Proﬁling of primary metabolites of Averrhoa carambola , Spondias dulcis and Syzygium malaccense fruits revealed underpinning markers during “on­tree” maturation and ripening stages

: The study aimed to proﬁle and quantify sugars and organic acids metabolites in carambola, June plum and otaheite fruits during three diﬀerent “on tree” stages: immature, green­mature and ripe stages. Metabolites were proﬁled and quantiﬁed by gas chromatography­mass spectrometry (GC­MS). Results showed that glucose, fructose, galactose, arabinose, and the sugar alco­ hol myo­inositol were detected in all fruits, while sucrose was detected in carambola and June plum only. Organic acids identiﬁed in all fruits were malic acid, citric acid, propanoic acid, and acetic acid. Comparatively, June plum showed the highest content of total sugars and carambola the lowest, while the highest total in organic acids content was found in otaheite and the lowest in carambola. On the other hand, most sugars increased during ripening of the three fruits, while organic acids decreased. Total sugars increased by 37%, 8% and 46% in ripe carambola, June plum and otaheite, respectively. Total organic acids decreased by 20% and 49% in ripe carambola and otaheite, while they slightly increased by 3% in ripe June plum. Furthermore, sugars/organic acids ratio in all fruits increased during maturation and ripening stages. Principal component analysis (PCA) showed two main groups of highly scoring metabo­ lites, while the hierarchical cluster analysis (HCA) showed that the metabolites were grouped into three main clusters. Conclusively, results showed that glu­ cose, fructose, malic acid and tartaric acids were the key marker metabolites of the maturation and ripening stages of the three fruits.


