Improved functional and nutritional properties of tomato fruit during cold storage.

The use of synthetic antioxidants has been associated with serious concerns for human and environmental health. During ripening stages, tomato fruit is exposed to different abiotic stresses which not only influence its nutritional, mechanical, and functional properties at harvest, but also affect the quality and shelf life of the fruit during storage. This study investigated the pattern of changes in dietary antioxidants during various ripening stages of tomato fruit (cv. Red Rose) and their impact on storage behavior of the fruit during cold storage. Tomato fruits were harvested at mature green, breaker, turning, pink, light-red and red stages of maturity. Then, they were analysed for flesh firmness, soluble solids content, titratable acidity, total sugars, pH, dry matter content, lipophilic (lycopene, β-carotene, and total carotenoids), and hydrophilic (ascorbic acid, phenolic and flavonoids) antioxidants. Additional fruits were harvested at each maturity stage and divided into three equal lots, then were subjected to low-temperature (10 ± 1 °C) storage with 80 ± 5% RH, for 7, 14, and 21 days. Flesh firmness, and the levels of dietary antioxidants were analysed following the subsequent storage periods. The results revealed that the peak of hydrophilic antioxidants such as ascorbic acid, phenolic compounds, and flavonoids was between the 'pink' and the 'light-red' stages of fruit maturity. Whereas tomatoes harvested at the 'red' stage of maturity had the highest levels of lycopene and β-carotene. Both the stage of fruit maturity at harvest and duration of cold storage influenced flesh firmness, organoleptic and functional properties of 'Red Rose' tomato fruit. In conclusion, the results of the current investigation have practical implications in formulating foods with improved functional properties at processing industries.

Introduction

Tomato (Solanum lycopersicum L.) fruit is not only an integral part of cuisines all across the globe but also serve as a staple food for many nations. The cultivated world area and the total production of tomato during 2018 were 4.76 M ha and 182.25 Mt, respectively (FAOSTAT, 2020). Being a rich source of vitamins, minerals, phenolic content, flavonoid groups, dietary fibers, proteins and a large number of antioxidant compounds, tomato helps us effectively in fighting against many types of cancer (Tilahun et al., 2017). In addition, it reduces the risk of developing hypertension and other cardiovascular diseases (Cheng et al., 2017). Tomato is one of the rich sources of natural antioxidants. Therefore, the beneficial properties of tomatoes are mainly attributed to a diverse range of antioxidative, chemo-preventive and anti-proliferative activities of its dietary antioxidants (Çelik et al., 2017). These compounds are known to decrease the adverse effects of reactive oxygen species (ROS) produced during the normal metabolic reactions such as cellular respiration and photosynthesis, due to environmental stresses or UV irradiation (Foyer, 2018). These ROS cause serious oxidative damages to lipids, DNA, carbohydrates and proteins in our bodies (Yang et al., 2019).

The accumulation of antioxidants in fresh biological systems are significantly influenced by different genetic and environmental factors including genotype (Coyago-Cruz et al., 2017, Flores et al., 2017), growing conditions and cultivation practices (Anton et al., 2017, Klunklin and Savage, 2017), storage conditions and duration of storage period (Tilahun et al., 2017). Stage of fruit maturity at harvest is one of the most important factors that determines the levels of dietary antioxidants in tomatoes (Nour et al., 2015). During ripening, the fruit is exposed to a diverse range of oxidative stresses which sequentially results in significant changes in the levels of antioxidant compounds and consequently to their antioxidant capacity at harvest as well as during storage (Wang et al., 2019, Loayza et al., 2020).

To address the growing demand of natural, preferably plant-based, antioxidants around the globe, it appears appropriate to equip the relevant industries with the knowledge of pattern of changes in nutritional and functional properties of fresh produce during growth, development and ripening and how these changes affect their quality and shelf life during storage. The present investigation focused on analyzing the activities of dietary antioxidants at various ripening events in ‘Red Rose’ tomato fruit, grown under greenhouse conditions. The storage behavior of tomato fruit in relation to its organoleptic and textural properties was also observed. The outcomes from this investigation may also help to provide a systematic base of information to produce/formulate tomato-based food or food products with better functional traits.

Materials and methods

Chemicals, reagents and standards

Reference compounds (gallic acid, quercetin, L-ascorbic acid, β-carotene, glucose) and Folin-Ciocalteu (FC) reagent were sourced from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other chemicals/reagents and organic solvents used in this study were of analytical grade and were procured from the local supplier (Somatco Scientific Supplies Co., Riyadh, Saudi Arabia).

Plant material and sampling

Tomato seeds cv. Red Rose, sourced from Reimer Seeds, Saint Leonard, MD, USA, were sown on September 20, 2017 and 40-day old seedlings were transplanted in plastic pots (pot size: 25 cm; growing medium: sand and poultry manure 1:1). Plastic pots were placed in the on-campus greenhouse of the Protected Cultivation Unit of King Saud University (24.7256° N, 46.6153° E) in Riyadh (Saudi Arabia). They were spaced at around 50 cm apart with 100 cm between the rows. The design of the experiment was completely randomized design. During the entire investigation, crop inputs such as fertilizers, water, and plant protection products were applied to the plants following standard cultural practices. Based on the exterior color of the fruit, 160 tomatoes were harvested at each of mature green (MG), breaker (BK), turning (TG), pink (PK), light-red (LR) and red (RD) stages of maturity (California Tomato Commission, 2008). Ten fruits from each replication (r = 4) were immediately analyzed for flesh firmness, soluble solids content (SSC), titratable acidity (TA), total sugars, pH, dry matter (DM) content, lipophilic (lycopene, β-carotene, and total carotenoids), and hydrophilic (ascorbic acid, phenolic and flavonoids) antioxidants. The remaining fruits (120) harvested at each maturity stage were divided into three equal lots which were subjected to low-temperature (10 ± 1 °C) storage with 80 ± 5% RH, for 7, 14, and 21 days. Flesh firmness, and the levels of dietary antioxidants were analyzed following the subsequent storage periods.

