Abstract
The artificial cultivation of Mengzi pomegranate has been conducted on a large area for more than 30 years in Yunnan, China. As the quality of pomegranate cultivars have degraded seriously, new cultivars have gradually been introduced to meet market demand. Comparative quality evaluation of different pomegranate varieties is beneficial to guide cultivar improvement and cultural program modification. The present study investigated the differences of physicochemical characteristics of the introduced Tunisian soft-seed pomegranate in different climate zones in Yunnan, China. Meanwhile, the differences between native cultivars (Tian guan yan (TGY) and Tian lv zi (TLZ)) and introduced cultivar were also compared. It was found that the Tunisian soft-seed pomegranate grown in Huize (Tunisian pomegranate in Huize, TH) in the temperate plateau monsoon climate belt had the highest fruit weight, % of seed, TAC, TPC, TSS, amino acids, and Mn, and had the lowest organic acids and Na. While grown in the subtropical monsoon climate area Shiping (Tunisian pomegranate in Shiping, TS), the Tunisian pomegranate fruits had the highest amounts of Cu. Commonly, there were significant correlations between cultivation climate regions and fruit properties. The contents of the TSS, TAC, TPC, flavor amino acids and organic acids varied with planted climate zones. The introduced Tunisian soft-seed pomegranate has comparable levels of physicochemical characteristics with the local main cultivars in the same planted climate region. Tunisian soft-seed pomegranate of higher quality can be obtained in Huize area. Collectively, the climate difference and cultivar shift have a significant effect on pomegranate production in Yunnan, and pomegranate with good quality can be obtained by using proper cultivars in optimized climate zone.
Keywords: Fruit quality, Bioactive ingredients, Climates, Cultivar, Multivariate analysis
1. Introduction
Pomegranate (Punica granatum L.) fruit has an important economic value in the agricultural sector [1], because it contains numerous nutritional components, which have health benefits, and could be processed into fruit derivatives to meet market demands [[2], [3], [4]].
China, as the main production country of pomegranates, has the planting area of more than 120,000 ha, with a production exceeding 1.2 million t yearly. Yunnan province is one of the main production areas of pomegranate in China, and the cultivation is mainly in Jianshui county [5]. The main cultivars of this area are ‘Tian Guan Yan’ (TGY) and ‘Tian lv zi’(TLV), and the pomegranate fruits are widely exported to Russia, Vietnam, Myanmar, Thailand, South Korea, Singapore, Dubai and other countries and regions [6]. The TGY is an early-maturing cultivar, with nearly spherical fruit shape, thick red peel and reddish seeds (Fig. 1 A). The TLZ is a medium-ripening cultivar, with green peel, large fruit size and light-yellow seeds (Fig. 1 B). The trunks of the two local cultivars are natural spread out outwards, the embracement, after 30 years of cultivation, become tall branched. As the two cultivars have poor condition of ventilation and penetrating light in prosperous fruit stage, the quality of fruit is decreasing gradually, and lose their core market competitiveness [7]. In recent years, under the background of creating the high-grade "green food brand" in Yunnan Province, the cultivation range of pomegranate has been expanded, and new cultivars of pomegranate have been introduced and domesticated locally. Due to the demand and consumption habits, Tunisian soft-seed pomegranate with strong sweetness and dark red peel was introduced into China (Fig. 1C), and has become the most popular cultivar, leading to the sale decline of native Mengzi pomegranate cultivars [8].
Fig. 1.
The three studied pomegranate cultivars. A, B, C, D and E represent TGY, TLZ, Tunisian Soft Seed Pomegranate, Tunisian Soft Seed Pomegranate from Huize County (TH), and Tunisian Soft Seed Pomegranate from Shiping County (TS), respectively.
Pomegranate plants are known to be very sensitive to climatic conditions [9], so intensive researches have focused on the relationship between the growth environment and fruit quality of different cultivars. Studies showed that pomegranate fruit quality was strongly influenced by cultivar difference and the planted environmental condition [10,11], such as growing altitude [12], radiation [13], cropping system [14], local climatic [15,16], and soil factors [17]. These factors lead to the unique fruit characteristics and qualities of different production areas.
Environmental conditions and cultivars appeared to be the main factors to affect the chemical composition of pomegranate fruit. Pomegranates were mainly cultivated in limited areas with temperate climates. Schwartz (2009) studied the differences of the pomegranate fruit in Mediterranean climate and desert climate conditions [15]. It was indicated that environmental conditions significantly influence pomegranate fruit quality and the accumulation of beneficial compounds. In addition, another report had evaluated the bioactive constituents and physical property differences among pomegranate cultivars grown in the subtropical region, and it was demonstrated that genetic factors were the main determinants of bioactive ingredients concentrations [18].
Yunnan province has a diversity of climate types due to the vertical change of terrain and the different climatic zones. The dominant factors affecting the quality of pomegranate fruit at different climatic zones are still uncertain. Therefore, our present research reported the physical and chemical characteristics of cultivars in different environmental conditions, and also compared the effects of climate and cultivar difference on fruit quality by screening the characteristic quality of pomegranate. The research result may guide the cultivar shift and production area selection for pomegranate cultivation.
