Results and discussions

The gradient elution system with C30 stationary phase applied in this study for the quantification of carotenoids and tocopherols provided good resolution, precision, and repeatability (Supplementary material Figure S1). Carotenoids were quantified without saponification, as most of the monohydroxy carotenoids in studied samples were found non-esterified in preliminary studies (data not shown). In the quality control study, the mean contents of major carotenoids, all-E-lutein, all-E-β-carotene, and all-E-lycopene (11.5, 21.3 and 12.2 μg/g, respectively) in certified reference material (CRM) (BCR-485), were found in accordance with certified values (12.5, 23.7 and 13.8 μg/g, respectively). The mean recovery percentage for α-tocopherol was 94.2%. Thus, these carotenoids and α-tocopherol data validated the accuracy of applied extraction methods from the tomato fruits. In HPLC method validation of carotenoids and tocopherols, the low coefficients of variation (% CV; always below 3.80 and 0.80 for peak areas and retention times, respectively) were recorded for intra-day (n = 6) and inter-day (n = 6 × 2) values assessed for peak area and retention time. The linear calibration curves also showed a high coefficient of correlation between area counts and standard concentrations (R ²; >0.992–0.994). With regard to these parameters, the currently used HPLC method shows good accuracy, linearity, precision, and stability. Using this approach, all-E- lutein, all-E- β-carotene, and all-E- lycopene were identified as the major carotenoids in tomato fruits of different ripening stages, on the basis of retention time with authentic standards and also by comparing the peak spectra recorded with a diode-array detector (DAD) during the analysis. The chromatograms (470 nm) and the peak spectra of major identified peaks were shown in Supplementary material Figure S1. The other minor carotenoids were not quantified due to the unavailability of standard compounds. During the ripening process, profiles of all the studied metabolites altered significantly (p < 0.05). All-E-lycopene content increased from the breaker (0.21 μg/g FW) to the red stage (30.6 μg/g FW), while all-E-lutein was slightly increased during initial ripening stages and then decreased significantly, with highest values during the pink stage (Fig. 2).

The content of all-E-lutein, all-E-β-carotene, all-E-lycopene, and α-tocopherol in tomato fruits of different ripening stages. Values above bars displaying the data labels. Values are mean ± standard deviation from six replicates (triplicate extractions with duplicate analysis). Different superscript letters indicate statistically significant differences among the various ripening stages (p < 0.05)

Regulation of carotenoid biosynthesis and high-accumulation lycopene during tomato fruit development is widely studied (Ronen et al. 1999; Moco et al. 2007; Su et al. 2015). Moco et al. (2007) observed an increase in β-carotene, reduction in neoxanthin contents, while lutein was virtually constant during tomato fruit development. In the present study, we have also recorded an increase in all-E-β-carotene and all-E-lycopene contents during ripening. However, all-E-lutein showed a profile that was slightly different from these carotenoids. This carotenoid first increased up to the pink stage (stage 4) and then decreased to the red stage. This may be due to the difference in cultivars, as, during tomato fruit ripening, the cultivar-specific pattern of accumulation of β-carotene is reported (Bhandari and Lee 2016). Authors recorded the higher β-carotene content in pink and light red stages of cherry tomatoes. However, cultivar Dafnis showed a higher level of β-carotene content in red stage than in other stages. During tomato fruit development, the mRNA levels for the lycopene-producing enzymes phytoene synthase (PSY) and phytoene desaturase (PDS) increase significantly, while the mRNA levels of the genes for the lycopene β- and ε-cyclases, which convert lycopene to either β- or δ-carotene, respectively, decline and completely disappear (Ronen et al. 1999). Thus, lycopene is produced as major carotenoid in cell-localized phytochromes and confers the red color to ripe tomato fruits.

In nature, vitamin E (tocochromanols or tocols) consists of four tocopherols (α-, β-, γ-, and δ-tocopherol) and four tocotrienols (α-, β-, γ-, and δ-tocotrienols), well-known for its potent antioxidant and anticancer activities (Saini and Keum 2016). Among various tocols, α-tocopherol was recorded as the major tocopherol in tomato fruits of various developmental stages. The level of α-tocopherol increased during ripening in all tissues, though its increase was largest between light red to red stages (Fig. 2). Similar trends of α-tocopherol accumulation were previously recorded in tomato fruits during development (Moco et al. 2007). α-Tocopherol plays a significant role in cell signaling toward environmental and developmental signals (Traber and Atkinson 2007). In non-climacteric fruit (such as grape, Vitis vinifera), tocopherol contents decline gradually along with its development (Horvath et al. 2006), whereas in climacteric fruits (such as mango and tomato), exhibits an inverse pattern (Singh et al. 2011). The intense respiration and ethylene production in climacteric fruits are associated with an oxidative phenomenon with increased hydrogen peroxide content, lipid peroxidation, and protein oxidation (Jimenez et al. 2002). As a result, within this oxidative environment, the maintenance or increase in tocopherol levels by activation of the tocopherol biosynthetic pathway is clearly advantageous to balance their levels (Quadrana et al. 2013). Additionally, the chlorophyll degradation during ripening also favors the tocols biosynthesis by utilizing chlorophyll degradation-derived phytyl-diphosphate in tocopherol biosynthesis (Almeida et al. 2015). Tomato fruit ripening is also reported to increase the total phenolics, flavonoids, vitamin C, and antioxidant potential (Periago et al. 2009).