Analysis of fruit

Flesh firmness

The firmness of tomato flesh was measured on their pared surfaces with the digital penetrometer, (Model BKD020; WEL, Willow Bank Electronics Ltd., Napier, New Zealand) fitted with an 8-mm plunger. The results were expressed in Newton (N).

Discriminative properties

The soluble solids content (SSC) of tomato juice freshly extracted from the composite fruit samples in each replication (r = 4) were estimated with the portable refractometer (Model: FG-103, Chincan brand, Hangzhou Weiku Co. Ltd., Zhegiang, China) as described earlier (Abbasi et al., 2019) and the results were expressed as °Brix. TA of tomato juice from each replication was gauged using the standard method (AOAC, 1990) while the acidity (pH) of the juice was determined by the pH meter (AE150 Benchtop pH Meter, Waltham, MA, USA).

Soluble sugars content in tomato pulp was estimated by the method described by Dey (1990). Briefly, the pulp (0.5 g) was incubated in 10 mL of ethanol (90%) at 60 °C for 1 h in a 25-mL conical flask. Following incubation, the volume was adjusted to 25 mL with 90% ethanol. In a glass test tube, 1 mL of phenol reagent and 5 mL of H2SO4 were thoroughly mixed with the aliquot (1 mL) by vertical agitation. Optical density (OD) of the supernatant was recorded at 485 nm (Ultrospec 2000 UV/VIS, Amersham Pharmacia Biotech, Little Chalfont, UK). Soluble sugars content was estimated against the standard curve plotted using the glucose solution used as the reference standard. Dry matter (DM) content of tomato sample was determined by the percent difference of their masses before and after drying in an oven at 60 °C to a constant mass.

Dietary antioxidants

Five fruits from each replication (r = 4) were cut into halves to remove their seeds and placenta. The pericarp slices of fruit were sheared (Burden, 2012) in a waring blender (Sanyo Food Drink Blender; Sanyo Electric Co., Ltd. Osaka, Japan), into a fine homogenate which was divided into two groups. The first group was immediately used to estimate ascorbic acid, lycopene, β-carotene, and total carotenoid contents of tomato samples. The second lot was freeze-dried and the samples (r = 4) were stored in small air-tight/water-proof plastic containers at –20 °C until needed for further protocols to estimate total phenolic and flavonoid contents.

Lipophilic antioxidants (carotenoids)

Lycopene and β-carotene contents in tomato pulp were simultaneously estimated by the method described by Nagata and Yamashita (1992) with minor modifications. Briefly, the pulp (1 g) was homogenised with 5 mL of acetone-hexane (4:6) solution in a plastic test tube. ODs of the clear supernatant were recorded at 663, 645, 505, and 453 nm. Lycopene and β-carotene contents, reported as microgram per gram of fresh mass (µg g−1, fm), were calculated by the following equations (Nagata and Yamashita, 1992):

Total carotenoid content (TCC) of tomato pulp was separately measured by a modified method from Malik and Singh (2006). Briefly, the tomato homogenate (2 g) was macerated in 12% ethanolic KOH and was saponified in the water bath (37 ± 1 °C; 30 min). By vigorous shaking, the carotenoids were extracted in petroleum ether. The mixture was allowed to stand for 10 min to produce two distinct layers. The yellow layer at the top was collected in a conical flask. The OD the supernatant from yellow-colored fraction was recorded at 450 nm. TCC of tomato pulp (µg g−1 fm) was calculated by the extinction coefficient of pure β-carotene (i.e., 2500 in petroleum ether).

Hydrophilic antioxidants

Ascorbic acid content

Ascorbic acid (AsA) content in tomato pulp was estimated by the method described by Hans (1992). The pulp (5 g) was homogenised with 5 mL of 1.0% hydrochloric acid and the mixture was centrifuged at 10,000 rpm for 10 min. The OD of the supernatant was recorded at 243 nm. AsA content was estimated in comparison with the standard curve against L-ascorbic acid used as the reference standard.

Phenolic and flavonoid compounds

Extraction

The plastic containers were submerged in tap water for 1.5 h to get the samples thawed. The fruit homogenate (1 g) was then extracted by dynamic maceration in 10 mL of absolute ethanol using an electric shaker {(Precise Shaking Incubator, Model: ThermoStable IS-20); Daihan Scientific Co., Gangwon-do, South Korea) set at 200 rpm for 16 h at 25 °C. Following maceration, the extract was filtered through Whatman No. 2 filter paper. The residue was re-extracted using absolute ethanol and the supernatants were pooled. Using absolute ethanol, the total volume was adjusted to 10 mL. The supernatant was stored at – 20 °C until needed (used within 3 weeks).