2. Material and methods
2.1. Plant material
The Pomegranate fruits were collected from the areas of subtropical monsoon climate (Mengzi, Shiping) and temperate plateau monsoon climate (Huize) in Yunnan province, China (Fig. 2). Mengzi (103° 40′60″E, 23° 32′ 59″ N) and Shiping (102° 61′ 12″ E, 23° 62′ 00″N) belong to the subtropical monsoon climate, having intense sunlight and mild winters with the minimal temperatures not lower than 0 °C. In Mengzi and Shiping, the "Tian Guan Yan"(TGY) and "Tian lv zi"(TLZ) are the mainly local traditional cultivars, and the pomegranate trees are 30 years old. The introduced variety, Tunisia soft-seed pomegranate, was planted both in Shiping (TS) (Fig. 1 E) and Huize (TH) (Fig. 1 D), and the trees are 6 years old. All the pomegranate trees were grown in regular orchards in full sun conditions and organic fertilization was applied. In the subtropical monsoon climate area, the harvest date of pomegranate fruits was on August 27, 2021, and the other was on September 26, 2021. The farmers make harvest decisions based on fruit size, color, taste and texture.
Fig. 2.
Location map of sampling sites in the two climate zones in Yunnan, Province.
2.2. Physical properties
Ten mature, healthy fruits were weighed by an electronic balance (±0.01 g sensitivity), and the fruit weight was average value. Fruit length and diameter were measured independently using a Vernier Caliper. Fruit shape index was the ratio of length and diameter. The size of the fruit is expressed by volume (mm3), using the displacement method to measure the volume of the pomegranate fruit. After that, the peel was removed and arils were separated by hand, the edible rate (%) was calculated as the ratio of total aril weight and total fruit weight. Fruit peel thickness was measured using a Vernier Caliper, and the fresh weight of 100 seeds was weighed. The pomegranate arils were divided into two parts, used for juice and crushing, respectively. The fruit juice was obtained by squeezing pomegranate seeds wrapped in gauze, and the juice yield was determined using the weight of juice to the total weight of the fruit. Fruit juice was used for the analysis of total soluble solids (TSS), titratable acidity (TA), pH, total anthocyanin content (TAC), total phenolic content (TPC), and organic acid content. The edible part of pomegranate was crushed and analyzed for protein, amino acids, and mineral elements.
2.3. Determination of TSS, TA, protein, and juice pH
The TSS was determined by an automatic refractometer (WYA-Z; INESA, China), the data was expressed as percentage of TSS (at 20 °C). An acid-base titration method was used for determination the fruit TA, and the result was expressed as the percentage of citric acid equivalents the juice. The TSS/TA ratio was calculated from the TSS and TA values. The pH of juice was measured by a digital Thermo-science pH (at 20 °C). The concentration of proteins of the fruit was determined by the Kjeldahl method.
2.4. Determination of TAC and TPC
TAC and TPC were measured according to the pH-differential method and colorimetric Folin–Ciocalteu method, respectively [18]. The TAC and TPC were expressed cyanidin 3-glucoside equivalents as mg/100g and the Gallic acid equivalents as mg/100g of fresh weight, respectively.
2.5. Determination of amino acids
The acid hydrolysis method was used for processing samples. Briefly, crushed fresh arils were hydrolyzed with 6 M HCl in sealed, evacuated glass tubes at 110 °C for 22 h. After hydrolysis, the mixtures were filtered through filter paper into a 25 mL volumetric flask, filled with distilled water to the volume, then the hydrolysate was through a 0.22 μm microfiltration membrane. The amino acids in the hydrolysis solution were determined by the amino acid analyzer (S-433D; Sykam, Germany). The contents of amino acids were calculated by the calibrating curve with standard amino acids and expressed as mg/g fresh sample.
2.6. Determination of mineral elements
The mineral content of crushed fresh arils was analyzed by ICP-OES. Wet digestion procedures were applied to the samples. Samples were digested by the mixture solution of 5 ml HNO3 (65%) and 5 ml H2O2 (30%) for the completed dissolution of the minerals. They were heated by stirring at 80 °C on the hot plate for 3 h. The undissolved solid phase was filtered through blue band filter paper. Then, the obtained filtrates were diluted with high-purity deionized water to 25 ml volumes. The selected elements in the solutions were analyzed simultaneously by an ICP-OES instrumenthe operating conditions of the ICP-OES instrument were the RF power of 1450 W, the coolant plasma gas flow of 13.5 L/min, the auxiliary gas flow of 1.0 L/min, the nebulizer flow of 0.8 L/min, the sample aspiration rate of 2.0 mL/min and the polychromator temperature of 15 °C. The standard solutions for the calibration curve in the mineral analyses were prepared for 100, 200, 400, 800, 1000, 1200, 1600, 2000 μg/L by diluting the multi-element stock solutions (1000 μg/L) in 0.1 mol/L HNO3 solution. The rinsing solvent was the 2 mol/L HNO3 during the ICP-OES measurement.
