Phytochemical and metabolic changes associated with ripening of Lycopersicon esculentum

Abstract

Four tomato genotypes GT-2, GT-6, GT-7, and DVRT were analysed for phytochemical and metabolic changes at different stages of ripening. Reducing sugar and soluble protein content reached up to 93.90% and 26.78% from green to red ripened phases, respectively, with varying maturity stages. The tomato fruit maturation stages were found to substantially alter the total phenol content (P < 0.05), with an overall increase of 8.9% total phenol content from the green to red ripening stage of the tomato while the ascorbate level grew 11.36% from green to orange stage and then slightly declined (1.68%) at red ripened stage. Lycopene concentration climbed up to 36.59% from green to red ripened stage. HPTLC analysis confirmed Arg, Cys, Val, Leu, Tyr, and Thr decreased as the fruit ripened, whereas Ala, Pro, Ile, Lys, and His increased. The non targated compound analysis with GC-MS/MS established the presence of hydroxymethylfurfural and proline during fruit ripening.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-95255-9.

Introduction

The quality of tomato fruit is primarily influenced by pre- and post-harvest elements. The overall nutrient composition of the fruit has been found to be strongly impacted by ripening procedures and storage temperatures¹. Since numerous physicochemical changes that lead to quality loss occur at this point, the tomato, a climacteric fruit, has a relatively limited postharvest life. Senescence, postharvest infections, transpiration, and accelerated ripening are only a few of the variables that shorten storage life. The main issue affecting tomatoes’ postharvest shelf-life, especially in tropical areas where the temperature is high, is increased respiration, which has the effect of hastening fruit ripening and degrading fruit quality². The shelf life of different tomato types varies according to how they are stored. This can last anywhere between 4 and 8 days at room temperature. The commercial shipping of this fruit over great distances is severely constrained by this brief window³. Due to their soft and succulent character, tomatoes are particularly prone to spoiling when they are mature. 20–25% of tomatoes produced in tropical nations are lost during harvest, transit, storage, and marketing⁴. The quality and marketability of tomato fruit are influenced by the fruit’s colour, chemical makeup, texture, and flavour, which are in turn influenced by the presence of lycopene, beta-carotene, ascorbic acid, and soluble solids. The quality of tomato fruits varies depending on the cultivar, ripening stage, growing environment, and climate⁵. When fruits ripen, substances within the fruit cause changes. Fruit ripening is a complex developmental process that involves biochemical changes such as an accumulation of pigments, sugars, and organic acids, the breakdown of chlorophyll and starch, and the solubilization of cell wall structure, as well as metabolic changes such as an increase in biosynthesis and respiration. These biochemical and metabolic changes have a significant impact on the nutritional value, colour, aroma, and flavour of fruit⁶. The irreversible breakdown of numerous components is known as ripening. The hue of the fleshy fruits generally changes as they ripen. A decrease in chlorophyll and an increase in pigments like anthocyanin, carotenes, and/or xanthophylls are what cause this change in hue⁷. The pigment system of tomato peel has chlorophyll content which reduces rapidly until the fruit ripens. In contrast, low carotenoid content from fruit formation increased rapidly until the fruit is fully ripening. The starch content decreases and reducing sugar content increases rapidly with ripening. Total organic acid and vitamin C increased continuously with ripening and decreased slightly at the full ripe stage. The pectin and tannin content decreases until the fruit is fully ripening. The catalase activity gradually increases and riches maximum at the ripe stage then decreases at over-ripened fruits. An experiment conducted to record the change of biochemical parameters during ripening revealed that the variety NHP11 (Product of Nong Hung Phu Company Limited, Vietnam), produced 0.72–0.19 mg/g chlorophyll, 0.013–0.48 mg/g carotenoids, 0.85–3.29% reducing sugar, 1.05–0.50% starch, 22.73–31.13 g/100 g ascorbic acid and 1.36–0.62% pectin content from 7 days old fruit initiation stage to 50 days matured fruit⁸. Another important compound is hydroxymethylfurfural (HMF), which can be found normally in processed tomatoes yet, it can also be seen in fresh fruit and may be synthesized during different physiological processes like photosynthesis and transpiration⁹. By considering the above facts, this experiment was conducted to determine the quality of different tomato genotypes at various ripening stages.