Total phenolic content (TPC)

Total phenolic content (TPC) in the fruit sample was estimated by the Folin-Ciocalteu (FC) colorimetric assay based on chemical reduction of the reagent (Singleton and Rossi, 1965) as described by Surana et al. (2016) with minor modifications. Gallic acid was used as the reference standard. To prepare the reaction mixture, 1 mL of the stock solution of each sample extract (i.e. 1 mg mL−1) was mixed with 1 mL of each standard (gallic acid) solution, 1.5 mL of Folin-Ciocalteu reagent and 10 mL of distilled water in a 20-mL volumetric flask. The mixture was allowed to react at 22 ± 1 °C for 10 min. Sodium carbonate (Na2CO3) solution (4 mL) was then added to the mixture and the volume was made up with distilled water. The reaction mixture was re-incubated for 90 min at 22 ± 1 °C for another 10 min. OD of the clear supernatant was recorded at 765 nm against the blank. Exactly the same procedure was followed for gallic acid solutions with known (20, 40, 60, 80, and 100 mg·L-1) concentrations to construct the standard curve (r2 = 0.997). The results were expressed as microgram gallic acid equivalents per gram of fresh mass (µg GAE g−1 fm). All samples were analyzed in triplicate to ensure the validity of the procedure.

Total flavonoid content (TFC)

Total flavonoid content of tomato pulp was estimated following the modified method of Nour et al. (2015). Briefly, 0.5 mL of tomato ethanolic extract (i.e. 0.5 mg mL−1) was diluted with ethanol (1:10 ratio) in a glass test tube. To prepare the reaction mixture, 0.1 mL of AlCl3 (10%), 0.1 mL of molar aqueous solution of potassium acetate and 4.3 mL of ethanol were added to the extract and the mixture was allowed to react for 40 min at 22 ± 1 °C. OD of the supernatant was recorded at 415 nm. The standard curve was plotted with various known concentrations of quercetin standard. The results were expressed as microgram quercetin equivalents per gram of fresh mass (µg QE g−1 fm).

Data analysis

Data were subjected to Analysis of Variance (ANOVA) following PROC GLM procedure using SAS 2.9 software and least significant differences (Fisher’s LSD) were calculated at P ≤ 0.05. All the assumptions of ANOVA were checked to ensure the validity of statistical analysis.

Results and discussion

Discriminative properties

Among the important quality traits during ripening of green-house tomatoes are soluble sugars, acidity, pH, antioxidant composition, colour and firmness (Bui et al., 2010). No significant difference in SSC, TA, and SSC/TA ratio was observed in tomato fruit harvested at various stages of fruit ripening (Table 1). However, soluble sugars content, active acidity (pH) of the juice, and dry matter content of tomato fruit were significantly (P ≤ 0.05) affected by the stage of fruit maturity at harvest. Soluble sugars content in tomato pulp was significantly increased from 10.72 g kg−1 at MG stage to 25.38 g kg−1 at RD stage of fruit maturity. A gradual increase in soluble sugars content in tomato pulp was observed between the period from MG to LR stages of fruit maturity whereas the fruit harvested at LR and RD stages of maturity were similar in soluble sugar content (~25 g kg−1) in their pulp.

Table 1

Changes in physico-chemical characteristics of ‘Red Rose’ tomatoes as affected by different stages of fruit maturity at harvest.

Stages of fruit maturity	SSC (°Brix)	TA (g CA L−1)	SSC : TA	Soluble sugars (g kg−1)	pH	Dry matter (%)
Mature green	4.27 ± 0.07	4.27 ± 0.11	10.00 ± 0.30	10.72 ± 0.38 d	4.25 ± 0.03 d	7.60 ± 0.17b
Breaker	4.20 ± 0.09	4.17 ± 0.11	10.07 ± 0.18	14.00 ± 0.48 cd	4.25 ± 0.03 d	7.85 ± 0.14 a
Turning	4.09 ± 0.05	4.00 ± 0.20	10.32 ± 0.49	16.18 ± 0.61c	4.34 ± 0.03c	7.02 ± 0.24c
Pink	4.21 ± 0.06	4.15 ± 0.15	10.17 ± 0.34	21.25 ± 0.61b	4.36 ± 0.02c	5.95 ± 0.17 e
Light-red	4.26 ± 0.10	4.12 ± 0.09	10.35 ± 0.30	25.03 ± 0.78 a	4.44 ± 0.02b	6.82 ± 0.16 d
Red	4.30 ± 0.11	4.00 ± 0.11	10.77 ± 0.27	25.38 ± 0.33 a	4.83 ± 0.03 a	6.82 ± 0.12 d
LSD (P ≤ 0.05)	ns	ns	ns	2.84	0.144	0.76

SSC = soluble solids content, TA = titratable acidity, CA = citric acid. The mean values followed by different letters in each column are significantly different at P ≤ 0.05.

The juice extracted from tomato fruit harvested at various stages of fruit ripening exhibited significant difference in its active acidity expressed in terms of pH values. Slight but gradual increases in active acidity of tomato juice were observed during the course of this study. The pH value of tomato juice was increased from 4.25 at MG to 4.83 at the final (RD) stage of fruit ripening, in the present investigation. During the 1st week of cold storage, the average active acidity of tomato juice was 5 ± 0.1, irrespective of the stage of fruit maturity at harvest (Table 2). The stage of fruit maturity at harvest and duration of cold storage individually exhibited robust influence on active acidity of tomato juice during cold storage. Additionally, a strong interaction (P ≤ 0.01) of the stage of fruit ripening at harvest and duration of cold storage was observed for pH value of tomato juice. On the other hand, Ratanachinakorn et al., (1997) reported that soluble solids, titratable acidity and pH were not different between ripening stages. In general, stages of ripening at harvest is one of the major factors affecting tomato sensory quality attributes (Carli et al., (2011).