Introduction
Carambola (Averrhoa carambola) belongs to the family Oxalidaceae (*) Corresponding author: noureddine.benkeblia@uwimona.edu.jm originated in Asia but has since developed a toler ance for tropical climates (Shui and Leong, 2006). Presently, carambola is cultivated extensively in India and China (Narain et al., 2001), but Malaysia is the largest exporter (Abdullah et al., 2007). Work has been done to improve carambola cultivars in the United States in the 1930s, and since then, the fruit became more popular, while some sub species of star fruit exist in the Caribbean, Central America and tropical West Africa (Neto et al., 2009). Unripe carambola is moderately sour, but when ripe it is very sweet and used for juice, fruit salads, chutney, stewed fruits, garnish drinks and dishes, or adding it to fruit smoothies. Physiologically, carambola does not exhibit climacteric ripening behaviour even though it continues to synthesise carotenoids and develop its yelloworange colour (Warren, 2009).
June plum (Spondias dulcis Forst. Syn. Spondias cytherea Sonn.), a drupe belonging to the family Anacardiacea, is native to the Society Islands of the South Pacific ranging from Melanesia to Polynesia and was first introduced to Jamaica in the year 1782, and later in 1792 by Captain Bligh (Graham et al., 2004 a). Although the fruit is well known, there are no named cultivars, but both forms exist, one is with a thick mesocarp and a more pleasant taste, and another has long spines, a woody endocarp with a pungent and resinous taste (Daulmerie, 1994). Ripe June plum is eaten mostly raw but is also used in making refreshing drinks, jam, chutney, sauce or served with meat and seafood. When harvested green and mature, June plum ripens, and studies showed that respiratory pattern is typical of a climac teric fruit (Daulmerie, 1994;Graham et al., 2004 b).
Otaheite (Syzygium malaccense) is a berry and belongs to the family Myrtaceae. It is thought to be native to the IndoMalay or Southeast Asian region (Whistler and Elevitch, 2006). Otaheite is, however distributed in many tropical countries throughout the world, particularly in Africa and South America (Oliveira et al., 2011). Other English common names for this fruit include: Malay apple, mountain apple, pomerac, and rose apple (Batista et al., 2017). Two colour forms exist: one which produces red flowers and fruits and another, less common variety, which produces white flowers and fruit (Whistler and Elevitch, 2006). Ripe otaheite fruit is not very sweet, and often eaten raw. However, in some tropical countries, it is stewed with sugar to make jam or wine and refreshing drinks. No study is recorded on whether otaheite is a climacteric or nonclimacteric fruit except the work of Basanta (1998) who reported that otaheite is a nonclimacteric fruit.
Ripening can be defined as the total changes of fruit tissue metabolism, leading to the production of an attractive fruit which can be consumed, aiding in the release and dispersal of the seed (AdamsPhillips et al., 2004). The ripening process is characterized by softening of fruit tissue and an increase in volatile compounds as well as pigments such as carotenoids and flavonoids which results in a more appealing fruit (Giovannoni, 2001). The concentration of sugars and organic acids in fruits varies depending on the fruit variety and the environmental conditions of the par ent plant (Haruenkit, 2004). Overall, there is a gener al decrease in organic acids and an increase in sugar content as fruit development progresses, due to decarboxylation of organic acids and breakdown of stored carbohydrates to produce sugars (BatistaSilva et al., 2018). According to Etienne et al. (2013), using advanced technologies, i.e. proteomics, transcrip tomics and metabolomics, studies have shown evi dence of a shift from the accumulation of organic acids to sugar synthesis in the final stage of fruit development in several species of fruit. Thus, the res piratory pathways commonly involved in the reduc tion of fruit sugars are glycolysis, oxidative pentose phosphate (OPP) pathway, and the tricarboxylic acid (TCA) pathway (Tucker, 2012).
Because most fruits reach their best sensorial and commercial quality attributes when they ripen on the plants, the correct maturity for harvest of fruits impacts their postharvest shelflife and quality attributes during storage (Thompson, 2003). Gene expression resulting from natural processes and trig gering fruit ripening induces many metabolic pro cesses leading to the formation of hundreds and even thousands of different metabolites (Pech et al., 2013).
Although extensive literature is readily available on the metabolic changes during the maturation, ripening and senescence of fresh crops, few researches reported on changes in metabolite pro files during postharvest ripening and senescence (Benkeblia, 2016). However, a limited work was car ried out on the metabolites variation during the development and ripening of peach (Lombardo et al., 2011), strawberry (Zhang et al., 2011), pear (Oikawa et al., 2015, and pitaya (Wu et al., 2019), while scarce work was reported on some tropical fruit (Fabi et al., 2010).
In the present work, in order to explore the varia tion of the metabolic profile of three tropical fruits commonly consumed in the tropics, we performed a profiling study of primary metabolites which are the main indicators of the maturation and the ripening of fruits. For this purpose, we selected three fruits namely carambola (sweet type), June plum and ota heite. One of the goals of this study was to assess how metabolically different the "on tree" maturation and ripening stages of these three fruits are and to find out if there is any particular metabolic profile which could be associated with these two stages of the fruits. On the other hand, by evaluating the metabolomic pattern at both maturation and ripen ing stages of the three fruits. Overall, this study is aiming to explore a part of the chemical potential of carambola (sweet type), June plum and Otaheite which will aid in the future to know the primary metabolites of these fruits that may correlate to dif ferent stages and determine which metabolites might be used as maturation and ripening markers.

Fruits collection
For the purpose of the present study, three physi ological stages of the fruits were investigated: green immature, mature, and ripe stages (Fig. 1). The colour and softness of the fruits were the two criteria used for discriminating the different maturation and ripening stages. A period varying from seven to ten days elapsed between each harvesting (sampling) stage. The commercial (optimal) harvesting stage of carambola is stage 3 (ripe), while for June plum and otaheite is stage 2 (mature). The fruits carambola, June plum, and otaheite of the three stages of each fruit were collected from three trees of same loca tion. The fruit June plum was collected from a farm in St. Elizabeth. Otaheite fruits were collected from a local farm in Mona, Kingston, and carambola samples were collected from Orange River Research Station in St. Mary. The three stages differentiated and sam pled based on their size and colour. For each stage, three samples were collected from three different trees, and each sample consisted of at least six fruits. Fruits collected were controlled for absent of any defect, wound or disease. Immediately after being collected, fruits were placed in plastic bags, and the bags were placed on ice in a cooler and transported to the laboratory within few hours. Then, fruits were washed with mild detergent and rinsed thoroughly, followed by seed removal, dicing or slicing and frozen for 48 hours at 20°C.

Freeze-drying
Prior to the extraction of the profiled sugars and organic acids, samples were freezedried in a Labconco freezedrier (Labconco Corp., Kansas, MO, USA). After six days and complete drying, samples were sealed in plastic bags under vacuum using a MULTIVAC C100 vacuum packer (MULTIVAC, Wolfertschwenden, Germany) and stored under dry ness in a desiccator until further use.

