## 4 Discussion  
The investigation of the ripening process is of paramount interest not only from a scientific point of view but also for the agri-farm industries facing the increasing consumer awareness about the healthiness and safety of their food [19]. In this regard, metabolic profiling has been applied to study the development of fleshy fruits such as tomatoes, grapes, strawberries, and peaches among others [20; 21; 22; 23]. Quite surprisingly, there are considerably fewer studies on the metabolic variations in roots, especially edible ones, although both development and ripening can have a significant influence on their phytochemical composition, as demonstrated by investigations on targeted compounds such as carotenoids, auxins, and plant hormones or on different cultivars [24; 25; 26; 27; 28]. Furthermore, the chemical composition of the raw material is affected by factors such as different cultivars, geographical origin, growing sites, and soil, seasonal, and climatic differences [15; 29]. This source of intrinsic variability is further increased by the choice of the harvest time, which should be standardized when evaluating the potentiality of a food in terms of either nutrient content and health benefits, or different utilization of the raw material or derived products such as, for example, natural colorants or source of bioactive compounds. PCA of the purple carrot composition as a function of time showed a clear clustering of the samples along PC1 with a separation of the carrots harvested in September, October, and November (Figure 3). The model was affected by a sharp increase in the free amino acids stored in the roots during maturation. This increase suggests that different metabolic processes are involved in root development compared to fruit ripening since an overall decrease in amino acid levels coupled with the induction of catabolic transcripts has been observed during fruit maturation [30]. Furthermore, while fruits accumulate sugars during ripening, sucrose, glucose, and fructose levels did not change significantly in carrot roots during the three sampling times. It is known that the regulation of amino acid catabolism varies among plant species, tissues of the same species, and their developmental stages [30]. In addition, amino acids are involved in many physiological processes such as energy production, redox state regulation, resistance to abiotic and biotic stresses, as well as plant growth and development, and are precursors of many different secondary products such as phenylpropanoids [31]. Amino acid content has also been linked to the sensing of nitrogen status, alterations in exudate composition, and inhibition of the level of root tissues [32; 33]. Therefore, the observed high increase in free amino acid content in carrot roots during maturation could be the result of both physiological processes during growth and seasonally linked environmental changing conditions. In this regard, the sharp increase in the levels of the amino acids leucine, isoleucine, and tryptophan from October to November could suggest that these compounds act as chemical biomarkers of full maturation for purple carrots (Table 1). The observed increase in the precursors of anthocyanins, especially aromatic amino acids and phenylpropanoids during ripening is in agreement with the increase in anthocyanins observed both in different cultivars of purple carrots reported in the literature and in relation to the harvest time [34; 35]. The concomitant increase during ripening of compounds possessing co-pigmentation enhancing properties, like the uncolored caffeic acid and NAD, could act as color stabilizers further increasing the value of carrot by-products in relation to the harvest time.