Table 2

Effects of maturity stages at harvest and storage duration on flesh firmness and physicochemical characteristics of ‘Red Rose’ tomato fruit during low-temperature storage.

Stages of fruit maturity	Storage duration	Soluble solids content	Titratable acidity	SSC: TA	Soluble sugars	pH	Dry Matter content	Flesh Firmness
	(days)	(°Brix)	(g CA L-1)	(ratio)	(g kg−1)		(%)	(N)
Mature green	0	4.27 ± 0.07	4.27 ± 0.11	10.00 ± 0.30	10.72 ± 0.38	4.25 ± 0.03	7.60 ± 0.17	25.35 ± 1.13
	7	4.38 ± 0.04	3.77 ± 0.07	11.65 ± 0.29	13.85 ± 0.77	5.03 ± 0.05	7.45 ± 0.22	15.67 ± 0.59
	14	4.24 ± 0.02	3.45 ± 0.06	12.30 ± 0.19	14.92 ± 0.89	4.17 ± 0.03	7.10 ± 0.14	11.57 ± 0.28
	21	4.24 ± 0.01	3.45 ± 0.04	12.25 ± 0.16	18.42 ± 0.80	4.25 ± 0.02	6.90 ± 0.21	8.17 ± 0.11
Mature green		4.28	3.74	11.55	14.48	4.43	7.26	15.19
Breaker	0	4.20 ± 0.09	4.17 ± 0.11	10.07 ± 0.18	14.00 ± 0.48	4.25 ± 0.03	7.85 ± 0.14	22.97 ± 0.47
	7	4.39 ± 0.06	3.80 ± 0.13	11.60 ± 0.28	16.52 ± 0.77	4.99 ± 0.04	7.95 ± 0.16	14.57 ± 0.28
	14	4.07 ± 0.04	3.42 ± 0.11	11.92 ± 0.28	19.90 ± 0.53	4.17 ± 0.04	7.22 ± 0.20	9.77 ± 0.25
	21	4.30 ± 0.05	3.45 ± 0.15	12.52 ± 0.40	22.08 ± 0.88	4.26 ± 0.03	7.27 ± 0.19	6.17 ± 0.09
Breaker		4.24	3.71	11.53	18.13	4.42	7.57	13.37
Turning	0	4.09 ± 0.05	4.00 ± 0.20	10.32 ± 0.49	16.18 ± 0.61	4.34 ± 0.03	7.02 ± 0.24	16.0 ± 0.57
	7	4.31 ± 0.03	3.27 ± 0.17	13.27 ± 0.68	20.88 ± 1.05	5.00 ± 0.08	7.25 ± 0.21	11.50 ± 0.34
	14	3.95 ± 0.05	3.02 ± 0.11	13.15 ± 0.54	22.55 ± 1.02	4.18 ± 0.02	7.12 ± 0.12	8.47 ± 0.35
	21	4.16 ± 0.03	3.37 ± 0.13	13.75 ± 0.43	19.83 ± 0.83	4.33 ± 0.03	6.95 ± 0.45	5.25 ± 0.17
Turning		4.13	3.42	12.62	19.86	4.46	7.09	10.31
Pink	0	4.21 ± 0.06	4.15 ± 0.15	10.17 ± 0.34	21.25 ± 0.61	4.36 ± 0.02	5.95 ± 0.17	16.17 ± 0.50
	7	4.39 ± 0.05	3.67 ± 0.07	12.00 ± 0.31	22.85 ± 1.25	4.91 ± 0.05	6.20 ± 0.20	10.97 ± 0.18
	14	4.29 ± 0.03	3.50 ± 0.06	12.27 ± 0.22	20.95 ± 0.73	4.44 ± 0.09	6.42 ± 0.27	8.25 ± 0.15
	21	4.28 ± 0.02	3.47 ± 0.04	12.30 ± 0.21	18.98 ± 0.41	4.41 ± 0.02	6.87 ± 0.22	4.70 ± 0.13
Pink		4.29	3.70	11.69	21.01	4.53	6.36	10.02
Light-red	0	4.26 ± 010	4.12 ± 0.09	10.35 ± 0.30	25.03 ± 0.78	4.44 ± 0.02	6.82 ± 0.16	9.85 ± 0.35
	7	4.52 ± 0.03	3.65 ± 0.06	12.40 ± 0.19	22.63 ± 1.31	5.00 ± 0.05	7.25 ± 0.20	6.70 ± 0.30
	14	4.08 ± 0.04	3.12 ± 0.04	13.10 ± 0.23	27.33 ± 2.01	4.36 ± 0.03	7.27 ± 0.31	4.12 ± 0.14
	21	4.31 ± 0.05	3.15 ± 0.06	13.72 ± 0.30	23.25 ± 1.00	4.46 ± 0.02	7.65 ± 0.15	3.15 ± 0.13
Light-red		4.29	3.51	12.39	24.56	4.57	7.25	5.96
Deep red	0	4.30 ± 0.11	4.00 ± 0.11	10.77 ± 0.27	25.38 ± 0.33	4.83 ± 0.03	6.82 ± 0.12	5.20 ± 0.51
	7	4.42 ± 0.05	3.85 ± 0.07	11.50 ± 0.27	29.48 ± 0.30	5.09 ± 0.02	6.67 ± 0.24	3.62 ± 0.34
	14	4.31 ± 0.04	3.55 ± 0.06	12.17 ± 0.25	31.38 ± 0.78	4.68 ± 0.03	6.75 ± 0.20	2.62 ± 0.16
	21	4.30 ± 0.03	2.82 ± 0.08	12.05 ± 0.13	29.50 ± 0.69	4.81 ± 0.03	6.92 ± 0.37	1.95 ± 0.11
	Deep red	4.33	3.56	11.62	28.94	4.85	6.79	3.35
LSD (P ≤ 0.05)		ns	ns	ns	2.84	0.144	0.76	1.31
Maturity		ns	ns	ns	**	**	**	**
Strg. duration		**	**	**	**	**	ns	**
Maturity × Strg. Duration		**	**	**	**	**	ns	**