Extraction of sugars and organic acids metabolites
Sugars and organic metabolites were extracted by the method described by Broeckling et al. (2005) with some modifications. In an Eppendorf tube, 300 mg of freezedried samples were mixed with 0.75 mL HPLC grade water containing 26 μg/mL Ribitol was added as internal standard and the tubes vortexed. After equilibrating to room temperature. The tubes were incubated in a shaker for 10 min at 80˚C, fol lowed by incubation at room temperature for c.a. 45 minutes. Afterwards, the tubes were cooled to 4˚C and centrifuged at 10 000 rpm for 15 minutes, the supernatant collected, and the pellet discarded. To the collected supernatants, 250 µL were mixed with 100 mL absolute EtOH and the samples dried under vacuum until dryness and stored at 20°C until GCMS analysis.

Derivatization
Prior to GCMS analysis, samples were derivatised as described by Broeckling et al. (2005) with some minor modifications. The dry residues were mixed with 80 μL of BSTFA+1% TMCS (Sigma Aldrich, St Louis, MI, USA) and 20 µL pyridine, vortexed and cen trifuged for 10 seconds at 10 000 rpm. Afterwards, the mixtures were incubated for 20 minutes at 85˚C. After incubation and equilibrating to room tempera ture, 200 μL isooctane (2,2,4 trimethylpentane) (Sigma Aldrich, St Louis, MI, USA) were added to the mixture, vortexed and followed by a centrifugation at 10 000 rpm for 10 seconds. From the mixtures, 100 μL were transferred to a 300 μL glass insert for the GCMS analysis.

GC-MS analysis of sugars and organic acids
The samples were analysed by GCMS using an Agilent 7890B gas chromatograph coupled to an Agilent 5977A mass spectrometer scanning in the m/z range from 40 to 550. The column used was an HP 5MS (5% Phenyl Methyl Polysiloxane, 30 m × 250 µm × 0.25 μm) with helium as the gas carrier at a constant flow rate of 1.0 mL/min. The samples were injected at a 15:1 split ratio. Initially, the inlet line was held at 260˚C and the transfer line was held at 280˚C. Separation was achieved with an initial tem perature program of 40˚C for 2 min, then ramped up at 4˚C per minute to 240˚C and held for 1 minute. The temperature was then increased to 10˚C per minute to 315˚C.
In order to produce the concentration curve, a mixture containing 250 µL of 26 µg/mL of ribitol was used. To the mixture, 20 μL of pyridine and 80 μL of BSTFA containing 1% TMCS was added, vortexed and injected into the GCMS. Prior to the injection, 50 µL of the mixture were taken and 50 μL of isooctane were added. This was repeated by adding each time 50 μL of isooctane until 6 concentration curves were produced. From the curve a scatter plot with a trend line was generated. The concentrations of the differ ent profiled sugars and organic acids metabolites were calculated from the generated trend line. The generated MS files were extracted, and the deconvo lution and identification of the metabolites was car ried out by using Agilent MSD Chem Station (Version F.01.01.2017) along with NIST library (Version 11 MS Mass Spectral Library) and AMDIS (Version 2.66) soft ware.

Statistical analyses
For the analysis of each sample (three fruits for each ripening stage and for each species), six samples were analysed, and the data were averaged. The data were analysed and compared by running analysis of variance (ANOVA), Tukey's Honestly Significant Difference (HSD) Post Hoc test using SPSS software package (version 22.0, (IBM Corp., New York, USA). The significance level of all statistical hypotheses testing procedures was predetermined at P < 0.05 and 0.01. For the classification, clustering, and regression, PCA (principal component analysis) and HCA (hierarchical cluster analysis) were performed using SPSS software package (version 22.0, IBM Corp., New York, USA), while the clustered heatmap was generated using ClustVis free software (https://biit.cs.ut.ee/clustvis/).