A large number of studies have been conducted to analyze the temporal changes in the contents of carotenoid and tocopherol during the ripening of tomato fruits. This study extends their results by analyzing the fatty acid composition during the various developmental stages of tomato fruits. The GC–FID analysis revealed the presence of 10 major fatty acids (C13–C24) in the unripe and ripened fruits (Supplementary material Figure S2). Among all the ripening stages, linoleic acid (C18:2n6c) was found in the highest quantity (42.3–49.2%), followed by oleic (C18:1n9c; 20.1–26.6%) and palmitic acids (C16:0; 16.6-17.7%) (Table 1). Similar compositions of fatty acids were reported in red ripened tomato fruits cultivated in greenhouses (Guil-Guerrero and Rebolloso-Fuentes 2009). The improvement in contents of linoleic and α-linolenic acids were previously reported in mature green tomato fruits stored at non-chilling and chilling temperatures (2 and 15 °C, respectively) for 12 days (Whitaker 1991). To our knowledge, no reports are available on the changes of fatty acid compositions during ripening of tomato fruits on the vine; however, this phenomenon has been widely studied in other climacteric fruits, including mango (Lalel et al. 2003) and avocado (Villa-Rodríguez et al. 2011). Lalel et al. (2003) studied the production of aroma volatiles during fruit ripening of ‘Kensington Pride’ mango and found that most of the fatty acids increased during fruit ripening. The linoleic and linolenic acids were also found to increase during ripening. However, their proportion (% fatty acid to total fatty acids) was first increased during initial ripening stages (0–6 days) and then decreased in further ripening stages (7–10 days). Mango fruits were ripe on the seventh day of the ripening period, which corresponded to the fruit being eaten soft and a skin color that was 75% yellow. The production of major aroma volatiles, monoterpene, and sesquiterpene hydrocarbons was also found concordant with fatty acids. Thus, it is considered that fatty acids are the primary precursors of volatile compounds responsible for plant aromas, modulated by ethylene and storage conditions during fruit ripening (García-Rojas et al. 2016). Villa-Rodríguez et al. (2011) studied the variations in fatty acid content during ripening of ‘Hass’ avocado and observed significant increase in total content of monounsaturated (MUFAs) and saturated fatty acids (SFAs) and a decrease of polyunsaturated fatty acids (PUFAs) during the ripening period, resulting in decrease of PUFAs: SFAs ratio. Contrasting to this, in the present study, PUFAs: SFAs ratio was increased significantly during ripening, from 1.89 in green fruits (stage 1) to 2.19 in red fruits (stage 6). Therefore, according to these results, it appears that the nutrition value of tomato fruit increased during ripening. α-Linolenic acid and linoleic acid (ω-3 and ω-6 FAs, respectively) are the essential fatty acids (EFAs) for the human diet. Though in tomato fruits, fatty acids are the minor component, the high proportion of PUFAs in fully ripened fruits can add their health benefits. Interestingly, the oleic acid proportions correlated inversely with linoleic (r = −0.945) and α-linolenic acid (r = −0.904) during all the stages of ripening. The contents of linoleic acid were lowest (42.3%) in fruits of stage 3 (turning) and increased in red ripened fruits (49.3%). This is probably due to the enhancement of enzymatic activities of desaturases, especially Δ12-desaturase responsible of linoleic acid biosynthesis, using 18 carbon fatty acids as substrate stimulation. Aidi Wannes et al. (2009) studied the variations in essential oil and fatty acid composition during Myrtus communis fruit maturation and found that linoleic acid proportions correlated inversely with palmitic and oleic acids during all the stages of ripening. In another study, SFAs and PUFAs decreased significantly, and MUFAs increased during maturation of coriander fruit (Msaada et al. 2009). Results of this study and also previous studies indicate that the variation in the fatty acid composition of fruit during ripening/maturation might be useful in understanding the source of nutritionally and industrially important phytochemicals.

Composition of fatty acids in tomato fruits of six ripening stages

Green (Stage 1), breaker (Stage 2), turning (Stage 3), pink (Stage 4), light red (Stage 5), and red (Stage 6). Values are percentages of the fatty acids in the total lipids (fresh weight basis), from an average of six replicates (triplicate extractions with duplicate analysis). Superscript letters indicate statistically significant subsets among different fruits of the various ripening stages (p < 0.05)

RT retention time