Comparative analysis of fruit quality parameters and volatile compounds in commercially grown citrus cultivars

2.1 Plant material

Seven citrus species and varieties were examined in this study. Species and varieties are listed as follows; Kumquat: Fortunella margarita (L.) Swingle, Limequat: X Citrofortunella sp, Moro blood: C. sinensis (L.) Osbeck, Alacalı kalamondin: X Citrofortunella microcarpa (Bunge) Wijnands Citrus madurensis Lour, Lemonquat: X Citrofortunella sp., pink lemon: C. limon L. Burm.f, Indio mandarinquat: X Citrofortunella sp. Fruits were harvested at the commercial maturity stage from 10-years-old trees. A total of 10 fruits were harvested from different trees with three replicates. The fresh hand-pressed fruit rind and juice of the experimental samples were utilized for further chemical analysis.

2.2 Colour measurement

The fruit colour of citrus species and varieties was measured using the Minolta chromometer (Minolta Co., model CR-400, Tokyo, Japan), with values recorded for L*, a*, and b*. These values were then used to calculate the chroma (C) and hue (h°) values, representing the outer colour of the fruits [10].

2.3 Total soluble solids

The percentage of total soluble solid content of fruit juice (%) was determined by a hand refractometer.

2.4 Total phenol and antioxidant capacity

The radical scavenging activity of DPPH (2,2-diphenyl-1-picrylhydrazyl) was performed as described by Brand-Williams briefly [11] with slight modifications. The radical scavenging activity expressed as % DPPH inhibition was calculated using the following equation:

where Abs sample represents the absorbance of the reaction in the presence of the sample (sample dilution + DPPH solution), Abs blank is the absorbance of the blank for each sample dilution (sample dilution + DPPH solvent), and Abs control is the absorbance of the control reaction (sample solvent + DPPH solution).

The total phenolic content of juice was determined colourimetrically using Folin Ciocalteu’s reagent [12]. Results were described as milligrams of gallic acid equivalent (GAE)/100 g of weight (mg/GAE 100 g).

2.5 Volatile compounds

Volatile compounds were extracted from both the peel and pulp of citrus fruits. Approximately three fruits were randomly selected for each replicate. One gram of the homogenized avocado fruit sample, from two different ripening stages, was weighed, and 1 mL of calcium chloride (CaCl₂) solution was immediately added. The samples were placed in headspace vials and incubated at 40°C for 30 min. Two types of SPME fibres were used for volatile extraction: grey fibre: DVB/CAR/PDMS (divinylbenzene/carboxen/polydimethylsiloxane). Red fibre: PDMS (polydimethylsiloxane, 100 µm). Blue fibre: CAR/PDMS (carboxen/polydimethylsiloxane, 58 µm). The adsorbed aroma compounds from the peel and pulp were analyzed using a Shimadzu GC-2010 Plus GC/MS [13].

2.6 Extraction of sugars and organic acids

For sugars and organic acids extraction, approximately 500 mg of fruit juice homogenated was used for each sample, and triplicate analysis were conducted for each sample.

2.7 Sugars

Glucose, fructose, xylose, and total sugar contents fruit samples were determined using the high-performance liquid chromatography (HPLC) technique according to the method developed by Cristosto [14].

2.8 Determination of organic acids

Organic acids in citrus fruit juice were determined by the HPLC analysis by Bozan et al. [15]. The malic, citric, succinic, fumaric, and l-ascorbic acid contents in citrus juice samples were determined.

2.9 Statistical analysis

The experiment followed a completely randomized block design with four replications for each of the seven different varieties. Data were analyzed using the SPSS statistical software package (version 23.0; SPSS Inc., Chicago, IL, USA). Results were expressed as the mean ± standard error and subjected to one-way analysis of variance. Statistical significance was determined at p < 0.05. [16].