Profile and sugar contents of carambola, June plum and otaheite fruits
The profiling of sugars showed that seven saccha rides and one sugars alcohol were detected in caram bola, June plum and otaheite fruits (Table 1). Glucose, fructose, sucrose, galactose, arabinose, and myoInositol have been detected in carambola, June plum and otaheite, however, mannose and xylose were not detected in June plum while sucrose and mannose were not detected in otaheite. Overall, fructose, sucrose, galactose, in carambola and June plum, and glucose, fructose and galactose in otaheite increased during maturation and ripening stages, while the other sugars varied differently in the three fruits.
Interestingly the highest levels of glucose, fruc tose and sucrose were observed in June plum, high est levels of galactose and xylose in otaheite, and the highest levels of arabinose and myoinositol in carambola. Results also showed that glucose and fructose were the most predominant monosaccha rides in carambola and June plum, while in otaheite glucose, fructose and galactoses were predominant.
On the other hand, a significant increase in total sug ars was noted in carambola and otaheite, while in June plum sugars content increased slightly. The total increase of sugars averaged 38% and 45% in caram bola and otaheite, respectively, but in June plum increases averaged 9%.
Statistically, total sugar contents in carambola and otaheite were significantly different and their con tents in immature fruits were significantly different in comparison with mature and ripe carambola. Immature carambola and otaheite had 35% and 36%, and 28% and 46% less total sugars than the mature and ripe stages, respectively. However, statistical analysis showed no significant difference in sugar contents of June plum during the three maturation and ripening stages.

Profile and organic acids contents of carambola, June plum and otaheite fruits
With ten different acids identified in carambola, June plum and otaheite fruits, eight were detected in June plum, six in carambola and four in Otaheite (Table 2). Overall, malic acid, oxalic acid, propionic acid and acetic acids were the most abundant organic  On the other hand, the ratio of sugars/organic acids plays an important role that can characterise the ripe stage of fruits. During the different stages, the ratio of sugars/organic acids maintained a signifi cant rising trend especially in carambola and ota heite. In carambola, the ratio was 6.47, 9.42 and 11.45 in immature, mature and ripe, respectively. In June plum, the ration was 9.13, 8.17 and 9.59, in immature, mature and ripe, respectively. In otaheite, the ratio was 3.07, 5.93 and 8.75, in immature, mature and ripe, respectively.
Statistical analysis showed that total organic acid contents of carambola was not significantly different between either immature and mature, or mature and ripe stages. Malic acid content varied significantly during the development and ripening of carambola and otaheite, but not significantly in June plum. Malic acid was also the main organic acid accumulating in carambola and otaheite and its level was significantly different among the three stages of the maturation and ripening of the two fruit. The statistical analysis also showed that citric acid and acetic acid contents did not show significant difference among the three developmental stages of carambola. Comparatively, total sugars and total organic acids during the three stages showed different correlations. In carambola and June plum, weak correlation (R 2 =0.18 and R 2 = 0.33, respectively) was observed between sugars and organic acids contents, however, a moderate correla tion (R 2 = 0.56) was observed between sugars and organic acids contents of otaheite during the three stages.

Factoring and clustering of the profiled metabolites
The principal component analysis of the data sets revealed two individual clusters that seem to be gov erned by the developmental and the ripening stages of the fruits (Fig. 2). The analysis showed that in PC 1, the metabolites in carambola with the highest load ing scores were glucose (0.99), galactose (0.99), xylose (0.97), sucrose (0.96), myoinositol (0.93) and mannose (0.92), while in PC 2 ascorbic acid (0.99), fructose (0.98), citrate (0.95) and acetic acid (0.98) had the highest loading scores. In June plum, the highest loading sores metabolites in PC 1 and PC 2 were oxalic acid (0.90), acetic acid (0.85), sucrose (0.84) and gluconic acid (0.80), and malic acid (0.89), fructose (0.89), arabinose (0.82, galactose (0.82) and sucrose (0.77), respectively. In otaheite, the metabo lites with the highest scores were galactose (0.98), myoinositol (0.98), malic acid (0.96), tartaric acid (0.96), acetic acid (0.89), citric acid (0.79) and propanoic acid (0.74), and arabinose (0.99) and xylose (0.99), respectively. Overall, principal compo nents analysis (PCA) of samples based on the devel opment and ripening stages revealed a difference between grouped metabolites. As suggested by the PCA in the figure 2, profiled metabolites were then divided into two classes, and loading values of sugars and organic acids of fruits samples were found most ly in quadrant PC2+, illustrating the discriminated metabolites reflecting the development and ripening of fruits. Hierarchical cluster analysis (HCA) was applied to a data set of the profiled and detected metabolites during the three stages of the three fruits. The den drograms (Fig. 3) show that the profiled metabolites were quite homogeneous and tend to be distributed into three groups. According to the dendrograms of the HCA, at the distance of three, the metabolites can be grouped as shown in Table 3. Interestingly, three metabolites have been classified within the same groups of the three fruits. Myoinositol, citric acid and arabinose were classified in group 1, group 2 and group 3, respectively. The clusters of the differ ent metabolites in the three fruits showed that the metabolites were quite clearly hierarchically separat ed, and these results were also clearly depicted by the PCA (Fig. 2) and the HCA (Fig. 3) which show the distribution of the metabolites into three main clus ters. Furthermore, the heatmap (Fig. 4) also shows that June plum concentrates the highest levels of ten metabolites, while carambola and otaheite concen trate the highest levels of four and six other metabo lites, respectively.
Indeed, primary metabolites profiling led to the identification 8, 6 and 5 sugars, and 6, 8 and 5 organ ic acids in carambola, June plum and otaheite, respectively. On the other hand, our results showed that the key marker metabolites of the maturation and ripening of the three fruits are glucose and fruc tose in carambola and otaheite, while in June plum glucose, fructose, galactose and sucrose were the key marker metabolites of the three different stages. Similarly, malic and tartaric acids were the key organ ic acids metabolites of the maturation and ripening  of carambola and otaheite fruits, while malic acid was the key marker metabolite of June plum matura tion and ripening.