2.7. Determination of organic acid
The measurement of organic acid content was conducted according to the previous study with minor modifications [19]. About 5 g of fresh juice sample was put into a 50 mL centrifuge tube, added 30 mL of distilled water, then put into an ultrasonic bath for continuous 30 min ultrasonic extraction at room temperature. After using distilled water to set the volume, the solution was then centrifuged at 10,000 rpm for 10 min (5417R centrifuge; Eppendorf, Hamburg, Germany), and the supernatants were filtered with a 0.22 μm filter membrane. A Waters e2695 HPLC system was used to analyze individual organic acids. Calculation of the concentrations was based on the external standard method. Dilutions 1:0, 1:1, 1:2 and 1:4 of an aqueous solution containing 1 g/L of each of the organic acid standards (citric, malic, tartaric acids and ascorbic) was used to fit a standard curve (peak area versus concentration in mg/L) with linear regression for each individual compound. The injection volume was 10 μL, and organic acids were detected by their absorbance at 210 nm using a UV detector, whereas ascorbic acid was detected at 254 nm. A Waters C18 column (4.6 × 250 mm, 5 μm) was used to conduct chromatographic separation, and the column temperature was maintained at 30 °C. The mobile phase was 0.1% phosphoric acid solution, with a flow rate of 1.0 mL/min. All chemical and organic acid standards were purchased from Sigma, and were dissolved in distilled water.
2.8. Statistical analysis
For each cultivar, five trees have been sampled. For each tree, four fruits were collected from each four geographical orientations of the tree (North-N, South-S, East-E and West-W respectively). Each cultivar was replicated three times for a total of 60 pomegranate samples. The data was expressed as mean ± standard deviation (SD), analysis of data were performed using the Graph prism statistical program (version 8.0 for Windows). Differences between the mean values of the samples were analyzed using one-way analysis of variance in Tukey's honestly significant difference test. The spearman correlation matrix method was used to study correlations between altitude and other characters. The variables with significant differences (P＜0.05）in fruit physical and chemical properties were carried out Z-score normalization, and perform of the principal component analysis (PCA) and the heat map with dendrogram in Origin 2021 (Northampton, Massachusetts, USA) to study on the cultivation altitude and cultivars effects of fruits quality.
3. Results
3.1. Physical properties
The physical parameters, such as fruit weight, length (L), diameter (D), length/diameter (L/D), volume, peel thickness, 100-seed fresh weight (% seed FW), edible rate, and fruit juice rate, were analyzed and the results were showed in Table 1. The fruit weight differed significantly among the three cultivars. The introduced variety, Tunisia soft-seed pomegranate (TS and TH), had relative higher fruit weight (331.50 and 474.37 g, respectively) than that of the hard seed pomegranate ‘TLZ’ (302.52 g) and ‘TGY’ (239.40 g). For the fruit length (L) and diameter (D), ‘TLZ’ cultivar was the highest, while ‘TGY’ was the lowest. And the introduced cultivar was obtained with the highest volume and fruit length/fruit diameter ratio (L/D) in temperate plateau monsoon climate (in Huize area). The L/D of the three cultivars were ranged from 0.93–0.96, which was correlated to the Florida pomegranate cultivar (from 0.89 to 1.21) [18]. The peel thickness varied from 3.21 to 3.75 mm, which was lower than other pomegranate cultivars planted in other areas [18,20]. However, there were no statistical differences of L, D, L/D, volume, and peel thickness among the three cultivars. The 100-seed fresh weight (% seed FW) ranged from 40.28 to 68.17 g, and the introduced variety planted in the temperate plateau monsoon climate (TH) was the highest, while the local variety ‘TGY’ was the lowest. Samples of ‘TGY’ and ‘TH’ had the highest edible rate (68.53% and 66.39%, respectively), while ‘TS’ was the lowest (61.47%). The fruit juice rate ranged from 58.41 to 63.46%. ‘TH’ and ‘TLZ’ showed the highest juice rate, while ‘TGY’ was the lowest (58.41%).
Table 1.
Physical properties of the studied pomegranates.
| Parameter | Subtropical monsoon climate |
Temperate plateau monsoon climate |
||
|---|---|---|---|---|
| TGY (local) | TLZ (local) | TS (introduce) | TH (introduce) | |
| Fruit weight (g) | 239.40 ± 20.38c | 302.52 ± 15.80b | 331.50 ± 40.96b | 474.37 ± 9.50a |
| Length (L) (cm) | 7.32 ± 0.08b | 8.18 ± 0.56a | 8.17 ± 0.60a | 7.65 ± 0.81b |
| Diameter (D) (cm) | 7.91 ± 0.32b | 8.70 ± 0.96a | 8.59 ± 0.60a | 7.93 ± 0.73b |
| Length/diameter (L/D) | 0.93 ± 0.04 | 0.94 ± 0.04 | 0.95 ± 0.05 | 0.96 ± 0.02 |
| Volume (cm3) | 246.50 ± 13.44 | 289.00 ± 19.80 | 357.83 ± 104.03 | 383 ± 17.17 |
| peel thickness(mm) | 3.65 ± 0.49 | 3.75 ± 0.35 | 3.31 ± 0.41 | 3.21 ± 0.04 |
| % seed FW | 40.28 ± 0.45c | 66.03 ± 2.44a | 53.81 ± 4.57b | 68.17 ± 1.66a |
| edible rate (%) | 68.53 | 63.87 | 61.47 | 66.39 |
| % of juice(whole) | 58.41 ± 5.15b | 63.20 ± 4.87a | 62.42 ± 6.25a | 63.46 ± 5.08a |
Different letters in rows indicate significantly different values at 0.05 level.