Strg. = storage, CA = citric acid, N = newton (force), ns = non-significant, ** = highly significant (P ≤ 0.05).

At harvest, stages of fruit ripening had significant effects on dry matter content of tomato pulp (Table 1). The least dry matter content (5.95%) was recorded for the pulp taken from the tomatoes harvested at PK stage of fruit ripening whereas the fruit harvested at MG or BK stages of fruit ripening had significantly higher levels of dry matter content (7.60% and 7.85%, respectively) in their pulp, compared to those harvested at the later stages of fruit ripening. During cold storage, the least average dry matter content (6.36%) was also recorded for the fruit harvested at the PK stage of fruit maturity, irrespective of the storage duration while we observed a strong interaction between the stage of fruit maturity at harvest and duration of cold storage for dry matter content in tomato pulp. Carli et al. (2011) reported that there were high correlations among pH, dry matter and soluble sugar content of tomato fruits.

Flesh firmness

As expected, tomato fruit harvested at MG and BK stages of ripening were significantly firmer (flesh firmness: 25.35 and 22.97 N, respectively) than those harvested at later stages of fruit ripening (Fig. 1). No significant difference in firmness was observed in tomatoes harvested at TG and PK stages of fruit maturity. Tomato fruit harvested at RD stage of maturity exhibited the least firmness (3.35 N) at harvest. During storage, flesh firmness of tomato fruit harvested during the period between BK and PK stages of ripening remained above 10 N for one week only and then declined rapidly whereas it stayed below 10 N for the fruit harvested at LR and RD stages of fruit ripening, during the entire period of cold storage (Table 2). Reduced flesh firmness is attributable to breakdown of cell-wall and middle lamella polysaccharides accompanied with higher enzymatic activity (Fraschina et al., 1998). Bui et al., (2010) determined the strength-deformation curves and firmness of tomatoes as a function of maturity. They indicated that the puncture force decreases during ripening stages with significant increase in puncture deformation. In general, tomato fruit firmness is reduced as ripening progressed either on-vine or during postharvest period. (Tilahun et al., 2017).

Fig. 1

Changes in flesh firmness of ‘Red Rose’ tomatoes as affected by the stages of fruit maturity at harvest. MG, BK, TG, PK, LR, and RD represent the mature green, breaker, turning, pink, light-red, and red stages of fruit maturity, respectively. Vertical bars represent mean values ± SE of 4 replicates (r = 4).

Lipophilic antioxidants/carotenoids

Oxygen-free carotenoids or simply ‘carotenes’ are photosynthetic pigments but unlike plastid pigments, they absorb light from the regions in ultra-voilet, violet, blue and yellow regions of the absorption spectrum. Carotenes transfer the light energy they absorb to chlorophyll a for photosynthesis and protect plant tissues from damaging effects of ultraviolet irradiation. Analysis for carotene in present investigation focusses on three important fractions of carotenes: lycopene, β-carotene and total carotenoids (Fig. 2, Fig. 4 b). A rapid rise in accumulation of carotenes was observed in tomato fruit harvested between TG and LR stages of maturity. Tomatoes harvested at RD stage of ripening had the highest levels of lycopene (811.9 µg g−1, fm), and total carotenoids (896.8 µg g−1, fm) whereas β-carotene peaked (96 µg g − 1, fm) in the pulp of tomato fruit harvested at PK stage of ripening.

Fig. 2

Changes in lycopene and total carotenoid contents in ‘Red Rose’ tomatoes as affected by the stages of fruit maturity at harvest. MG, BK, TG, PK, LR, and RD represent the mature green, breaker, turning, pink, light-red, and red stages of fruit maturity, respectively, Lyco = lycopene, TCC = total carotenoid content, fm = fresh mass, Vertical bars represent means ± SE of 4 replicates (r = 4).

Fig. 4

Changes in β-carotene (a) and ascorbic acid (b) contents in ‘Red Rose’ tomatoes as affected by the stages of fruit maturity at harvest. MG, BK, TG, PK, LR, and RD represent the mature green, breaker, turning, pink, light-red, and red stages of fruit maturity, respectively. Vertical bars represent mean values ± SE of 4 replicates (r = 4).

The levels of lycopene and total carotenoids content in the pulp of tomato fruit harvested at BK-PK stages of ripening rapidly increased between the period from the first and second week of cold storage (Table 3). During storage, the average lycopene and total carotenoid contents in tomato fruit harvested at LR and RD stages of ripening remained in the range of 750–800 and 825–850 µg g−1 on fresh mass basis, respectively, regardless of the storage duration.

