TPC), total anthocyanin content (TAC), and antioxidant activity (DPPH and TOSC) of arils of pomegranate ‘Barski slatki’ grown at four locations.
Location
Aril PropertiesKaštela, CROMetković, CROMostar, BiHBar, MNE
Total phenol content (mg L−1 GAE)3101.9 * ± 35.7 a **2603.0 ± 124.1 b2725.5 ± 150.2 b2772.3 ± 108.3 b
Total anthocyanin content (mg/100 g)44.9 ± 5.5843.99 ± 4.6751.3 ± 6.7159.36 ± 4.03
DPPH (%)70.5 ± 0.867.7 ± 1.269.1 ± 1.369.3 ± 2.5
TOSC (umol Trolox eq/kg)414.3 ± 12.4425.8 ± 10.0420.3 ± 8.2436.4 ± 10.4
* Values are given as mean ± SE. ** Different lowercase letters in each row indicate a significant difference between locations at p ≤ 0.05 by LSD test.
Table 5. Aril color parameters (CIE L, a*, b*, C, and h°) of pomegranate ‘Barski slatki’ grown at four locations.
Table 5. Aril color parameters (CIE L, a*, b*, C, and h°) of pomegranate ‘Barski slatki’ grown at four locations.
Location
Color ParametersKaštela, CROMetković, CROMostar, BIHBar, MNE
L*32.95 * ± 0.57 a **35.47 ± 2.26 a32.91 ± 2.09 a26.36 ± 1.03 b
a*6.47 ± 0.41 c8.34 ± 1.83 bc10.66 ± 0.83 ab11.92 ± 1.62 a
b*12.07 ± 0.65 a14.56 ± 3.84 a4.20 ± 1.54 b8.61 ± 2.18 ab
C14.33 ± 0.31 ab17.23 ± 3.24 a11.62 ± 0.97 b15.06 ± 1.05 ab
h°58.27 ± 2.40 a56.90 ± 9.30 a19.96 ± 5.53 b36.07 ± 10.42 b
* Values are given as mean ± SE; n = 10. ** Different lowercase letters in each row indicate a significant difference between locations at p ≤ 0.05 by LSD test.
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Radunić, M.; Jukić Špika, M.; Gadže, J.; Goreta Ban, S.; Díaz-Pérez, J.C.; MacLean, D. Microclimate Condition Influence on the Physicochemical Properties and Antioxidant Activity of Pomegranate (Punica granatum L.): A Case Study of the East Adriatic Coast. Agriculture 2025, 15, 2210. https://doi.org/10.3390/agriculture15212210

AMA Style

Radunić M, Jukić Špika M, Gadže J, Goreta Ban S, Díaz-Pérez JC, MacLean D. Microclimate Condition Influence on the Physicochemical Properties and Antioxidant Activity of Pomegranate (Punica granatum L.): A Case Study of the East Adriatic Coast. Agriculture. 2025; 15(21):2210. https://doi.org/10.3390/agriculture15212210

Chicago/Turabian Style

Radunić, Mira, Maja Jukić Špika, Jelena Gadže, Smiljana Goreta Ban, Juan Carlos Díaz-Pérez, and Dan MacLean. 2025. "Microclimate Condition Influence on the Physicochemical Properties and Antioxidant Activity of Pomegranate (Punica granatum L.): A Case Study of the East Adriatic Coast" Agriculture 15, no. 21: 2210. https://doi.org/10.3390/agriculture15212210

APA Style

Radunić, M., Jukić Špika, M., Gadže, J., Goreta Ban, S., Díaz-Pérez, J. C., & MacLean, D. (2025). Microclimate Condition Influence on the Physicochemical Properties and Antioxidant Activity of Pomegranate (Punica granatum L.): A Case Study of the East Adriatic Coast. Agriculture, 15(21), 2210. https://doi.org/10.3390/agriculture15212210

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