By comparing these physical parameters, we demonstrated that the introduced Tunisian soft-seed pomegranate is comparable to the local varieties in the same climatic zone. And this introduced cultivar planted in temperate plateau monsoon climate areas has advantages compared with the same cultivar in the subtropical monsoon climate areas. This indicates that climatic zone has an effect on the physical properties of the Tunisian soft-seed pomegranates.
3.2. Chemical properties
The data of TSS, TA, TSS/TA, pH, TAC, and TPC were summarized in Table 2. The TSS was significantly different among the three cultivars (p＜0.05). The ‘TH’ was the highest, on the contrary, the ‘TGY’ was the lowest. The concentration of TA was ranged from 0.18% to 0.37%. The local varieties, ‘TGY’ and ‘TVZ’, were higher than the introduced Tunisian soft-seed pomegranate samples (‘TS’ and ‘TH’). The TSS/TA ranged from 39.58 to 89.04, and the introduced cultivar samples (‘TS’ and ‘TH’) was significantly higher than ‘TGY’ and ‘TLZ’. The ratio detected in ‘TS’ and ‘TH’ was consistent with the cultivar planted in Tunisia [20], but was higher than other pomegranate varieties in other areas [18,21]. The TSS/TA ratio is an indicator of the fruit taste and quality, and the introduced Tunisian soft-seed pomegranate obtained a relatively high of TSS/TA, so this variety becomes dominant in the introduced areas. The juice pH ranged from 4.21 (‘TGY’) to 4.69 (‘TH’), which is in accordance with some reports [1,14], but is higher than the cultivars grown in Montenegro (2.77) [17] and Florida (from 2.36 to 3.68) [18].
Table 2.
The TSS, TA, TSS/TA, pH, TAC and TPC content of the studied pomegranates.
| Parameter | Subtropical monsoon climate |
Temperate plateau monsoon climate |
||
|---|---|---|---|---|
| TGY (local) | TLZ (local) | TS (introduce) | TH (introduce) | |
| TSS(%) | 14.49 ± 0.01c | 15.26 ± 0.01 ab | 14.98 ± 0.46bc | 15.91 ± 0.24a |
| TA(%) | 0.37 ± 0.01a | 0.32 ± 0.01b | 0.20 ± 0.03c | 0.18 ± 0.01c |
| TSS/TA | 39.58 ± 0.29b | 47.99 ± 0.60b | 77.41 ± 13.23a | 89.04 ± 1.51a |
| pH | 4.21 ± 0.01b | 4.27 ± 0.01b | 4.50 ± 0.22 ab | 4.69 ± 0.08a |
| TAC (mg/100g) | 7.71 ± 0.84bc | 3.05 ± 0.25c | 10.60 ± 4.58b | 16.95 ± 0.24a |
| TPC (mg/100g) | 179.13 ± 21.90b | 36.93 ± 3.84c | 111.90 ± 41.61b | 267.36 ± 35.71a |
Different letters in rows indicate significantly different values at 0.05 level. TA, TSS, TSS/TA, pH, TAC and TPC content were from juice. TSS: total soluble solid, TA: titratable acidity, TAC: Total anthocyanin content, TPC: Total phenolic content.
Pomegranate fruit is rich in anthocyanins and phenolic, which are the main antioxidant substances. Anthocyanins are the key color molecules for the juice [22]. Both the total anthocyanin content (TAC), total phenolic content (TPC) were significantly different (P＜0.05) among cultivars. And the introduced variety sample (‘TH’) possessed the highest contents of the two parameters, while the local variety (‘TLZ’), cultivated in the subtropical monsoon climate, was the lowest. Overall, ‘TH’ has higher TSS, TSS/TA, TPC and TAC, and lower TA content than other variety samples. The levels of polyphenol and anthocyanin were consistent with some reports [20,23], but lower than the data reported by Shahkoomahally S [18], while higher than that reported by Feng L [21]. Temperature is an important factor affecting anthocyanin biosynthesis. Borochov-Neori H [24] reported that cooler temperatures enhanced anthocyanin content accumulation in pomegranate. As the average temperature of the temperate plateau monsoon climate belt area (Huize) is lower than that in the subtropical monsoon climate belt area (Shiping), the ‘TH’ has relatively higher levels of TSS, TSS/TA, pH, TA and TPC, comparing with ‘TS’. The same trend was also discovered in the Helow pomegranate cultivar planted in different areas of Oman [25].
The mineral contents in fruit arils were shown in Table 3. Generally, for all the three cultivars, K was the most abundant macro-element, followed by P, Mg, Ca, and Na. As for the micro-elements, the high to low order is Fe, Zn, Cu, and Mn. The contents of Ca, Cu, K, Mn, and Na were significantly different among cultivars. The introduced variety in the two climate belt areas had relatively low contents of Ca, K, and Mn, and possessed relatively high contents of Cu, Fe, and Na.
Table 3.