3.1 Colour

Results of fruit skin colour of citrus varieties are presented in Table 1. Varieties significantly differed in terms of rind colour (L*, a*, b*, Croma*, and Hue*). The highest L* (67.79), a* (14.38), b* (49.08), Croma (51.14), and Hue* (70.88) values were determined in Alacalı Kalamondin variety. On the contrary, the lowest L* (56.99) was determined in kumquat, whereas the lowest a* (−0.89) was obtained from limequat and the lowest b* (21.11), C* (22.35), and Hue* (30.74) values were determined in fruit samples of pink lemon. In a study conducted by Toker et al. [17], the average fruit skin colour brightness values L*, a*, and b* were determined as 8.92, 5.39, and 2.31, respectively. Also, Arena et al. [18] conducted a study on different varieties of citrus fruits: L*: 58.0–60.2, (a*: 53.3–33.9, b*: 65.1–61.2. Canan et al. [19] reported similar L*, a*, b* values for eight citrus varieties in their study in Turkey.

3.2 Total phenol and total antioxidant capacity

The citrus varieties evaluated in our study showed significant differences in the total phenol content (Table 2). The highest total phenol content was observed in pink lemon (550.28 mg/100 g GAE), followed by the Alacalı Calamondin (361.49 mg/100 g GAE) variety. The lowest total phenol content was recorded in Kumquat (92.37 mg/100 g GAE). Similarly, Xu et al. [25] reported total phenol content in five citrus varieties from China ranging from 751.82 to 1555.49 mg/L GAE. Fratianni et al. [20] measured the total phenol content of the Bergamot variety as 748 µg GAE/g in the fruit peel and 208 µg GAE/g in the juice. Da Pozzo et al. [21] reported the total phenol content in three bergamot varieties (Castagnaro, Fantastico, and Femminello) as 8.77, 14.00, and 17.10 mg GAE/g, respectively. Ghafar et al. [22] found the total phenol content ranging between 105.0 and 490.74 mg/L GAE in four citrus cultivars. In Tunisia, Tounsi et al. [23] determined the phenolic content of fruit juices from four citrus varieties (dark orange, lemon, blood orange, and mandarin) to be between 106.22 and 784.67 mg/L GAE. Our findings were slightly higher than those reported in some previous studies. These variations could be attributed to differences in analytical methods, cultural practices, environmental factors, climate conditions, and the specific citrus varieties analyzed.

There is a growing interest in the high levels of bioactive compounds found in fruits and vegetables, as these compounds are known to help protect against various diseases. Bioactive components have been shown to reduce free radical damage in cells caused by oxidative stress and are associated with a lower risk of major chronic conditions such as cancer, cardiovascular disease, obesity, and others. The total antioxidant capacity results for the citrus varieties examined in this study are presented in Table 2. Statistically significant differences (p < 0.05) were observed in the total antioxidant capacity among the citrus varieties. Among the cultivars evaluated, pink lemon had the highest total antioxidant content (92.74%), followed by the Alacalı Calamondin variety (92.32%). In contrast, the lowest value was observed in limequat (65.03%).

Tounsi et al. [23] conducted a study in Tunisia where the total antioxidant content of fruit juices of four (dark orange, lemon, blood orange, and mandarin) citrus varieties was found to be 16.6, 18.27, 22.67, and 26.67% in blood orange, dark orange, lemon, and mandarin, respectively. In another study, Rekha et al. [24] determined the total antioxidant capacity of four mature and immature citrus varieties (C. limon, C. reticulata, C. sinensis, and C. auntium) in their study in India. In the study, they were determined as C. limon (95.0%), C. reticulata (96.6%), C. sinensis (97.5%), and C. aurantium (88.8%) in the mature period. Xu et al. [25] determined the total antioxidant capacity of 15 varieties, including 7 types of tangerines, 4 types of sweet oranges, 1 lemon, 1 grapefruit, and 2 pummelo varieties, in their research at the Zhejiang Citrus Research Institute in China. In the study, the highest antioxidant value of the 15 citrus varieties selected at the ripe stage was found in Hybrid 439 (61.62%) and the lowest antioxidant value was found in Manju (23.69%). The values found in our study were close to the values of the study by Rekha et al. [24], while the values in the studies by Tounsi et al. [23] and Xu et al. [25] were found to be higher. It can be revealed that this is due to ecological conditions, genetic factors of varieties, harvest date, and cultural processes.