Table 3

Effects of maturity stages at harvest and storage duration on the activities of lipophilic and hydrophilic antioxidants in ‘Red Rose’ tomato fruit during low-temperature storage.

Stages of fruit maturity	s	Lipophilic antioxidants			Hydrophilic antioxidants
		Lycopene	Β-carotene	Total carotenoid content	AsA content	Total flavonoid content	Total phenolic content
	(days)	(µg g−1 fm)	(µg g−1 fm)	(µg g−1 fm)	(µg g−1 fm)	(µg QE g−1 fm)	(µg GAE g−1 fm)
Mature green	0	0.0 ± 0.0	32.8 ± 2.47	44.9 ± 2.53	5.3 ± 0.27	91.2 ± 4.9	141.1 ± 1.6
	7	0.0 ± 0.0	44.1 ± 2.5	72.0 ± 4.1	4.3 ± 0.2	113.7 ± 7.5	208.8 ± 8.5
	14	4.5 ± 0.2	66.9 ± 4.4	100.9 ± 5.7	3.5 ± 0.04	123.6 ± 7.7	174.3 ± 4.0
	21	9.1 ± 0.6	81.6 ± 3.9	110.8 ± 4.6	2.9 ± 0.2	181.8 ± 9.1	155.7 ± 4.2
Mature green		3.4	56.4	82.2	4.0	127.6	169.9
Breaker	0	16.7 ± 0.2	49.8 ± 1.3	132.9 ± 5.0	18.2 ± 0.4	114.6 ± 3.7	183.2 ± 9.1
	7	44.8 ± 2.4	61.9 ± 1.5	180.3 ± 10.6	15.2 ± 0.3	144.6 ± 4.3	218.6 ± 7.6
	14	88.9 ± 3.0	75.9 ± 2.0	243.8 ± 11.0	12.8 ± 0.14	191.3 ± 7.5	177.4 ± 6.6
	21	135.1 ± 7.2	88.7 ± 1.5	308.7 ± 12.0	9.6 ± 0.4	198.3 ± 5.4	156.8 ± 3.9
Breaker		71.4	69.1	216.4	13.9	162.2	184.0
Turning	0	99.0 ± 2.6	82.3 ± 5.0	234.6 ± 13.6	25.9 ± 0.8	172.8 ± 3.0	228.2 ± 11.3
	7	200.8 ± 5.6	99.5 ± 5.5	367.6 ± 5.1	21.3 ± 0.7	211.0 ± 9.0	265.7 ± 19.7
	14	307.8 ± 13.1	86.9 ± 2.5	495.6 ± 9.3	17.2 ± 0.5	202.7 ± 5.4	226.5 ± 19.0
	21	401.4 ± 11.1	77.5 ± 1.6	569.6 ± 18.5	13.7 ± 0.3	176.3 ± 8.7	196.4 ± 17.6
Turning		252.3	86.6	416.9	19.5	190.7	229.2
Pink	0	308.9 ± 10.7	96.0 ± 3.4	451.4 ± 27.8	33.5 ± 1.6	211.4 ± 5.5	302.4 ± 12.1
	7	708.8 ± 19.6	83.5 ± 3.7	665.7 ± 36.0	28.2 ± 1.4	215.2 ± 6.5	300.4 ± 6.7
	14	800.8 ± 17.2	80.6 ± 1.4	721.2 ± 11.4	24.5 ± 1.2	187.1 ± 9.0	262.0 ± 4.9
	21	825.9 ± 14.7	80.7 ± 1.1	806.9 ± 18.5	19.4 ± 0.6	176.9 ± 10.6	233.2 ± 4.6
Pink		586.9	85.2	661.3	26.4	197.7	274.5
Light-red	0	705.3 ± 16.6	84.0 ± 3.1	816.6 ± 22.9	43.2 ± 2.2	199.9 ± 7.3	285.6 ± 5.6
	7	788.9 ± 9.4	79.3 ± 2.1	896.6 ± 21.6	35.8 ± 2.1	205.1 ± 7.7	296.3 ± 6.3
	14	799.1 ± 5.4	69.9 ± 1.9	871.3 ± 5.1	28.8 ± 1.9	192.7 ± 4.2	259.9 ± 4.5
	21	763.9 ± 25.7	67.7 ± 1.8	823.1 ± 17.1	23.6 ± 1.8	175.2 ± 10.3	211.5 ± 9.9
Light-red		764.3	75.2	851.9	32.9	193.2	263.3
Deep red	0	811.9 ± 13.3	83.7 ± 4.3	896.8 ± 36.8	55.7 ± 1.6	198.3 ± 6.4	234.4 ± 12.9
	7	826.4 ± 12.4	77.9 ± 1.7	953.4 ± 20.2	47.3 ± 1.8	200.2 ± 5.9	263.4 ± 11.3
	14	800.3 ± 5.6	77.6 ± 2.6	723.8 ± 20.5	39.2 ± 2.0	179.4 ± 5.8	231.2 ± 13.1
	21	739.7 ± 23.5	73.4 ± 1.7	724.2 ± 12.3	30.7 ± 1.5	189.7 ± 14.2	174.5 ± 6.0
	Deep red	794.6	78.2	824.6	43.2	191.9	225.9
LSD (P ≤ 0.05)		67.02	13.98	109.46	1.84	33.74	50.45
Maturity		**	**	**	ns	**	**
Strg. duration		**	**	**	ns	**	**
Maturity × Strg. duration		**	**	**	ns	**	**

Strg. = storage, CA = citric acid, N = newton (force), ns = non-significant, ** = highly significant (P ≤ 0.05).