The mineral element content (mg/kg) from arils of the studied pomegranates.
| Parameter | Subtropical monsoon climate |
Temperate plateau monsoon climate |
||
|---|---|---|---|---|
| TGY (local) | TLZ (local) | TS (introduce) | TH (introduce) | |
| Ca | 105.90 ± 1.13a | 72.04 ± 0.94 ab | 49.16 ± 3.69b | 61.76 ± 33.15b |
| Cu | 0.42 ± 0.01b | 0.63 ± 0.01 ab | 0.78 ± 0.09a | 0.73 ± 0.29 ab |
| Fe | 1.25 ± 0.01 | 1.73 ± 0.02 | 2.10 ± 0.64 | 2.25 ± 0.89 |
| K | 1689.50 ± 17.68a | 1487.50 ± 19.09b | 1393.50 ± 136.44b | 1350.75 ± 25.67b |
| Mg | 125.40 ± 1.27 | 133.10 ± 1.70 | 139.90 ± 23.06 | 118.00 ± 35.93 |
| Mn | 0.65 ± 0.01 ab | 0.43 ± 0.01b | 0.66 ± 0.35 ab | 1.04 ± 0.25a |
| Na | 7.48 ± 0.08a | 8.30 ± 0.11a | 5.93 ± 0.40b | 1.77 ± 1.18c |
| P | 290.50 ± 2.97 | 317.85 ± 4.17 | 305.85 ± 31.48 | 296.15 ± 68.24 |
| Zn | 1.51 ± 0.02 | 1.70 ± 0.02 | 1.88 ± 0.25 | 1.41 ± 0.36 |
Different letters in rows indicate significantly different values at 0.05 level.
The protein in juice varied between 0.91% (TLZ) and 1.19% (TH) (Table 4). In general, the percentage of total proteins in pomegranate juice is usually low, ranging from <1.0 to 1.1% [22]. AlMaiman and Ahmad [26] reported juice of fully-ripe Saudi Arabian pomegranate contained 1.05% total protein, which is consistent with our result. Interestingly, we found that the same variety planted in different climate belts might have different protein levels. The protein in TH was significantly higher than TS, while samples TS, TGY and TLZ in the same climate region had the same protein level.
Table 4.
Protein (%) and Amino acid (mg/g) composition from arils of the studied pomegranates.
| Parameter | Subtropical monsoon climate |
Temperate plateau monsoon climate |
||
|---|---|---|---|---|
| TGY (local) | TLZ (local) | TS (introduce) | TH (introduce) | |
| Protein | 1.03 ± 0.01b | 0.91 ± 0.01b | 0.98 ± 0.11b | 1.19 ± 0.04a |
| Asp | 70.90 ± 0.28b | 230.50 ± 12.02a | 202.95 ± 96.51 ab | 115.50 ± 10.47 ab |
| Thr | 11.85 ± 0.07 | 13.35 ± 0.35 | 12.43 ± 1.69 | 13.58 ± 1.24 |
| Ser | 22.55 ± 0.07b | 19.90 ± 0.28b | 21.47 ± 4.66b | 33.15 ± 1.87a |
| Glu | 99.80 ± 0.28b | 27.05 ± 2.47c | 50.63 ± 40.26bc | 175.50 ± 9.68a |
| Gly | 28.70 ± 0.14 ab | 17.30 ± 0.28c | 20.10 ± 7.75bc | 32.05 ± 2.57a |
| Ala | 20.80 ± 0.14b | 19.35 ± 0.50b | 19.75 ± 3.98b | 28.35 ± 2.71a |
| Cys | 2.02 ± 0.01b | 1.85 ± 0.14b | 2.32 ± 0.22b | 6.63 ± 0.44a |
| Val | 17.85 ± 0.50 | 19.85 ± 0.64 | 18.98 ± 3.36 | 21.48 ± 2.11 |
| Met | 6.47 ± 0.07b | 3.89 ± 0.16c | 4.37 ± 0.83c | 8.16 ± 0.68a |
| Ile | 11.95 ± 0.07 | 13.15 ± 0.92 | 12.40 ± 2.50 | 11.67 ± 2.04 |
| Leu | 20.55 ± 0.21 | 20.75 ± 0.64 | 19.63 ± 4.47 | 22.25 ± 0.35 |
| Tyr | 9.12 ± 0.23 | 8.74 ± 0.57 | 8.12 ± 1.59 | 9.23 ± 0.92 |
| Phe | 13.20 ± 1.13 | 12.85 ± 0.35 | 11.90 ± 2.36 | 13.68 ± 1.23 |
| His | 17.35 ± 0.64 | 15.15 ± 0.35 | 14.97 ± 2.09 | 17.88 ± 1.48 |
| Lys | 15.05 ± 0.07 | 20.25 ± 0.78 | 16.75 ± 4.16 | 15.95 ± 1.54 |
| Arg | 30.45 ± 0.78 ab | 11.50 ± 0.28c | 19.10 ± 11.30bc | 37.63 ± 1.07a |
| Pro | 18.10 ± 0.02b | 24.10 ± 0.57a | 19.33 ± 1.79b | 14.98 ± 0.42c |
| Total | 416.71 ± 0.39b | 479.53 ± 15.37b | 475.20 ± 61.58b | 577.64 ± 32.92a |
Different letters in rows indicate significantly different values at 0.05 level.
