# Profiles of Volatile and Phenolic Compounds as Markers of Ripening Stage in Candonga Strawberries
## 3 Results and Discussion
### VOCs Compounds in Half-Red and Red Strawberries
The formation of C6 aldehydes originates from the lipid oxidation pathway, which involves lipoxygenase (LOX) and hydroperoxide lyase (HPL) enzymes. During ripening, the content of the two C6 aldehydes, namely hexanal (Ald1) and 2-hexenal (Ald 2), increases, paralleling the 25-200% increment in the activity of LOX and HPL enzymes and the availability of total linolenic acid as the precursor, which rises up to 200% [32]. Nonanal (Ald3) and decanal (Ald5), arising from the autoxidation of oleic acid, and benzaldehydeds (Ald4), deriving from Strecker degradation of aromatic amino acids, increase during oxidative processes [33]. Gene expression studies reported the existence of a specific enzyme, O-methyltransferase, that is responsible for the synthesis of mesifurane (F1). Additionally, a _Fragaria \(\times\) ananassa_ quinine oxidoreductase (FaQR) was documented to be involved in the formation of furaneel (F2). F1 and F2 increased considerably during ripening of strawberries, as the enzymes belonging to their biosynthetic pathway showed the maximum activity at the full red stage, allowing potential targets to engineer the strawberry flavor from breeding populations [31]. 3.2 Selection of the VOCs Correlated to Ripening Stage of Strawberry by Principal Component Analysis  
Principal component analysis (PCA) took into account AA, TPC, the semi-quantitative data of both individual phenolic compounds (Table 2) and VOCs (Table 3), to infer possible significant associations of strawberries at the two different ripening stages (half-red or red) and at the three harvesting times (Figure 3). The two components accounted for 94.8% of the variation in the dataset, since PC1 and PC2 explained 90.3% and 4.5% of the total variance, respectively. In the PCA plot, all red samples (Red-H1, Red-H2 and Red-H3) appeared in the left part of the score plot, while all the half-red strawberries (Half-red-H1, Half-red-H2 and Half-red-H3) were positioned on the right part of the graph, revealing a different distribution of the samples in the PCA quadrants (Figure 3A). Specifically, Half-red-H1 and Half-red-H2 samples clustered closely in the bottom right part of the plot, having positive PC1 and negative PC2 values (Figure 3A). These samples showed a significant correlation with three phenolic compounds (P5, P6 and P7) and with AC (Figure 3B). The direct association of Half-red-H1 and Half-red-H2 samples with the P5, P6 and P7 metabolites likely reflects the decline in the synthesis of flavonoids during the last stage of ripening, as demonstrated for other fruits. The decrease in flavonoids might correspond to their utilization for the downstream biosynthesis of other metabolites, or to the covalent association with other cellular components [34]. Furthermore, a higher contribution of flavonoids compared to other classes of phenolic compounds could explain the correlation with AA. On the contrary, Half-red-H3 fruit presented positive PC1 and PC2 scores (Figure 3A) and appeared statistically associated only with TPC (Figure 3B). The strawberry samples classified as Red-H1 and Red-H2, with negative PC1 and PC2 scores, were grouped closely in the bottom left part of the PCA plot (Figure 3A) and covariied with the same volatile metabolites: 39 VOCs comprising 14 esters (E9, E10, E13,E15, E16, E17, E18, E19 E20, E21, E23, E24, E25, E26), 4 aldehydes (Ald2, Ald3, Ald4, Ald5), 3 alcohols (Al1, Al2, Al4), 8 acids (Ac1, Ac2, Ac3, Ac5, Ac6, Ac7, Ac8, Ac9), 4 terpenes (T1, T3, T4, T5), 2 furanones (F2, F3), 3 lactones (L1, L2, L3) 1 other (O1) and 3 phenolic compounds (P2, P3 and P8) (Figure 3B). In the PCA score plot, Red-H3 strawberries presented negative PC1 and positive PC2, and were placed in the high left section of the PCA plot (Figure 3A). This sample showed a significant correlation with 2 phenolic compounds (P1 and P4) and 18 VOCs, including 12 esters (E1, E2, E3, E4, E5, E6, E7, E8, E11, E12, E14, E22), 1 aldehydes (Ald1), 2 alcohols (Al3 and Al5), 2-methylbutyric acid (Ac4), \(\beta\)-farnesene (T2) and mesifurane (F1) (Figure 3B).