3.3 Sugars and total soluble solid

The results of sugar and soluble solid contents content of citrus varieties are presented in Table 3. When our study was examined in terms of sugars (sucrose, glucose, and fructose), statistically significant differences were determined. The most important monosaccharide in the diet is fructose sugar and is known as the sweetest of all naturally occurring carbohydrates. The highest value was sucrose in all varieties, followed by fructose and glucose sugars. When citrus varieties were evaluated in terms of sugar components, Moro blood variety had the highest sucrose, glucose, and fructose contents (63.66, 31.91, and 37.99 g/kg, respectively), limequat variety had the lowest sucrose (9.57 g/kg) and fructose (2.98 g/kg), and the lowest glucose in (0.07 g/kg) Alacalı Kalamondin variety was found. Moro blood (33.58 g/kg) variety had the highest sugar content in terms of total sugar, while Alacalı Kalamondin (17.71 g/kg) variety had the lowest sugar content. The sum of sucrose, glucose, and fructose should generally be less than the total amount of soluble solid contents. In our study, the total amount of soluble solid contents was higher than the sum of sucrose, glucose, and fructose for each variety we studied. When our study was evaluated in terms of the total amount of soluble solid contents, statistically significant differences were determined compared to 0.05. Among the varieties, Moro blood (14.2%) variety was the highest in terms of total soluble solid contents, while pink lemon (5.4%) was the lowest.

In a study by Karadeniz [26], soluble solid contents values in C. reticulata, C. sinensis, C. paradisi, C. limon, and C. aurantium samples were 12, 11.5, 10.1, 7.4, and 10%, respectively. Again, Sdiri et al. [27] found that the total soluble solid contents of Safor, Garbi, Fortune, Kara, and Murcott cultivars were 14.5, 16.7, 15.7, 13.6, and 9.3%, respectively. In a study by Kafkas et al. [28] on citrus fruits, sucrose, glucose, and fructose values of citrus species were as 46.6, 25.3, and 29.6 g/kg, respectively. Kelebek and Selli in a study they conducted in Hatay Dörtyol [29] determined the sugar content in orange juice. They found that the major sugar was sucrose (46.40 g/kg). They also stated that glucose value, fructose value, and total sugar as 30.99, 33.05, 110.44 g/kg. In their study, Canan et al. [19] determined the sucrose value of ten varieties as 88.0–26.4 g/kg, the glucose value as 53.0–15.0 g/kg, and the fructose value as 49.0–17.2 g/kg. In this study, the sucrose content was generally higher than glucose and fructose contents, reflecting the significant contribution of soluble sugars and metabolites to overall fruit quality. While many of the findings align with those reported by other researchers, some discrepancies were observed. These differences are likely attributable to various factors, including the variety and maturity of the fruit, climatic and soil conditions, pressing pressure, the extraction solvent used, and the standard solutions employed during analysis.

3.4 Organic acids content

Organic acid content results of citrus varieties are presented in Table 4. When our study was evaluated in terms of organic acids, a total of three organic acids were detected, namely, l-ascorbic acid, citric acid, and succinic acid, and statistically significant differences were found compared to 0.05. When citrus varieties were evaluated in terms of organic acid components, citric acid was found to be the major acid. Ascorbic acid is an important nutrient with its vitamin activity, and it is also important because of its strong antioxidant properties. In our study, Moro blood (1.03 g/kg FW) cultivar had the highest l-ascorbic acid content, while limequat (0.10 g/kg FW) cultivar had the lowest. In terms of citric acid, pink lemon (58.91 g/kg FW) was the highest, while Moro blood (15.73 g/kg FW) was the lowest. Finally, when evaluated in terms of succinic, it was determined that kumquat (2.31 g/kg FW) was the highest and pink lemon (0.13 g/kg FW) was the lowest.