In addition, the total amino acids ranged from 416.71 (TGY) to 577.64 mg/100g (TH) (Table 4). TH had the highest protein and amino acid levels compared with other cultivars (P＜0.05). Aspartate acid, glutamic acid, and arginine were the major accumulated amino acids, followed by glycine, serine, alanine, leucine and proline. The result was in accordance with a previous study [27]. The contents of aspartic acid, glutamic acid, glycine, methionine, arginine, and proline were significantly different (P＜0.05) between TGY and TLZ when grown in Shiping. The TS and TH also had differences (P＜0.05) in the content of aspartic acid, serine, glutamic acid, glycine, alanine, cystine, methionine, arginine, and proline grew.
The organic acids of pomegranate juice were calculated based on mg/100g fruit juice in Table 5. The result showed that citric acid was the dominant organic acid, followed by malic acid and ascorbic, while trace amounts of tartaric acid were also detected (other organic acids were not detected). The similar result was also reported by Tezcan [28] and Türkylmaz [29]. Especially, the citric acid was also the dominant acid of Turkish and Indian pomegranate varieties [23]. However, malic acid was the major one in Tunisian pomegranate planted in other area [14].
Table 5.
Organic acids content of pomegranate juices.
| Parameter | Subtropical monsoon climate |
Temperate plateau monsoon climate |
||
|---|---|---|---|---|
| TGY (local) | TLZ (local) | TS (introduce) | TH (introduce) | |
| malic acid (mg/100g) | 27.14 ± 7.27a | 23.74 ± 1.97a | 8.22 ± 0.89b | 2.60 ± 0.66c |
| citric acid (mg/100g) | 43.88 ± 12.29a | 43.85 ± 2.52a | 13.30 ± 0.61b | 11.54 ± 0.15b |
| tartaric acid (mg/100g) | 0.71 ± 0.04 | 0.48 ± 0.04 | 0.67 ± 0.02 | 0.61 ± 0.05 |
| Ascorbic (mg/100g) | 9.14 ± 0.52a | 8.38 ± 0.33a | 3.42 ± 1.39b | 2.15 ± 0.07b |
Different letters in rows indicate significantly different values at 0.05 level.
Known from Table 5, the organic acid values significantly varied across the cultivars and the grown area. The citric acid, malic acid and ascorbic content in the local varieties TGY and TLZ were significantly higher (P＜0.05) than that in the introduced cultivar. Tartaric acid concentrations of pomegranate juice were found to range between 0.48 and 0.71 mg/100 g, with no significant differences between these cultivars (P＞0.05). These differences in organic acids were affected by climate and growth conditions. Schwartz reported the pomegranate cultivars grown in subtropical climate had higher level of citric acid and malic acid compared with that planted in the desert climate area [15]. Boussaa indicated that pomegranate grown in the full shade oasis had low malic acid content compared to the full sun exposure due to the low temperature [14]. In the present study, TH grown in temperate plateau monsoon climate, having relative lower average temperature, has the lowest organic acids compared to other cultivars grown in subtropical monsoon climate.
Overall, the TH had the highest levels of TSS, TAC, TPC and flavor amino acids, and possessed the lowest level of organic acids. It was indicated that the introduced variety grown in the temperate plateau monsoon climate environment had better quality compared with that in the subtropical monsoon climate area. The inclusion level of Tunisia soft-seed pomegranate was equivalent to the main local cultivars when grown in the same climate area. Collectively, the climate played an important role on the quality of pomegranate.
3.3. Multivariates analysis
In order to further investigate the relationship between growth environment and chemical composition, the spearman correlation analysis based on the physical and chemical parameters was carried out for the Tunisian soft-seed cultivar in both subtropical monsoon climate areas (Shiping) and temperate plateau monsoon climate environment (Huize), respectively. The results showed that there were significant correlations between cultivation climate regions and the physical and chemical parameters (supplementary material) except for Ca, Cu, Asp and juice yield. The climate regions showed strong correlations with TAC (r = 0.844), TSS (r = 0.656), TPC (r = 0.680), TA (r = −0.750), malic acid (r = −0.938), citric acid (r = −0.938), and ascorbic (r = −0.751), respectively. Mphahlele RR and Mditshwa A had reported that the fruit from drier and warmer environments had relative high levels of TSS, glucose, fructose, and phenolic contents [12,30], as the fruit trees could maximize the production of beneficial compounds and promote relative gene expression [16]. While our result showed that the pomegranate trees, planted in temperate plateau monsoon climate region with lower average temperature, accumulated relative high level of anthocyanin and polyphenol, but reduced level of organic acids, which is in accordance with Kalbani B's report showing that the low temperature is benefit for obtaining the superior fruit quality, such as juice volume, TSS, and maturity index [24].