Lycopene is an important intermediate of carotene biosynthesis in photosynthetic organisms and is a pre-dominant carotene in tomato fruit. It not only plays a vital role in red, orange and yellow pigmentation in plants and algae but also exhibits the strongest antioxidant activity and the highest ROS quenching ability among the 600 carotenes of natural origin (Maria Alda et al., 2009).

Lycopene is insoluble in water and can only be dissolved in organic solvents or oils. Lycopene content in tomato fruit depend on a number of factors: genotype, phonological stage, environmental conditions and cultural practices (Serio et al., 2007). Massive accumulation of carotenoids within the plastids of ‘Red Rose’ tomato fruit with advancement of fruit maturity may be attributed to massive degradation of chlorophyll during the time. With advancing maturity of tomato fruit, chloroplasts at mature green stage change into chromoplasts that are specialized plastids containing high levels of carotenoids including lycopene in the form of membrane-bound crystals (Tigist et al., 2015). However, the entire route of chromoplast formation on molecular level is yet to be elucidated.

Plastid pigments (chlorophyll and their derivatives) provide two main health benefits to the human body: (1) they block dietary iron-induced metabolites from generating desolation in the body, and (2) they serve as dietary sources of magnesium that promotes healthy pH level thereby helping useful bacteria to thrive (Alsuhaibani et al., 2017). The possible mechanisms by which dietary chlorophyll and their derivatives play their role in reducing the risk of cancers in human being include anti-oxidation process, chelation of pro-oxidant ions especially iron or stimulation of cellular defense systems (Hsu et al., 2013). Likewise, carotenoids are natural pigments, red, orange or yellow in colour, synthesised by various plants and micro-organisms. They have long been used in cosmetics, food and feed industries for their proven biological activities such as antioxidant and pro-vitamin A. Lutein, Lycopene, ɑ- and β-carotene, zeaxanthine and β-cryptoxanthin are some of the most important carotenoids that may compose human diet. The diet rich in lutein and zeaxanthin has been shown to reduce the risk of breast cancer and to lower the incidence of eye problems (Grudzinski et al., 2018). Consumption of lycopene reduces the risk of heart failure and prostate cancer (Rao and Agarwal, 2000). Foods supplemented with β-carotene have been demonstrated to reduce the risks related to many cardiovascular diseases and to protect from esophageal cancer (Kang et al., 2017, Zanfini et al., 2016)

Total phenolic content (TPC)

In the present study, TPC in tomato fruit was considerably low (141.1 µg GAE g−1) at MG stage of maturity (Fig. 3). However, the levels of TPC were significantly increased during the subsequent stages of fruit maturity, peaked (302.4 µg GAE g−1) at PK stage, and then started to gradually decline to 234.4 µg GAE g−1 at the final (RD) stage of fruit maturity.

Fig. 3

Changes in total phenolic and flavonoid contents in ‘Red Rose’ tomatoes as affected by the stages of fruit maturity at harvest. MG, BK, TG, PK, LR, and RD represent the mature green, breaker, turning, pink, light-red, and red stages of fruit maturity, respectively, TFC = total flavonoid content, TPC = total phenolic content,. Vertical bars represent mean values ± SE of 4 replicates (r = 4).

These results are in accordance with studies reported by Anton et al., 2017, Fuentes et al., 2013, Slimestad and Verheul, 2005, Mini, 2017 for various tomato cultivars. Tilahun et al. (2017), however, indicated that ‘TY Megaton’ and ‘Yureka’ tomatoes exhibited the highest levels of TPC at BK stage of fruit maturity. They also observed a declining trend of TPC levels with the advancement of fruit maturity following BK, in both of the cultivars, they studied. These variation in observations may be ascribed to the differences in genotypes studied, growth conditions, analytical protocols, and/or varying edaphoclimatic conditions. A detailed quantitative analysis of individual polyphenolic compounds in tomato fruit using advanced analytical methods and protocols may help in clarifying these variations.

TPC constitute most of the natural antioxidant in food material. Being ubiquitous in plants, phenolic compounds are present in our daily diet in gram quantities. The antioxidant activities of phenolic compounds are well established. They inhibit not only autoxidation of lipids but also possess an ability of retarding regular lipid oxidation possibly by inhibiting the activity of lipoxygenase enzyme. Due to their high antioxidant properties, consumption of food material containing phenolic compounds will surely remain the area of great interest especially in the functional food industry. Phenolic compounds have also been demonstrated to exhibit genotoxic and cytotoxic effects (Mendoza-Meza and España-Puccini, 2016) in animals, the mechanism of their ambivalent activities however remains to be further elucidated

Total flavonoid content (TFC)

