Significant increase for total soluble solids (TSS) and titrable acidity (TA) existed in the selected fruits in relation to maturity stages (Table 1). TSS (% °Brix) increased as the maturity progressed and Morus laevigata has the highest amount (15.62%) in the tested fruits, similar increasing trend was also observed for TA (%). According to Ercisli and Orhan [19], the levels of 15.9–20.4% for TSS and 0.25–1.40% for TA investigated at fully-ripened stage in white mulberry genotypes are in close agreement to our present data. Similarly, Koyuncu et al. [20] reported 13.11 to 16.23% TSS and 1.35–1.86% TA in black mulberry genotype; these values are also comparable with present values. In agreement with the present study trends, Dıaz-Mula et al. [21] also reported an increase in TSS and TA in different cultivars of cherry fruits analyzed from Spain as the maturity progressed. In a recent study by Chang and Chang [22], M. laevigata cultivar from Taiwan exhibited somewhat higher TSS (20.1%) as compared with M. laevigata species from Pakistan. A higher value of TSS is related to greater sweetness and superior eating quality of a fruit [22].

The effects of maturity on some sensory attributes such as texture, flavor and appearance of fruits are presented in Table 2. As fruit progressed from un-ripened to full-ripened stage, significant (p < 0.05) improvement in flavor, appearance and texture were recorded by the panelist. Taking into account data for flavor, appearance, texture, acidity and TSS (sensory and nutritive parameters), the fruits more appreciated could be “M. laevigata”, “korona” and M. macroura. From commercial view-point, in strawberry, mulberry and sweet cherry, appearance is considered to be an important quality index that has the strongest influence over price formation. Chitarra and Chitarra [23] reported a positive and significant correlation between TSS and flavor. Flavor of a fruit is characterized by several factors such as sweetness, acidity, and astringency, in combination with aroma, due to the volatile compounds involved. Generally higher values of TA in a typical fruit are negatively linked with the flavor and consumer acceptance [23].

Statistically significant (p < 0.05) variations in the amounts of three key sugars namely fructose, glucose and sucrose were found in strawberry, sweet cherry and mulberry fruits with regard to the three maturity stages (Table 3).

The total sugar contents (sum of sucrose, glucose and fructose) for ripe strawberries in the present study ranged from 4.8–5.4 g/100 g FW. These levels fell in the range of those investigated earlier, wherein the total sugar concentrations of 8.2 g/100 g FW [24] and 3.6 g/100 g FW [5] were reported. In the present study glucose and fructose were the predominant sugars for each of the strawberry varieties studied, while sucrose was usually present at lower concentrations, typically 5–10 times lower. Literature studies indicate that there is some variation, and little consistency with respect to the relative concentrations of the sugars. Basson et al. [5] reported that glucose (1.6–1.8 g/100 g FW) was the predominant sugar in ripe strawberry fruit of two cultivars Festival and Ventana while the sucrose (0.9–1.1 g/100 g FW) and fructose (0.9–1.2 g/100 g FW), with almost similar concentration, were lower relative to glucose. Castro et al. [24] studied the concentration of the sugars in ripe strawberries from the cultivars Camaraso and Selva and found that, in agreement with this study, fructose and glucose were the main sugars, 3.1–5.5 and 3.5–6.7 g/100 g FW, respectively and that of sucrose (0.6–0.9 g/100 g FW) was present at relatively lower level. Similarly, Sturm et al. [25] studied 13 strawberry varieties and reported sucrose and glucose to be the dominant sugars, the content of the former component were typically 10 times lower than the later.

In sweet cherry, fructose (2.34 g/100 g FW) was the predominant sugar followed by glucose (0.39 g/100 g FW) and sucrose (0.13 g/100 g FW). In agreement to the present study trend, Serradilla et al. [10] also reported an increase in the fructose level during ripening of sweet cherry. The total sugar content for ripe sweet cherry, 2.87 g/100 g FW, in the present study was lower than that reported by Serrano et al. [12].