In a study by Karadeniz [26], the average citric acid content in organic acid analyses of various citrus fruits was determined as follows (g/kg fresh weight, FW): C. reticulata: 0.92, C. sinensis: 1.32, C. paradisi: 1.96, C. limon: 5.51, C. aurantium: 4.87. Rekha et al. [24] reported l-ascorbic acid content in different citrus varieties ranging from 0.26 to 0.63 g/kg. Similarly, Violeta et al. [30] used HPLC analysis to determine organic acids in citrus fruits, reporting l-ascorbic acid at 0.636 g/kg and citric acid at 13.918 g/kg. In a study by Kafkas et al. [28] on citrus species, the total organic acid content was determined to be 5.48 g/kg, with a citric acid value of 0.56 g/kg. In addition, Kelebek and Selli [29] conducted a research in Hatay Dörtyol and quantified citric, l-ascorbic, malic, and succinic acids in orange juice. Their findings were as follows (g/kg): citric acid: 14.22, l-ascorbic acid: 0.47, malic acid: 3.91, succinic acid: 1.82, The results obtained in our study were comparable to those reported by some researchers but higher than those of others. These variations may be attributed to factors such as the morphological characteristics of the varieties, genetic factors, climatic and soil conditions, maturity stage, harvest time, the extraction solvents used, and the standard solutions employed in the analyses.

3.5 Volatile compounds

The volatile compounds in the fruit rind and juices of seven varieties were identified (Tables 5 and 6). The same extraction conditions were applied for all samples. The main volatile compounds found in the rind were terpenes – for blue SPME fibre: (Alacalı Kalamondin) 71.28% to (Kumquat) 97.53%, for grey SPME fibre: (Moro blood) 50.84% to (lemonquat) 95.4%, for red SPME fibre” (Moro blood) 51.48% to (lemonquat) 98.94%; esters – for blue SPME fibre: (Indio Mandarinquat) 0.61% to (Alacalı Kalamondin) 13.14%, for grey SPME fibre (limequat): 0.70% to (Moro Blood) 10.85%, for red SPME fibre: (Alacalı Kalamondin and Indio Mandarinquat) 0.69% to (pink lemon) 15.59%; aldehydes – blue SPME fibre: (lemonquat and limequat) 0% to (Moro Blood) 5.14%, for grey SPME fibre: (lemonquat and limequat) 0% to (Moro Blood) (18.23%), for red SPME fibre: (lemonquat and limequat) 0% to (Moro Blood) 19.2%; alcohols – for blue SPME fibre: (Kumquat) 0.31% to (Alacalı Kalamondin) 13.14%, for grey SPME fibre: (kumquat) 1.66% to (Moro Blood) 26.78%, for red SPME fibre (lemonquat) 0.15% to (pink lemon) 27.13%. Forty-one terpene hydrocarbon compounds, 12 alcohol compounds, 11 ester compounds, 1 acid compounds, and 5 aldehyde compounds were determined using blue SPME in the fruit rind seven citrus varieties. In addition, 37 terpene compounds, 12 alcohol compounds, 5 aldehyde compounds, and 10 ester compounds were found in the seven citrus varieties fruit rind using grey SPME fibre. With red SPME, volatile compounds were found in the fruit rind, and we found 37 terpene compounds, 12 alcohols compounds, 5 aldehyde compounds, and 8 ester compounds. The blue SPME fibre is found with more aromatic compounds than other SPME fibres in the fruit rind seven citrus varieties.