Materials and methods

Sample preparation

The present investigation was carried out on four different genotypes of tomato namely, GT-2, GT-6, GT-7 and DVRT (Fig. 1). The fruits were collected in the winter season from Regional Horticultural Research Station (RHRS), Navsari Agricultural University, Navsari (20° 57′ N latitude and 72° 54′ E longitude with an altitude of 11.98 m above the mean sea level). Fruits were collected from four genotypes chopped with a knife into 5 mm size and dried in a hot air oven at 65 ± 5 °C followed by grinding into dry powder and used for analysis. The fresh samples were also used for the analysis as and when needed.

Fig. 1.

Different genotypes of tomato under various maturity stages.

Total carbohydrate

Total carbohydrate was analysed by using spectrophotometer with the help of anthrone method. Tomato fruit samples were extracted by boiling in water bath at 85–90 °C for 3 h with 2.5 N HCl. After that the sample was cooled and reaction was carried by adding known amount of extract to 4 ml of chilled 2% anthrone reagent. The intensity of green colour of hydroxymethyl furfural formed was measured by using spectrophotometer at 630 nm and expressed as per cent basis of standard curve of glucose¹⁰.

Reducing sugar

The fresh fruits were extracted with 80% hot alcohol, evaporated to dryness and dissolved in distilled water. The extract was reacted with DNS solution followed by heating for 5 min and addition of 1 ml of 40% Rochelle salt solution. The absorbance was recorded at 510 nm and amount of reducing sugar was calculated from standard glucose solution (0 to 500 µg) and expressed in percent¹¹.

Soluble protein

Fresh fruits were extracted using phosphate buffer of pH 7.0 and the extract was reacted with alkaline copper solution (50 ml of 2% Na₂CO₃ in 0.01 N NaOH and 1 ml of 0.5% CuSO₄·5H₂O in 10% potassium sodium tartrate) followed by addition of FolinCiocalteau reagent. The absorbance of blue colour formed was recorded at 660 nm and amount of protein was calculated from standard curve of bovine serum albumin (200 µg ml^− 1). The amount of soluble protein was expressed in percent¹².

Phenol

The fruits was extracted with 80% methanol and the extract was further reacted with Folin–Ciocalteau reagent and Na₂CO₃ (20%). A blue colour was developed and absorbance wad recorded at 650 nm followed by estimation of phenol content against catechol as standard. the unit of expression of phenol content was mg 100^− 1g¹³.

Lycopene

Fresh tomato pulp was extracted with acetone and the extract was separated with petroleum ether using a separating funnel. The pool of the petroleum ether extracts was passed through anhydrous sodium sulphates and absorbance was recorded at 503 nm. The lycopene content was expressed as mg 100^− 1 g¹⁴.

Ascorbic acid

Ascorbic acid was determined by the 2,6 dichlorophenol indophenols (DCPIP) titration procedure¹⁵. Fresh samples were extracted by 4% oxalic acid. The extract was titrated against 2,6 dichlorophenol indophenol dye. Pure ascorbic acid standard solution was also titrated against DCPIP solution. The ascorbic acid content was expressed as mg 100^− 1g.

Antioxidant activity

Based on the scavenging activity of 1,1-diphenyl-2-picrylhydrzyl (DPPH) antioxidant activity of tomato fruits was estimated¹⁶. The quantity of samples that reduced the initial absorbance of DPPH solution by 50% was picked to determine the antioxidant activity. Using the following formulas, the proportion of the remaining DPPH radical (% DPPH rem) and antioxidant activity (%AA) were determined for all concentrations of the sample extract based on the absorbance values recorded for the sample (As) and the blank (A0):

The concentrations of the IC₅₀ were computed and represented as µg of samples per millilitre of extract, which is required to scavenge 50% of the DPPH radical in the solution being examined. The antioxidant concentration needed to scavenge 50% of the DPPH radical within the allotted time is known as the IC₅₀ value.

Alkaloid content

A known amount of dry powdered fruit extracted with methanol followed by evaporation of methanol on a rotary evaporator and resuspension in HCl. The extract was reacted with BCG (Bromocresol green) and then extracted with chloroform followed by recording of the absorbance at 417 nm. The alkaloid content was expressed as mg 100^− 1g¹⁷.

Titratable acidity content

A known amount of the fruits were extracted with distilled water and filtered. An aliquot (10 ml) was titrated against standard sodium hydroxide (0.1 N NaOH) using phenolphthalein as an indicator. The Titratable acidity was expressed in terms of percentage citric acid equivalent¹⁸.