Within the mulberry fruits, fructose was determined to be the principle sugar compound. Morus laevigata, M. macroura, the long mulberry fruits, contained 3.07 and 5.72 g/100 g fructose at un-ripened stage and 4.59 and 9.41 g/100 g at fully-ripened stage, respectively showing an increasing trend in the content of this sugar as maturity progressed from un-ripened to fully-ripened stage. The glucose content of these two mulberry fruits also increased with ripeness; however a reverse trend was observed for sucrose, the concentration of which decreased with ripening. Similar results were also recorded for small mulberry namely M. nigra and M. alba fruits investigated as part of this study (Table 3). Ozgen et al. [18] reported that M. nigra and M. rubra contained fructose in the range of 4.86–6.41 and 2.77–4.66 g/100 mL, glucose (5.50–7.12, 2.85–4.96 g/100 mL) and sucrose (0.01–0.07, 0.04–0.10 g/100 mL), respectively. Nevertheless, there are no detailed data on the sugars composition of Morus laevigata and M. macroura with which we can compare our present results.

The total contents of sugars among mulberry fruits at fully-ripened stage varied between 7.93 g/100 g (M. laevigata) to 14.39 g/100 g FW (M. macroura). These values are in line to those investigated earlier by Ahmad and Sadozai [26] for ripe black mulberry fruit (9%) from another province namely Khyber Pakhtunkhwa, Pakistan. In consistent with our present data, Elmac and Altuq [27] reported the total sugar contents to be varied between 11.3 and 16.2% in three black mulberry (M. nigra) cultivars from Aegean region of Turkey. The slight variations in the total sugar contents of different mulberry fruits may be due to differences in genotypes and agroclimatic conditions of the harvesting regions.

Overall, the contents of total sugars varied widely among the different fruits analyzed, the highest amount was determined in fully-ripened M. macroura (14.39 g/100 g) while the lowest in fully ripened sweet cherry (2.87 g/100 g) fruits. This can be supported by the literature which reveals that soluble sugar composition in fruits varies among species, varieties, and cultivars [28]. As far as is concerned about the effect of fruit maturation/ripening on the individual sugars, the concentration of glucose and fructose generally increased as the maturity progressed from unripe to ripened stage for all the tested fruits. The content of sucrose increased in strawberry and sweet cherry; while its concentration decreased regularly in mulberry cultivars as the maturity progressed. In agreement with the present investigation, some previous reports also reveal that the content of sugars (fructose, glucose and sucrose) varies with fruit maturity depending upon the cultivation regimes and harvest time [10,24,29].

A total sweetness index concept was used to assess fruit sweetness as sucrose equivalent (Suceq) (Table 3). Based on these results, M. macroura had the highest total sweetness index (19.95 Suceq) followed by M. nigra (11.19 Suceq) while sweet cherry had the lowest sweetness index (4.48 Suceq) at fully ripened stage. The sweetness index reported by Crespo et al. [6] in four strawberry cultivars (viz., Antea, Asia, Clery, Matis), ranging from 5.31–8.32, was within the range of this study values.

Statistically significant (p < 0.05) differences were found for malic, citric, ascorbic, and total acid contents of the fruits studied at different maturity stages (Table 4). For the strawberry varieties studied, citric acid (1200–1434 mg/100 g FW) was the dominant organic acid at the fully-ripened stage and was typically present at concentrations 10 times higher than the second most abundant acid, ascorbic acid (90–131 mg/100 g FW). Tartaric acid was detected at concentration 48–55 mg/100 g FW, while malic acid was present at much lower concentrations (0.7–2.6 mg/100 g FW). The presently determined concentration of tartaric acid in strawberry is quite comparable with that investigated by Koyunch and Dilmacunal [30] in “Dorit” (40 mg/100 g) and “Selva” (48 mg/100 g) cultivars of strawberry. Tartaric acid, which can not only inhibit the precipitation of melanin, but can, also moisten the skin, is useful against dry skin. Fruits rich in tartaric acid such as star fruit are undoubtedly good for maintain healthy skin. As the strawberry cultivars studied presently from Pakistan, do contain considerable amount of tartaric acid, these can be explored as a valuable moistening agent for cosmetics products.