Discussion and Conclusions
Although extensive literature is readily available, the variation of sugars and organic acids of many fruits including carambola and June plum at ripe stage, less and scattered work was done on the varia tion of the metabolites including sugars and organic acids in carambola, June plum and otaheite fruits during the development and ripening stages. On the other hand, most of the work carried out on caram bola targeted the postharvest physiology and bio chemistry of the fruit during storage.
In carambola, Campbell and Koch (1989) found that total soluble sugars concentration, mainly glu cose and fructose, increased during ripening and var ied between 22 and 27 mg/g fresh weight depending on the varieties, while Narain et al. (2001) investigat ed the variation of the chemical composition of carambola at three different ripening stages and found that total sugars increased from 2.91 to 5.60 g/100 g fresh weight. Later, Patil et al. (2010) report ed the composition of the fruit at three stages of maturity (young, halfripe and ripe), and they noted a tremendous increase of total sugars, oxalic acid and ascorbic acid by 100%, 89% and 65%, respectively. Similar increase by 33% and 90% of total sugars and ascorbic acid respectively were also reported by Ali and Jaafar (2012). Glucose, fructose and sucrose were reported to be the most predominant sugars in carambola (Mohd Zainudin et al., 2014;Benkeblia and López, 2015), however, Benkeblia and López (2015) reported an increase of glucose and fructose, but a slight decrease of sucrose in the ripe fruit com pared to the green one, while Mohd Zainudin et al. (2014) noted an increase of the three sugars.
There is almost no work reporting on the compo sition of June plum fruit during maturation and ripen ing except from the one of Benkeblia and López (2015). The authors investigated the variation of glu cose, fructose and sucrose in green and ripe June plum and found that in ripe fruit glucose and fructose increased in ripe fruit, while sucrose decreased signif icantly. Other scattered studies reported on the vari ation of sugars and organic acids in June plum fruit but at a specific stage. In a study carried out on immature green June plum, Franquin et al. (2005) investigated the composition at this early stage and found the concentrations of glucose, fructose, sucrose, citric acid, malic acid, oxalic acid and ascor bic acid were 1.5 (± 0.2), 1.2 (± 0.2), 3.1 (± 0.3), 0.9 (± 0.1), 0.2 (± 0.02,) 0.03 (± 0.01), and 52.0 (± 4.9) g/100 g fresh weight, respectively. In his study, Nahar et al. (1990) reported that 0.3% of fresh weight of the pulp is composed by free sugars, where glucose, fructose and sucrose were the most predominant (Nahar et al., 1990;Mahmood et al., 2012).
Similarly to June plum, few studies investigated the composition of otaheite during the maturation and ripening, however, few studies reported on ripe otaheite. Lu and Lin (2011) investigated the sugars in ripe otaheite and found that fructose yielded the highest content compared to glucose and sucrose which were detected at this ripe stage.
The untargeted profiling of primary metabolites during the maturation and ripening of fruits is a good approach to provide better insight into their metabolome changes during these stages. Different studies on metabolite analyses of fruits have focused on temperate and stone fruits such as tomato, peach, strawberry, and grape among many others, but scarce studies focused on tropical fruits. However, these studies revealed similar dynamic variations in the levels of sugars and organic acids, as well as many other primary and secondary metabolites dur ing fruits maturation and ripening (Oikawa et al.,   (2010) used transcriptomics markers to understand the matura tion and ripening programmes in mango (Mangifera indica L.) fruit. Among eighteen genes related to the fruit physiology and biochemistry, genes related to primary metabolism showed higher expression in comparison to that of the genes related to flavour production.