Amino acid profiling by HPTLC

Amino acids were extracted from fruits using 6 M HCl through incubation at 100 °C for overnight followed by evaporation to dryness and re-suspended in 0.02 M HCL solution. Re-suspended sample was again centrifuged and supernatant was collected, filtered followed by analysis by HPTLC. Extracted samples (8 µl) along with standard amino acid mixtures (5 µl) were sprayed as 1 × 6 mm bands on HPTLC aluminum plate 20 × 10 cm (preactuivated in 100 °C for 20 min) (Silica gel 60 F254 Merck) by CAMAG Linomat 5 applicator using nitrogen gas. Each set of standard amino acids were sprayed on HPTLC (Fig. 2; Table 1). Standard amino acids were collected from Hi-media and100 ppm solution was prepared in distilled water except tyrosine and phenylalanine (dissolved in 0.05 N HCl), and tryptophan (dissolved in 0.05 N NaOH). Solvent system was used n-butanol : water : acetic acid (8:2:2) and derivatized¹⁹). After separation the derivatization was done spraying with 0.2% ninhydrin (dissolved in acetone). Finally the HPTLC plates were photographed using CAMAG Reporstar 3 system and area % was calculated²⁰.

Fig. 2.

Chromatogram of amino acids generated through HPTLC.

Table 1.

List of standard amino acids and RF value detected by CAMAG 360 SCANNER 3 at 546 nm and its mixture.

Sr. No.	Name of amino acid	Name of mixture	Start RF	Max RF	End RF	% Area
1	dl-Alanine (Ala)	Mixture 1	0.17	0.21	0.27	45.39
2	Arginine HCl (Arg)		0.01	0.06	0.10	15.40
3	dl-Aspartic acid (Asp)		0.02	0.07	0.11	14.40
4	l-Glutamine (l-Glu)		0.11	0.14	0.17	24.81
5	l-Glutamic acid (Glu)	Mixture 2	0.11	0.16	0.18	17.80
6	l-Proline (Pro)		0.19	0.19	0.24	08.88
7	l-Histidine HCl (His)		0.02	0.06	0.09	23.56
8	dl-Isoleucine (Ileu)		0.37	0.47	0.52	49.76
9	l-Leucine (Leu)	Mixture 3	0.45	0.49	0.55	32.82
10	l-Lysine HCl (Lys)		0.09	0.15	0.20	15.56
11	dl-Methionine (Met)		0.35	0.41	0.44	35.45
12	Glycine (Gly)		0.09	0.15	0.19	16.17
13	dl-Serine (Ser)	Mixture 4	0.02	0.05	0.07	10.44
14	dl-Tryptophan (Try)		0.48	0.52	0.57	26.83
15	l-Tyrosine (Tyr)		0.40	0.44	0.48	28.09
16	dl-Valine (Val)		0.28	0.36	0.40	34.64
17	Cysteine (Cys)	Mixture 5	0.31	0.38	0.42	14.01
18	Hydorxy Proline		0.13	0.20	0.26	49.46
19	Phenyl alanine (PAL)		0.44	0.49	0.54	38.45
20	Threonine (Thr)		0.27	0.27	0.31	03.77
21	Asparagine (Asp)		0.02	0.05	0.07	08.32

GC-MS/MS analysis of nontargeted metabolite

The analysis of nontargeted metabolites from different ripening stages of tomato was done by GC-MS/MS. Samples were quenched with liquid nitrogen and pre cooled in – 80 °C followed by extraction with methanol: water (3:1 v/v) including 0.1% formic acid. The extract was subjected to phase separation by the use of water: chloroform (2:1) and each phase was evaporated to dryness with nitrogen evaporator and pyridine solution (20 mg ml^− 1) was added for re-suspension. Untargeted detection of compounds was done by TSQ- 9000 triple quadrupole GC MS/MS system (thermo). 1 µl of sample was injected into the injector at 280 °C using splitless mode. TG-5MS (30 m × 0.25 mm × 0.25 μm ) column was operated at a constant flow of helium gas at 1 ml min^− 1. A total of 32 min run time was employed (90 °C for 0–5 min, 180 °C for 5.1 to 8.6 min, 280 °C for 8.7 to 28.6 min, and 300 °C for 28.7 to 32 min). The detector was used in scan mode m/z 50–650. The EI source was kept at 310 °C.