In agreement with our present results, Holcroft et al. [31] also found citric acid as the principle organic acid in strawberry. Similarly, the study conducted by Castro et al. [24] on fresh Camarosa and Selva strawberry cultivars at fully ripening stage also reported citric acid (747 mg/100 g) to be the most abundant organic acid and malic acid (117, 20 mg/100 g) to be the least abundant of the acids studied, however, the concentration of malic acid was relatively higher compared to the present study. Organic acids content of strawberry fruit increased as the fruit maturity progressed; the most significant increase in the content of ascorbic acid was observed. Other authors have also reported marked increase in the ascorbic acid content of strawberries fruit during ripening [7,32,33].

Meanwhile, ascorbic acid was determined as the major organic acid (17 mg/100 g) in fully ripened sweet cherry fruit followed by citric acid, tartaric acid and malic acid. Usenik et al. [15], in contrast to present finding, determined malic acid with contribution 353–812 mg/100 g FW, as the principle organic acid among different cultivars of sweet cherry. Likewise, in another study by Souci et al. [34], malic acid was found to be the major acid in fully ripened cherry fruit.

For the mulberry species studied, citric acid (773–987 mg/100 g FW) was the most abundant organic acid found in M. laevigata and M. nigra at fully-ripened stage, however it was not detected in other Morus species. Ascorbic acid (17–305 mg/100 g FW) was the second main acid, determined to be 10 times higher in M. macroura and M. nigra than in M. laevigata and M. alba. Tartaric was also present in considerable amounts (10–118 mg/100 g FW) while malic acid was present at a smaller concentrations (2.6–4.1 mg/100 g FW). In contradictory to our present result, black mulberry genotype, grown in different agro climatic regions of Turkey, contained malic acid, in the range of 123–209 mg/g, as the major acid followed by citric acid (21–41 mg/g) [19]. Similarly, in another investigation, malic acid was determined to be the predominant acid with an average amount of 57.5 mg/g, while citric 8.2 mg/g and tartaric 3.4 mg/g were the other important acids found in M. nigra, black mulberry from Sutculer districts of Isparta, Turkey [20]. Nevertheless, there are no literature data on the organic acids analysis of Morus laevigata, M. macroura with which to compare the values of this study. Ozgen et al. [18] conducted a study on M. nigra accessions; citric acid was 92% of the total acids while malic acid was only 8%. The same ratios were 15 and 85%, respectively, for M. laevigata.

Overall, among the fruits tested, strawberry cultivars were found to be the main source of total organic acids contents (1300–1600 mg/100 g FW). As far as is concerned about the distribution of individual organic acids, ascorbic was found to be principle component in sweet cherry, M. nigra and M. macroura while citric acid in strawberry cultivars, M. laevigata and M. nigra imparting these species a superior nutritional value. Overall, during fruit development, the amount of selected organic acids increased in strawberry, sweet cherry and mulberry as maturity progressed.

Fruit samples of strawberry (Fragaria x ananassa Duch), cultivars (Korona and tufts), sweet cherry (Prunus avium L.) and mulberry species (Morus alba, M. nigra, M. macroura and M. laevigata) at un-ripened, semi-ripened and fully-ripened stage were collected from the orchards in the region of Lahore, Kalat and Faisalabad, respectively of Pakistan during April-July, 2009. The selection of the fruits at different maturity stages was based upon their colour and texture (Table 5). The fruit (2 kg for each category) were collected in polyethylene bags, transferred to the experimental lab and stored in a refrigerator at 5 °C overnight for further analyses.

