## 3 Results and Discussion  
### Integrated Metabolomics and Flavoromics of Germinated Sacha Inchi Seeds  
The germination percentage of sacha inchi seeds was \(\geq\)80%. The seeds began germinating after 2 days and reached the maximum germination percentage at 8 days. Metabolomics identified 63 (-65%) of a total of 95 metabolites at 0 DAG-8 DAG, similar to comparable metabolite profiling studies on mung bean [12] and soybean [25]. The 63 metabolites were categorized into 4 groups: 18 FAMEs (16 FAMEs and 2 hydrocarbons), 17 polar lipids (10 FFAs, 2 fatty alcohols, and 5 phytosterols), 6 sugars (3 organic sugars and 3 sugar alcohols), and 22 acids (17 amino and 5 organic acids). Flavoromics identified 35 (23%) of a total of 150 flavor compounds, similar to germinated brown rice (41 volatile compounds identified at 0 DAG-5 DAG [17]). The 35 flavor compounds were categorized into 7 groups: 9 aldehydes, 12 alcohols, 2 ketones, 8 volatile acids, 2 lactones, pyridine, pyrazine, and 2-methoxy-3-(1-methylethyl). Figure 2 shows the PCA biplot and loading of all identified metabolites and flavor compounds at different germination times. Polar and nonpolar metabolites were major contributors to the separation along principal component (PC) 1 (Figure 2A). The flavor compounds were prominently arranged at 4 DAG, 6 DAG, and 8 DAG, explaining 75% of the variation (Figure 2B). The PCA biplots of metabolites and flavor compounds displayed four distinct clusters along with the PC (Figure 2C). The first two PCs explained 81.67% of the total variation at 0 DAG-8 DAG, PC1 and PC2 demonstrated eigenvalues of 60.330 and 20.518, respectively, and explained 60.94% and 20.73% of the total variation, respectively. Metabolites and flavor compounds were categorized based on the difference in the germination times into four clusters. The differences between ungerminated seeds (0 DAG) and germinated seeds could be generally described on PC1. The 0 DAG seeds relied on cluster 1 (17 FAMEs, 5 phytosterols, and 8 flavor compounds), 2 DAG on cluster 2 (1 FAME, 5 FFAs, and 1 flavor compound), 4 DAG on cluster 3 (5 FFAs, 3 amino acids, and  
4 flavor compounds), and 6 DAG and 8 DAG on cluster 4 (all sugars, most of the acids, and 20 flavor compounds), indicating that most of the FAMEs and FFAs decrease, while most of the sugars, amino acids, aldehydes, alcohols, and volatile acids increase throughout germination in sacha inchi. To confirm PCA grouping, agglomerative hierarchical clustering analysis in similarity mode was performed based on 0 DAG-8 DAG (Figure 3). The dendrogram confirmed the similarity among the germination stages, differentiated into four groups: group 1 included ungerminated seeds (0 DAG); group 2, 2 DAG seeds; group 3, 4 DAG seeds; and group 4, 6 DAG and 8 DAG seeds. The coupled metabolomics-flavoromics of 0 DAG was closer to that of 2 DAG, whereas that of 4 DAG was closer to that of 6 DAG and 8 DAG, indicating that metabolic and flavor components change more rapidly at the final compared to the early stage of germination. Figure 2: Biplot of principal component analysis from metabolites (**A**), flavor compounds (**B**), and metabolites and flavor compounds (**C**) in sacha inchi seeds at different germination times (\(p\leq 0.05\)). FAME, fatty acid methyl ester; FFA, free fatty acid; DAG, days after germination. Heat map analysis was used to represent dynamic changes in specific compounds by relative concentrations at each germination time (Figure 4). Figure 4A shows a heat map of all metabolites. The FAME contents were higher on day 0 and significantly decreased during germination. Linolenic (50%), linoleic (36%), and oleic (11%) acids were most abundant in the lipid fraction, followed by stearic (4%) and palmitic (0.25%) acids at 0 DAG-8 DAG. Compared to ungerminated sacha inchi seeds, the palmitic acid content decreased by ~65.6-fold, followed by stearic (13.6-fold), linoleic (12.3-fold), linolenic (11.7-fold), and oleic (4.9-fold) acids.