Principal component analysis (PCA) was applied to distinguish the indicators of the three cultivars, and the principal components with eigenvalues greater than one were selected. Using the first two principal components, 67.9 and 19.1% of variances were obtained (Fig. 3). TA, Malic, citric, ascorbic acids, K, and Ca were the indicators for negative and positive loadings on PC1 and PC2. TA was significantly correlated with Malic, citric, ascorbic acids, K, and Ca (0.921, 0.943, 0.976, 0.872, 0.641), respectively. There were significant positive correlations between the three organic acids (Fig. 4). Pro and Asp were the variables with negative loading on PC1 and PC2, and they were positively correlated (0.647) with each other. The TAC, TSS, % of juice, Cu, and TSS/TA were positive and negative parameters loadings on PC1 and PC2. A significant positive correlation (0.731) was found between the total anthocyanin content (TAC) and TSS (Fig. 4). Generally, TSS represented the concentration of sugar in fruit. Studies have shown that anthocyanidins and sugar increased simultaneously in blueberry [31]. Gly, Arg, Met, Glu, Ser, Ala, Mn, TPC and protein were positive loads on PC1 and PC2. In addition, TPC and Mn were significant positive correlation with other amino acids and protein (Fig. 4).
Fig. 3.
Principle component analysis (PCA) bi-plot.
Fig. 4.
Correlation analysis. ★ means significant difference (P ≤ 0.05), red means positive correlation, blue means negative correlation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
The same positions of pomegranate cultivars in the PCA plot indicated their similar ingredient content. The cultivar TGY was characterized by having the highest values of malic, citric, ascorbic acids, TA, K, and Ca contents. Cultivar TLZ had the highest value of Pro and Asp contents. The TH had the highest Gly, Arg, Met, Glu, Ser, Ala, Mn, phenolic and protein contents. These characteristic indexes could be used as markers for evaluating and distinguishing different pomegranate varieties. Though the TS was close to the center of the bi-plot, it was high in Pro and Asp contents and low in malic, citric, ascorbic acids, TA, K and Ca contents. Thus, it is difficult to identify these varieties by constituent compositions.
Based on the PCA results (Fig. 5), Tunisian soft-seed pomegranates were remarkably low in organic acids, and high in amino acids, polyphenols, anthocyanins, and TSS content, so TS and TH could be distinguished by the first two PCs. The result demonstrated that climate had great influence on quality of Tunisian soft pomegranate. In addition, geographical origin also had a great influence. TH had characteristics of low Na and high Mn, while TS pomegranate has high Cu content. The results showed that both cultivars and environmental conditions affect pomegranate fruit ingredients.
Fig. 5.
Two-dimensional principles component analysis (PCA) score plot.
Based on the components of the cultivars, a hierarchical agglomeration cluster assessment was performed (Fig. 6). The first group was the soft-seed pomegranate TH. TH possessed the highest fruit weight, total anthocyanin, total phenolic, protein, TSS, TSS/TA, Mn, Ser, Glu, Met, Gly, Arg, and Ala contents, while had the lowest TA, Na, Pro and organic acids contents. The second cluster included the Tunisia soft-seed pomegranate TS and TLZ in subtropical monsoon climate areas. This cluster had medium to high fruit weight, TSS, TA, Cu, protein, Ser, Ala, and organic acids contents. Within this cluster, TLZ accumulated the highest levels of Na, Asp, and Pro, and obtained the lowest values of TAC and TPC. The third cluster included TS and TGY in subtropical monsoon climate areas. The characteristics were the highest TA, Ca, K, and organic acids, medium TAC, TPC, Asp, Glu, Ala, Pro content, and low fruit weight, % of juice and TSS. Both the second and third clusters grow in the temperate plateau monsoon climate region.
Fig. 6.
Cluster analysis of pomegranate cultivars based on physical and chemical properties of edible part. Abbreviations: TA, titratable acidity; TSS, total soluble solids.
PCA and cluster analysis divided the cultivars into two groups. The fundamental reason for the huge difference between the two groups was due to different climatic zones. It was indicated that the climate conditions and cultivars play an important role in the formation of fruit quality.
4. Discussion
In present study, three pomegranate cultivars from two different climate regions of Yunnan, China, were characterized by physical properties and chemical ingredients. The results showed a vast variance in terms of pomegranate chemical ingredients content among cultivars and climate conditions. In the same subtropical monsoon climate region, Tunisian soft-seeded pomegranate TS was high in fruit weight, % of seed, TPC, TAC, TSS and low in organic acids. While, the cultivar of TGY was highest in malic, citric, ascorbic acids, TAA, K, and Ca contents, and ‘TLZ’ had the highest values for Pro and Asp contents.
Pomegranates have broad genetic diversity, resulting in differences in their phytochemical composition [32]. A report has showed that cultivars contributed most to the variance in fruit size and skin color [33]. In fact, cultivars have different adaptability to environmental conditions, lead to physical properties and chemical ingredients of pomegranate fruit will vary from variety [10,21]. From our results, it can be seen that the influence of environment on the physical and chemical properties of cultivars were correlates, and not only affect a certain component.