Total soluble solids (TSS) were determined in the juice produced from fruits at each maturity stage using a digital refractometer Atago RX-7000cx (Atago Co. Ltd., Japan) at 25 °C and results expressed as % °Brix. Total acidity (TA) of the juice (1 mL of diluted juice in 25 mL distilled H₂O) was determined by potentiometric titration against 0.1N NaOH up to pH 8.1. All determinations were made in triplicate and the results expressed as g of citric acid equivalent per 100 g fresh weight.

Sensory analysis was carried out by a 15 member panel consisting of University students and staff who were trained in a one-hour training session to become familiar with the characteristics of the fruits. Panel evaluated the fruit samples using sorting-preference tests and hedonic scale. According to his/her preferences, panel were asked to sort the fruit sample on 9-point hedonic scale (1 = dislike extremely, 5 = neither like nor dislike, 9 = like very much), taking into account flavor/taste, appearance and texture. Each panel evaluated three samples of each cultivar at each maturity stage, previously randomized to avoid position bias.

Fresh samples of strawberry, sweet cherry and mulberry fruit (10 g) were homogenized with 20 mL of distilled water using a commercial blender (Ika-Labortechnik). The fruit puree was clarified by centrifugation at 3000g for 10 min. The supernatant was filtered through 0.25-μm Millipore filters. Samples were prepared in triplicate, stored in a refrigerator and analyzed within 12 h of preparation.

Chromatographic analysis of sugars was carried out on an Agilent 1100-series HPLC system equipped with a quaternary pump (G1311A), vacuum degasser (G1379A), auto-sampler/auto-injector (G1313A ALS), column compartment (G1316A Colcom) and a refractive index detector (model RID10A). The sugars (glucose, fructose and sucrose) were separated on a Bio-Rad Aminex HPX-87K 300 × 7.8 mm column (Cat. #1250142). A Bio-Rad Guard column containing the same stationary phase as the separation column was fitted to the front of the separation column. The mobile phase was ultra pure H₂O at a flow rate of 0.6 mL/min. The sample volume injected was 20-μL. A refractive index detector maintained at 68 °C was used for detection purposes. The sugars were identified on the basis of comparison of their retention times with those of pure standards (Sigma-Aldrich) and quantified using standard calibration curves (in the range of 1 mg/100 mL to 5 mg/100 mL). Agilent Chem Station software was used to process the chromatographic data.

A sweetness index was used to estimate the total sweetness perception described by Baldwin et al. [35] and Obando-Ulloa et al. [36] as sucrose equivalents (Suceq) according to the formula:

HPLC analysis of organic acids was performed on an Agilent 1100- series HPLC system equipped with a Quaternary pump (G1311A), vacuum degasser (G1379A), auto-sampler/auto-injector (G1313A ALS), column compartment (G1316A Colcom) and diode array detector (DAD) (model G1315B DAD). The separation was carried out using a Hibar^® RP-C18 column (250 mm × 4.6mm; 5 μ particle size) from Merck Company (Merck KGaA, 64271 Darmstadt, Germany) thermostated at 30 °C. The mobile phase consisted of 5% methanol in 25 mM potassium dihydrogen phosphate (pH was adjusted to 2.10 by dilute phosphoric acid), and was delivered at a flow rate of 1.0 mL/min. The mobile phase was filtered through Nylon membrane filter (47 mm, 0.45 mm) and degassed by sonication before use. Target compounds were detected at 215 nm. Quantitative measurements were based on the external standard calibration 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 (malic, citric, tartaric and ascorbic acids) were used to prepare the standard curve (peak area versus concentration in mg/L). Calibration curves had correlation coefficients of 0.99 or greater. An Agilent Chem Station was used to process chromatographic data.

Univariate analysis of variance (ANOVA) was performed in each individual fruit trait (sugar or organic acid) using Minitab 2000 Version 13.2 statistical software [37]. The stage of maturity was the factor and a probability value of p < 0.05 was considered to denote a statistically significant difference.