Statistical analysis

The experimental data was analysed following CRD with Different genotypes. The observations for each parameter were taken in five replications. The appropriate standard error was calculated for specific maturity stages. The critical difference at 5% level of probability was worked out to compare the treatment means, where treatment effects were significant.

Results and discussion

The total carbohydrate content was observed to be affected during fruit maturity stages and lowered down from green to red ripening stage up to 20.09%. Reducing sugar as well as soluble protein content increased with different maturity stages up to 93.90% and 26.78% from the green to red ripened stage (Fig. 3). In the present experiment the total carbohydrate content reduced with fruit ripening (Fig. 3). It was observed from earlier study that, the total carbohydrate is accumulated during fruit development as starch, sucrose, glucose or fructose²¹. In the early stage of fruit development, starch accumulates and with maturity, it is degraded by the activity of the enzyme sucrose synthase, which converts the starch into sucrose which is hydrolyzed into the hexose sugars such as glucose and fructose in the presence of enzyme invertase. It was also observed that the total carbohydrate content was reduced with tomato fruit ripening²². The highest carbohydrate content was obtained from green followed by a breaker and lowest at the turning and red stage. Red tomatoes had the largest amount of soluble protein compared to all other fruit maturity stages since the soluble protein content increased marginally with fruit maturity. The same result as soluble protein content slightly increased with tomato fruit maturity was found in earlier studies²³. The total phenol content was observed to be significantly affected (P < 0.05) by tomato fruit maturity stages with an overall increase of 8.9% total phenol content from the green to red ripening stage of tomato. The Lycopene content in different individual maturity stages had increased up to 36.59% from the green to the red ripened stage. The result showed that the ascorbate content increased by 11.36% from the green to orange stage subsequently and then slightly decreased (1.68%) at a red ripened stage in all the genotypes of tomato. Antioxidant activity was decreased from green to red ripened stage subsequently in the case of all the genotypes up to 36.16%. Genotype GT7 proved to be the most antioxidant-rich one with an IC₅₀ value of 34.77 µg/ml (Fig. 4). The results obtained here for total phenol (Fig. 4) content agree with other studies²⁴ and observed that the amount of total phenol increased as the fruit ripened, and cultivars and cultivation method both had a significant impact on changes in phenolic component concentration. Phenolic compounds accumulate during ripening and act as antioxidant compounds due to their chemical structure. Phenylalanine ammonia-lyase, a key enzyme of phenolic synthesis, is commonly induced during pattern-triggered immunity, which is activated after the recognition of a microbe-associated molecular pattern²⁵. This experiment indicated that the lycopene content is increased with the tomato fruit maturity. The same result was also observed as lycopene content was higher at the orange and red stages as compared to the green and yellow maturity stages of tomato fruit²⁶. Change in lycopene content is varied with the different accession of tomato. The reduction of chlorophyll content along with the increase of the carotenoids content of fruit development is suitable for the process of tomato development and reflects the true colour of the fruit when ripe. An increasing pattern of lycopene accumulation in all cultivars of tomatoes during the ripening process was mainly due to the transition of chloroplast into chromoplast was also established from earlier studies²⁷. In our experiment it was observed that ascorbate content increased with fruit maturity (Fig. 4). It was further noted that the observed variations could be attributed by the differences in cultivars or growing conditions, implying that significant effects on ascorbic acid are caused not just by ripening stages, but also by tomato genotypes²⁷. Previous studies also established the same result as vitamin C content continuously increased from immature 22.73 g 100 g^− 1 to half-mature stage 33.68 g 100 g^− 1 and then decreased at full ripen stage 31.12 g 100 g^− 18. The change in the ascorbic acid content is mainly due to oxidation and reduction during ripening. It was reported earlier that, the antioxidant capacity of tomatoes can be mainly attributed to some bioactive components, such as lycopene, ascorbic acid, and phenolic compounds²⁸. These antioxidant compounds can be classified as hydrophilic (flavonoids and ascorbic acid) and lipophilic (lycopene, β-carotene) antioxidant compounds. The same result as the total antioxidant activity of tomato fruit from mature green to over-ripe stage, significantly increased was found from other studies²⁹.

Fig. 3.