Anthocyanins and polyphenols accumulation are regulated by ambient temperature. Anthocyanins are responsible for the color of pomegranate skin and flesh tissues and the accumulation of anthocyanin is positively correlated with the expression level of genes involved in anthocyanin biosynthesis [16,33]. Borochov-Neori has reported that cooler temperatures enhanced anthocyanin accumulation and found that anthocyanin accumulation had a strong inverse correlation with harvest date temperatures, and phenolics were significantly increased in the coolest season [34]. Plants can react to elevated and low temperatures by altering anthocyanin synthesis. Too high a temperature can inhibit biosynthesis and cause degradation of anthocyanin, and a low temperature can increase anthocyanin production [35]. Temperature has an effect on both anthocyanin synthesis and degradation rates. Ripening fruit under hot temperatures, virtually all the anthocyanins were glucosylated, which can increase the stability of anthocyanins [36]. In our study, ‘TH’ fruits, which had a cooler climate in the Huize area, anthocyanins, and polyphenols were higher, which was consistent with the above result. In contrast to these results, Attanayake reported that in drier and warmer environments, it could promote anthocyanin synthase gene expression in pomegranate fruit and anthocyanin accumulation. This may be due to differences in the cultivar response to the environment [16].
Pomegranate juice mineral content varied significantly in response to growing location, climate and soil conditions [14]. In our result, fruits harvested from TGY and TLZ, located in Mengzi, exhibited high levels of mineral elements except Cu, Fe and Mn. On the other hand, the highest levels of Cu, Fe and Mn were founded in fruits obtained from TS and TH in Shiping and Huize, respectively. Furthermore, our results agreed with previous studies that K and Fe are the most abundant macro- and micro-minerals in fruit aril, respectively [1,10,37]. Fawole and Umezuruike reported that nitrogen was the predominant macro-element in ‘Ruby’ cultivar from South Africa [38] and the other assayed elements, such as Al, Ba, Ni and Zn were variable within cultivars [39].
Organic acids and the total titratable acidity value of pomegranate juice were affected by climate and growth conditions. Generally, sour cultivars are mostly grown in northern cold regions, while sweet cultivars are mostly found in southern hot dry regions [22]. In contrast to our results, grown in northern cooler 'TH' had lower titratable acidity and organic acid content than 'TS' grown in southern warmer conditions. This difference may be due to the harvest date and environment. 'TH' was almost one month later than 'TS'. In addition, warmer conditions have enough light and promote photosynthesis. In active photosynthesis conditions, the tricarboxylic acid (TCA) cycles in mitochondria are transformed into a partial cycle supplying citrate, resulting in malate and citrate accumulating in plants [40]. In addition, K+ in the soil affect the metabolism and storage of organic acids, K+ supplied to the fruit by the sap is necessarily accompanied by the equivalent organic anions, to buffer the excess of organic cations absorbed from the soil [41]. Thus, significant positive correlation between K+ and organic acids.
The reason for the variations in amino acid contents might be due to the cultivars, and geographic and climate influences. Li and Sun have reported the differences in the pomegranate cultivars from Xinjiang and Shandong areas of China, and it was suggested that planted regions and genotype were significant influences on the accumulation of amino acids in pomegranates [42,43]. Halilova and Yildiz analyzed proline content in three Turkish cultivars' juice in two successive years, and found that in the drier and hotter second year, the proline content was 3-fold increased than that in the first year [44]. In this study, we also found that grown in hotter subtropical monsoon climate TGY, TLV and TS cultivars aril juices have lower total amino acid content but higher proline than that in cooler temperate plateau monsoon climate region TH.
As in the cooler climate region, the Tunisian soft-seeded pomegranate has superior quality than that in the warmer climate regions. There were significant correlations between climate zones and physicochemical properties. The highest content of TSS, TA, TP and flavor amino acids, and low levels of organic acid were found in the soft-seed pomegranate ‘TH’, which grown in temperate plateau monsoon climate. Our results are similar with the reports by Mphahlele, who studied the relationship between physicochemical contents and altitudes of 'Wonderful' pomegranate in South Africa [12]. These results indicate that the microclimate differences which are formed by the altitude have an impact on the quality of pomegranate. It may be that the high-altitude environment is conducive to the accumulation of beneficial components of pomegranate fruit. Due to different cultivars responding differently to climate, studies on pomegranate fruits' physicochemical changes need to be studied with more cultivars and climates in different years to determine their impact, and the appropriate planting regions and management technology.
The influence of Environmental conditions on the quality of pomegranate fruit has been confirmed in several experiments. However, for a better understanding of the adaptation of pomegranates to different climates, detailed studies that focus on comparing similar cultivars at different climates are still less. In future garden trials, different cultivars are grown in the same place, together compared with the cultivar in the original regions, combined multi-year data, to determine cultivars × environmental interactions effects on fruit quality traits.
5. Conclusions
This study provided evidence that relevant differences in the physical and chemical properties of pomegranate fruits were induced by the differences of cultivars and climate regions. Tunisian soft-seed pomegranates in the temperate plateau monsoon climate had better fruit quality, indicating that the optimized climate condition may help to improve pomegranate quality.
Author contribution statement
Xingyong Liu: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.
Lijuan Du，Benlin Yin: Performed the experiments.
Xukun Yang: Contributed reagents, materials, analysis tools or data.
Luxiang Wang: Analyzed and interpreted the data.
Yunmei Wang: Conceived and designed the experiments.
Funding statement
This work was supported by the Major Science and Technology program (Agriculture) in Yunnan ProvinceFoundation of China (202102AE090051).
Data availability statement
Data included in article/supplementary material/referenced in article.
Declaration of interest’s statement
The authors declare no conflict of interest.
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Data Availability Statement
Data included in article/supplementary material/referenced in article.