However, regardless of the origin and environ mental zones, the maturation and ripening of fruits are complex and highly coordinated processes. Globally, the increase in sugar and decline in organic acids are one of the main changes associated with these processes (Giovannoni, 2001;Klee and Giovannoni, 2011;Osorio et al., 2013;BatistaSilva et al., 2018). During maturation and ripening of fruits, organic acids contents are inversely related to sugar contents. The rising trend of sugars is due to photo synthates import or starch degradation, while organ ic acids that accumulated in young fruits strongly decrease by being converted to other organic acids (Carrari et al., 2006;Beauvoit et al., 2018). Although environmentally different from tropical fruits, there have been a number of different studies reporting similar metabolic changes that occur in temperate fruits during maturation and ripening stages (Fait et al., 2008;Osorio et al., 2011Osorio et al., , 2012 (Kurt et al., 2017;Liang et al., 2011;MunõzRobredo et al., 2011), loquat (Eriobotrya japonica Lindl.) (Amorós et al., 2003), mango (Mangifera indica L.) (Mokhtar et al., 2014), melon (Cucumis melo L.) (Wang et al., 1996), pome granate (Punica granatum L.) (NuncioJáuregui et al., 2014) and wolfberry (Lycium barbarum L.) (Zhao et al., 2015) among other reported fruits. Furthermore and in agreement with our finding, glucose, fructose, and sucrose were found to be the most predominant among mono and disaccharides, while malic, citric and tartaric acids were predominant organic acids (Wang et al., 1996;Liang et al., 2011;Mahmood et al., 2012;Kurt et al., 2017;Yang et al., 2021).
Indeed, the relative levels of sugars and organic acids in fruits are of great importance for harvesting time and are one of the determinants of the organoleptic quality attributes of fruits particularly sweetness (Itai and Tanahashi, 2008). Furthermore, the postharvest quality attributes of fruits, their shelflife and even processed products are strongly associated to their sugars and organic acids levels (Matsumoto and Ikoma, 2012;Aprea et al., 2017). In order to preserve freshness and reduce economic losses, it is of great importance to understand the metabolic changes occurring during maturation and ripening which might contribute to accelerate fruits senescence and perishability after harvesting. In this sense, metabolomic profiling of key metabolites responsible for quality attributes such as sugars and organic acids can be a powerful tool for further understanding the biochemical basis of pre and postharvest physiology and have the potential to play a critical role in the identification of the path ways affected by fruit maturation and ripening (Allwood et al., 2021;Pott et al., 2020;Tian et al., 2021).
The data presented here indicates that the pro filed metabolites varied significantly during the matu ration and ripening of the fruits. Glucose, fructose, galactose, sucrose and myoinositol were found pre dominantly in all the fruits and during the three stages, except sucrose in otaheite. Comparatively, June plum showed the highest content of total sugars and carambola the lowest, while the highest total in organic acids content was noted in otaheite and the lowest in carambola. On the other hand, most sugars increased during ripening of the three fruits, while organic acids decreased. Interestingly, the multivari able analysis showed than all the metabolites were clustered into three main clusters, and myoinositol, citric acid and arabinose shared group one, group two and group three, respectively. From the different profiled sugars and organic acids, our results are sug gesting that glucose and fructose are the marker metabolites of the maturation and ripening of caram bola and otaheite, while the ripening marker metabolites in June plum are glucose, fructose, galac tose and sucrose. Because this study represents the first report on the profiling of sugars and organic acids in carambola, June plum and otaheite, it might be interesting to profile the secondary metabolites mainly phenolics and volatiles, and their variation during the maturation and ripening of these fruits.