Total carbohydrate, reducing sugar and soluble protein content of tomato under various maturity stages.

Fig. 4.

Lycopene, phenol ascorbate content and antioxidant activity of tomato under various maturity stages.

Titratable acidity content was decreased from the green to red ripened stage in GT-2 (64.29%) and GT-6 (60%) genotypes, but in the case of GT-7 and DVRT genotype, titratable acidity content reached its maximum values at yellow stage and increased up to 5.36% and 4.36% as well as continuously decreased at orange (49.15% and 43.75%) and at red ripened stage (26.67% and 29.63%) (Fig. 5). Pectin content was reduced from green to red ripened stage subsequently in case of all the genotypes up to 45.76%. GT-6 genotype revealed the lowest value (10.98%) while GT-2 genotype was proved to be the best genotype containing 13.20% on average in four different maturity stages (Fig. 6). Alkaloid content was reduced but not significantly from green to red ripened stage in case of all the genotypes. The highest alkaloid content was found in DVRT (4.12 mg/100 g) while GT-2 was proved to be the lowest (3.28 mg/100 g) alkaloid-containing genotype among the others (Fig. 6). The decrease of titratable acidity content in tomato fruits in the present study (Fig. 5) is supported by the experiment of Tilahun et al.³⁰. Titratable acidity was found to be at its peak at the breaker stage and then started to decrease with the advancement of fruit ripening³¹. The result indicated that the pectin content decreased with the fruit ripening in all the genotypes of tomato (Fig. 6). The pectin content was found to be responsible for the softening of the tomato fruit during ripening and its content reduced with the ripening³². The result indicated that the highest amount of alkaloid content found at the green stage and with maturity it was declined. It was observed that the alkaloid content was higher at the immature stage while the red stage contained a very low amount of alkaloids and tomatine was the main alkaloid found in tomatoes³³. It was found that representative solanum alkaloid and steroidal glycoalkaloids (SGAs) are higher in value at the immature stage which is toxic and decreases with ripening³⁴.

Fig. 5.

Titrable acidity of tomato under various maturity stages.

Fig. 6.

Pectine and alkaloid content of tomato under various maturity stages.

Among twenty different protein-forming amino acids, some of the specific amino acids like Arg, Ala, Cys, Val, Leu and Tyr had been found most frequently in four types of tomato genotypes under different ripening stages. The other amino acids like Thr, Pro, Ile, PAL, Lys, Ser, His and Asp had also been found in some ripening stages. The maximum types of amino acids were found in GT7 and DVRT. Among different amino acids, Arg, Cys, Val, Leu, Tyr, Thr were reduced during ripening while Ala, Pro, Ile, Lys, His were increased during ripening. According to area per cent, Ala occupied the highest position and prominent amino acid in different tomato genotypes (Table 2; Fig. 7). GC-MS/MS analysis confirmed the presence of Hydroxymethylfurfural and proline mostly. In most of the cases these compounds had been increased during ripening (Table 3).

Table 2.

Amino acid profiling of different tomato genotypes under different ripening stages. (Individual amino acid and % Area was detected by CAMAG SCANNER 3 at 546 nm)