Fruit and vegetables are rich sources of numerous classes of plant secondary metabolites that have a polyphenolic structure and are commonly referred to as flavonoids which are associated with a broad spectrum of health promoting functions, They constitute an indispensable component of a wide range of pharmaceutical, nutraceutical, and cosmetic products and a variety of traditional medicines. In plant kingdom, flavonoids exhibit antioxidant, photo-receipting, light screening, antimicrobial, visual attracting, and feeding to repellants activities. In animals, they exhibit miscellaneous useful biological activities including antioxidant, anti-inflammatory, antiviral, antiallergenic, vasodilating, and a variety of other functions (Brodowska, 2017). During the last a couple of decades, however, most studies on the biological activities of flavonoids have been focussed on their compelling antioxidant properties by which they are able to reduce the formation of tissue damaging ROS or to scavenge them efficiently. Tomato fruit exhibited the least TFC (91.2 µg QE g−1) in their pulp at MG stage of maturity (Fig. 3). TFC in tomato pulp peaked (211.4 µg QE g−1) at PK stage of fruit ripening. The fruit pulp from tomatoes harvested during the period from PK-RD maturity stages had TFC in the range of 198–212 µg QE g−1, on fresh mass basis. Flavonoids and their derivatives are ubiquitous in plants and many of them have been used in traditional eastern medicines for thousands of years. Among the exhaustive list of the pharmacological activities in a mammalian body, their antioxidant and antiproliferative properties stand out. Flavonoids possess anti-inflammatory, antiallergic, antiviral, anticarcinogenic, antimicrobial, antiulcer, hepatoprotective, antidiabetic, vasorelaxant, and antineoplastic properties that exhibit many beneficial effects on gastrointestinal and central nervous systems and have been shown to play protective roles in many cardiovascular, cataracts and liver diseases (Georgiev et al., 2014, Santini and Novellino, 2017, Tapas et al., 2008).

TFC in the pulp from tomato fruit harvested during the period between TG-RD stages of ripening peaked following the 1st week of storage and remained in the range of 200–215 µg QE g−1, on fresh mass basis (Table 3). After one week of cold storage, the levels of TFC started to decline gradually. A strong relationship between the stages of fruit ripening at harvest and duration of storage was observed for accumulation of TFC in ‘Red Rose’ tomato fruit grown under greenhouse conditions.

The pattern of changes in flavonoid content at various stages of tomato ripening as depicted in the present study was in line with that reported by Garcia-Valverde et al. (2013) in various tomato cultivars. Bhandari and Lee (2016) reported that most of the flavonoid groups such as quercetin, rutin, and naringenin peak during the time from pink to light-red stage of maturity in tomato fruit, however, depending on genotype and growth conditions. As a thumb rule, higher the accumulation of individual flavonoid constituents in a biological system, higher would be its total flavonoid content. Substantially higher quantities of total flavonoid content recorded in the present investigation compared with those reported by earlier studies may be due to the difference of genotype and/or analytical methods.

Ascorbic acid (AsA) content

Ascorbic acid, also known as L-ascorbic acid or simply vitamin C, is not only a common co-factor in a variety of biochemical reactions but also an essential nutrient for many mammals including humans. Being one of the most powerful reducing agents, it is capable of rapidly scavenging reactive oxygen species produced in the body during normal cellular respiration. It has been demonstrated to exhibit a variety of cyto-protective functions in the human body. As a powerful free radical scavenger in the blood plasma, it efficiently protects our cells from potentially damaging free radicals. It helps to form, regenerate and maintain connective tissue in the bone, blood vessels and in the skin (Ivanov et al., 2016) and thus protects us from many cardiovascular diseases and a variety of cancers. Quadros et al. (2016) demonstrated that AsA helps in absorption of iron, prevents scurvy, decreases total and LDL cholesterol and provides support to our immune system. Prevention of DNA mutation induced by oxidation, protection of lipids from per-oxidative damages and repair of oxidized forms of amino acid to maintain the integrity of proteins in our body are some of the most important cytoprotective functions that explain the potential mechanisms of vitamin C in the body (D'Aniello et al., 2017, Karim and Kadowaki, 2017).

The AsA content varied in homogenate samples from tomato fruit harvested at various stages of maturity (Fig. 4b). The results revealed that at MG stage of fruit ripening, tomato pulp contained the lowest level (5.3 µg g−1) of AsA content that gradually increased to its peak (55.7 µg g−1) at the final stage (RD) of fruit maturity. We observed the trend of decreasing levels of AsA content in tomato pulp immediately after subjecting the fruit to cold storage, regardless of the stage of fruit maturity at harvest (Table 3). It appears that when once harvested, there is no further accumulation of AsA content in tomato fruit.

These observations are in line with those reported by Kotíková et al. (2011) in eight and Bhandari and Lee (2016) in seven tomato cultivars. These authors demonstrated that ascorbic acid content continued to increase from BK to RD stages of fruit maturity. This pattern of changes in AsA content may be ascribed to oxidative deprivation of ascorbic acid as the rate of cellular respiration increases with the onset of ripening (N’Dri et al., 2010) which is actually a characteristic physiological phenomenon in climacteric type of fruit and vegetables including tomato.

Conclusion

The results obtained from the current investigation showed that the stage of fruit maturity substantially influenced the texture, quality, and functional properties of ‘Red Rose’ tomatoes grown under greenhouse conditions, both at harvest and during cold storage. In addition, the harvesting operation of tomato crop can be carried out from breaker to light-red stage of fruit ripening for tomato-based processing industries. The reason is to address the consumer’s demand of foods with improved functional properties in terms of health promoting dietary antioxidants. However, for retaining the organoleptic and functional properties of tomato fruit to their optimum level during long-term storage, harvest process can be finalized till the beginning of the light-red stage of ripening.