The volatile compounds in the fruit juice of seven variety were identified (Tables 5 and 6). Extraction conditions were identical for all samples. The main volatile compounds found in the juice were terpenes for blue SPME fibre (Moro blood) (92.96%) to (kamkat) (99.15%), for grey SPME fibre (Moro blood) (70.23%) to (limequat) (99.69%), for red SPME fibre (pink lemon) (83.88%) to (kamkat) (97.62%); esters for blue SPME fibre (kamkat) (0.14%) to (Moro blood) (2.64%), for grey SPME fibre (kamkat) (0.23%) to Moro Blood (7.69%), for red SPME fibre (limequat) (0%) to (pink lemon) (8.87%); aldehydes for blue SPME fibre (lemonquat, limequat, and kamkat) (0%) to (Moro blood) (2.72%), for grey SPME fibre (lemonquat, limequat, and kamkat) (0%) to (Moro blood) (10.33%), for red SPME fibre (lemonquat, limequat, and kamkat) (0%) to (pink lemon) (0.94%); alcohols for blue SPME fibre (kamkat) (0.33%) to (limequat) (2.90%), for grey SPME fibre (limequat) (0.8%) to (Moro blood) (12.49%), for red SPME fibre (kamkat) (6.82%) to (pink lemon) (27.13%). Twenty-nine terpene hydrocarbon compounds, 12 alcohol compounds, 10 ester compounds, 1 acid compound, and 2 aldehyde compounds were determined using blue SPME in the fruit juice seven citrus varieties. On the other hand, 13 alcohol compounds, 5 aldehyde compound, 10 ester compounds, 1 acid, and 44 terpenes were found in the seven citrus varieties fruit juice using grey SPME fibre. With red SPME, volatile compounds were found in the fruit juice, such as 11 alcohols, 3 aldehyde compounds, 8 ester compounds, 1 acid, and 45 terpene compounds. The grey SPME fibre is found with more aromatic compounds than other SPME fibres in the fruit juice seven citrus varieties. As shown in Tables 5 and 6, the amounts of esters, aldehydes, acids, alcohols, terpenes from aroma compounds varied among varieties. Compounds such as decanal, myrtenal, nonanal, octanal, and undecanal were detected in both fruit juice and rind as aldehydes. Decanal compound was found to have the highest ratio in both rind and juice (Tables 5 and 6). In addition, 3-cyclohexen-1-ol, 4-methyl-1, alpha-terpineol, linalool, santalol alpha, and farnesol were detected in both fruit juice and fruit rind as alcohols. Linalool compound was found to have the highest ratio in both rind and juice (Tables 5 and 6). Moreover, acetic acid 2-ethylhexyl ester, geranyl acetate, and linalyl acetate were detected in both fruit juice and rind as esters. While acetic acid 2-ethylhexyl ester compound was found at high rate in fruit juice, geranyl acetate compound was found at the highest rate in fruit rind (Tables 5 and 6). On the other hand, 1,3,6-octatriene, 3,7-dimethyl, alpha-amorphene, alpha-terpinolene, benzene, methyl, beta-myrcene, Dl-limonene, pseudolimonene, and germacrene-D were found in both fruit juice and fruit rind as terpenes. We found the same number of aroma compounds in all SPME fibres. Among all the volatile compounds of the juice, limonene had a remarkably high content for blue SPME fibre (41.46–71.71%), for grey SPME fibre (0–86.58%), and for red SPME fibre (60.01–90.37%). On the other hand, when we examine the fruit peel, limonene among all its volatile compounds had a remarkable high content for blue SPME fibre (0–78.89%), for grey SPME fibre (0–82.74%), and for red SPME fibre (0–80.57%).

Giovanelli et al. [31] figured out that limonene was one of the main components in all citrus fruits with different percentages. Still, it characterized C. myrtifolia > C. mitis > C. japonica as 95.6% > 75.2% > 71.6%, respectively. The other compounds were inevitably present in low contents. Among them, geranyl acetate (0.49–3.65%), linalyl acetate (0.28–6.21%), methyl benzoate (0.3–0.88%), linalool (0.19–3.29%), linalyl acetate (0.28–6.21%), and alpha-pipene (0.77–1.55%) were identified in the peel of Chinotto. Besides, the other compounds in the pulp of quinotto are linalool (0.07–0.16%), alpha-pinene (0.04–0.49%), alphe-terpinolene (0.05–0.51%), and beta-myrcene (1.31–6.86%). Guney et al. [32] conducted an aroma analysis of five kumquat cultivars Fortunella margarita (Lour.) Swingle, F. crassifolia Swingle, F. obovata Hort. ex Tanaka, F. hindsii (Champ. ex Benth.) Swingle, and limequat (Citrus aurantifolia × F. japonica (Thumb.) under the climatic conditions of Adana. They identified 39 aroma components, with d-limonene (67.78–88.72%) being the dominant compound. Similarly, Gupta et al. [33] identified 111 volatile aroma compounds in 2018 and 101 in 2019, with 91 compounds found to be common across both years. Their study detected 31 terpene compounds in 2018 and 34 in 2019, with limonene, pinene, terpinene, copaene, elemene, and humulene emerging as the predominant terpenes in the juice. In addition, 14 acid compounds were identified as common to both years. Notable acids include nonanoic acid, butyric acid, linoleic acid, and cis-vaccenic acid, which are considered significant. Our findings were consistent with these previous studies as well [31–33]. These differences in results could be because of the maturity stage, cultivation treatments, genetic origins, or climatic conditions.