Sr No.	Genotypes	Ripening stages of tomato	Total No. of identified amino acid	% Area of amino acids
				Arg	Ala	Cys	Val	Leu	Tyr	Thr	Pro	Ileu	PAL	Lys	Ser	His	Asp
		Green	6	+ 16.33	+ 35.45	+ 8.11	+ 10.67	+ 13.22	+ 8.26	−	−	−	−	−	−	−	−
2		Yellow	7	+ 13.42	+ 39.55	+ 5.96	+ 7.13	+ 9.66	+ 7.13	−	+ 13.41	−	−	−	−	−	−
		Orange	7	+ 9.82	+ 42.19	+ 5.89	+ 8.2	+ 9.44	+ 6.17	−	+ 17.97	−	−	−	−	−	−
4		Red	7	−	+ 43.22	+ 4.65	+ 6.71	+ 7.35	+ 5.85	−	+ 21.27	−	−	−	−	−	−
5	GT6	Green	7	+ 15.51	+ 46.63	+ 2.02	+ 8.37	+ 8.32	+ 12.39	+ 4.74	−	−	−	−	−	−	−
6		Yellow	6	+ 12.97	+ 49.93	−	+ 7.40	+ 7.60	+ 11.16	+ 4.35	−	−	−	−	−	−	−
7		Orange	6	+ 11.56	+ 52.30	−	+ 6.69	+ 7.04	+ 10.50	+ 3.26	−	−	−	−	−	−	−
8		Red	5	+ 9.85	+ 58.35	−	+ 5.17	+ 6.43	+ 9.18	−	−	−	−	−	−	−	−
9	GT7	Green	9	+ 12.99	+ 12.62	+ 8.62	+ 11.03	+ 16.41	+ 10.82	−	+ 14.95	+ 10.22	+ 1.54	−	−	−	−
10		Yellow	9	+ 9.23	+ 26.94	+ 8.52	+ 10.1	−	+ 8.81	−	+ 16.92	+ 13.82	+ 0.44	+ 2.35	−	−	−
11		Orange	8	+ 4.75	+ 29.7	+ 8.24	+ 9.4	+ 13.59	+ 1.79	−	−	−	−	+ 4.3	+ 14.29	−	−
12		Red	7	−	+ 32.55	+ 7.82	+ 9.27	+ 8.13	−	−	−	−	−	+ 4.66	−	+ 10.87	+ 17.59
13	DVRT	Green	9	+ 9.45	−	+ 8.30	+ 10.39	+ 20.86	+ 13.96	−	+ 21.94	+ 8.82	+ 0.99	+ 3.24	−	−	−
14		Yellow	7	+ 8.21	+ 28.35	+ 7.92	+ 9.30	+ 18.68	+ 13.63	−	−	−	−	+ 3.85	−	−	−
15		Orange	7	+ 6.18	+ 30.01	+ 5.30	+ 8.15	+ 17.01	+ 8.99	−	−	−	−	−	−	+ 10.38	−
16		Red	6	−	+ 32.01	+ 5.23	+ 7.37	+ 12.07	+ 1.43	−	−	−	−	−	−	+ 13.28	+ 15.71

+ = Presence, - = Absence.

Fig. 7.

Amino acid profiling of tomato genotypes (GT-2 and GT-6) on HPTLC 356 aluminum plate 20 × 10 cm (Silica gel 60 F254 Merck).

Table 3.

List of major compounds found by nontargeted metabolite analysis in GC MS/MS.

Sr No.	Genotypes	Ripening stages of tomato	Peak No.	Ret time (min)	Peak name	Area count*min
1	GT2	Green	3	7.669 5	Hydroxymethylfurfural	5,034,348
2			4	7.847	3-n-Propyl-2-pyrazolin-5-one	1,722,338
3			12	8.917	l-Proline, 5-oxo-methyl ester	2,402,347
4			14	9.108 4	4-Amino-1,5- -pentandioic acid	109,800
5			15	9.262	Estra-1,3,5(10)-trien-17β-ol	1,914,090
6			23	10.101	Glycerol, 2-TMS	3,198,255
7			24	10.164	4-Methylcyclohexaneacetic acid, trimethylsilyl ester (stereoisomer 1)	4,939,075
8			27	10.513	Desulphosinigrin	3,429,507
9		Yellow	4	7.713 5	Hydroxymethylfurfural	11,259,152
10			14	8.917	l-Proline, 5-oxo-, methyl ester	5,562,950
11		Orange	3	7.676 5	Hydroxymethylfurfural	14,758,258
12			11	8.920	dl-Proline, 5-oxo-, methyl ester	3,945,773
13		Red	2	7.676 5	Hydroxymethylfurfural	7,326,738
14			10	8.917	dl-Proline, 5-oxo-, methyl ester	6,835,639
15	GT6	Green	3	7.672	5-Hydroxymethylfurfural	9,495,642
16			11	8.920	dl-Proline, 5-oxo-, methyl ester	3,521,313
17			24	10.161	Glycerol, 2-TMS	2,752,603
18		Yellow	3	7.676	5-Hydroxymethylfurfural	11,843,833
19			11	8.920	dl-Proline, 5-oxo-, methyl ester	7,751,222
20		Orange	2	7.672	5-Hydroxymethylfurfural	22,597,637
21		Red	3	7.669	5-Hydroxymethylfurfural	24,166,018
22	GT7	Green	3	7.673	Hydroxymethylfurfural	3,873,928
23			10	8.913	dl-Proline, 5-oxo-, methyl ester	8,517,779
24		Yellow	4	7.672	Hydroxymethylfurfural	7,066,191
25			12	8.920	dl-Proline, 5-oxo-, methyl ester	11,205,558
26		Orange	3	7.672	5-Hydroxymethylfurfural	7,627,110
27			12	8.920	l-Proline, 5-oxo-, methyl ester	2,719,375
28			22	10.090	Glycerol, 2-TMS	3,775,861
29			26	10.516	Desulphosinigrin	2,245,351
30		Red	3	7.672	5-Hydroxymethylfurfural	33,897,955
31	DVRT	Green	3	7.672	5-Hydroxymethylfurfural	7,008,204
32			11	8.917	l-Proline, 5-oxo-, methyl ester	4,743,610
33			24	10.171	Glycerol, 2-TMS	4,223,854
35		Yellow	3	7.669 5	Hydroxymethylfurfural	11,365,740
36			11	8.917	l-Proline, 5-oxo-, methyl ester	5,862,547
37			13	9.118	Oxacycloundecane-2,7-dione	4,134,521
38			23	10.157	(5-Ethyl-1,3-dioxan-5-yl)methanol, TMS	8,830,665
39		Orange	3	7.669	5-Hydroxymethylfurfural	24,914,375
40		Red	3	7.669	5-Hydroxymethylfurfural	30,525,355
41			11	8.917	dl-Proline, 5-oxo-, methyl ester	8,586,823

The total free amino acid increased fivefold in the ripened tomato due to the high proteolytic activity³⁵. Although the free amino acid pool released by tomato pericarp proteins increased in ripening fruits, in vitro degradation of endogenous proteins was reduced. They found that the non-specific protease activity was connected to an increase in exopeptidase activity during ripening. It is worth noting that the peptidases found in that case may have distinct intracellular localizations. It has been proposed that the intracellular movement of amino acids across vacuole and cytosol is a crucial factor in their accumulation in ripening fruits³⁶. Variations in the content of various components are to be predicted when the fruits strengthen their taste and flavour during ripening. Free amino acids are crucial non-volatile molecules that are essential to the overall flavor of many foods, particularly glutamate and aspartic acid³⁷. In our experiment also it was found that aspartate was present in red ripened tomatoes in GT7 and DVRT while in other three stages like green, yellow, and orange fruit there were no traces of aspartate.

Hydroxymethyl furfural is a type of organic compound that is produced by the dehydration of reducing sugars. The reducing sugar content increased with the different maturity stages of tomato fruit ranging from 0.76 to 4.04 g 100 g^− 138, while the reduced sugar content increased with the fruit maturity ranging from 2.23 to 6.33%³⁹. The increase in soluble protein content found in tomato fruit during maturity coincided with increased activity of pectin esterase, polygalacturonase, and cellulase enzymes⁴⁰. The same result as soluble protein content slightly increased with tomato fruit maturity was observed earlier²². The concentrations of proline increased parallelly with sugar accumulation in grapes during later stages of ripening⁴¹.

Conclusion

Complex carbohydrates and stored proteins were broken down into relatively basic molecules like monosaccharides (reducing sugar) and soluble proteins throughout fruit maturation. Lycopene and phenol content are both more abundant in the red ripening stage. As the fruits have a stronger taste and flavour while ripening, variations in the content of various components are to be expected. In a nutshell, it can be conferred that, orange to red ripened tomato is suitable for consumption purposes. The expression of ripening-related enzymes like pectin esterase, and polygalacturonase may studied further and correlated with metabolite changes during ripening. The future study of the mode of action in respective enzymes and related metabolic pathways may lead to longer self-life in tomatoes.

Electronic supplementary material

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Author contributions

Conceptualisation and design of experiment (Nilima Karmakar); Analysis of biochemical parameters and bioactive compounds (Priyankaben Antala, Ankita Chakote, Kiran Suthar, Diwakar Singh, Kelvin Gandhi, Susheel Singh); Data analysis (Nitin Varshney, Nilima Karmakar, Ajay Narwade); Manuscript writing (Nilima Karmakar, Nitin Varshney and Kamlesh Patel).

Funding

The authors are obelized to Navsari Agricultural University, Gujarat, India, for financial support to carry on the research work.

Data availability

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Declarations

Competing interests

The authors declare no competing interests.

Research involving human participants and/or animals

This article does not contain any studies with human participants or animals performed by any of the authors.