## 3 Results and Discussion
### Metabolic Profile Changes during Coffee Fermentation
#### 3.3.2 Volatile Compounds  
Figure 5 shows the evolving pattern of VOCs over a 24 h fermentation, highlighting the metabolic impact of both plant-derived (25 VOCs) and microbial-derived (13 VOCs) compounds [2; 24; 38; 40; 41; 42; 43; 2; 44]. The content of each VOC identified throughout the fermentation is also presented in the Supplemental Material, Table S1. The fluctuating levels of volatiles across the time points suggest that microbial activity intensifies as fermentation progresses, with a distinct profile emerging at 24 h. A variety of microbial-associated volatile compounds, including 2-butenal, hexanal, 4-heptanol, hexanoic acid, ethenyl ester, 2-octenal, benzaldehyde, 3-methylbenzaldehyde, and 6-Methyl-hept-2-en-4-ol, were exclusively identified at the end of the fermentation (Figure 5). This could be due to the cumulative effects of enzymatic activities and microbial growth over time, leading to the production of new volatile compounds or the transformation of existing ones [2]. Although members of the Enterobacteriaceae family are often associated with the production of off-flavors in coffee fermentations [15; 17; 22], _Enterobacter_, _Kluyvera_, and _Pantoea_ have been identified as part of the microbiota responsible for flavor formation in fermented foods. This is due to their metabolic versatility, which can contribute to specific esters and alcohol generation [45; 46]. It is important to note that the metabolic pathways of these microorganisms are influenced by the coffee bean's substrate, the fermentation environment, and interactions between microbial communities [11; 12; 23; 46]. The specific metabolites produced during coffee fermentation are often the result of complex microbial consortia rather than individual species [5]. Hence, while these attributions are educated guesses based on typical metabolic products, precise identification requires direct analysis and isolation of compounds produced by each microorganism during fermentation.  
Figure 5: Volatile organic compounds detected during coffee fermentation in the subtropical zone. The differentiation between plant- and microbial-derived based on a comprehensive analysis of studies referenced in [2; 17; 22; 38; 40; 41; 42; 44].  
From the plant metabolites, it is notable that caffeine remains relatively constant throughout the fermentation, which is expected as caffeine is a stable alkaloid during fermentation. The role of caffeine in coffee plants is multifaceted, influencing not only the taste but also contributing to various ecological functions within the plant [48]. During the processing of coffee, including the fermentation stage, the presence of caffeine remains a prominent characteristic of the beans, contributing significantly to the final product's sensory profile [40]. Other plant-derived volatiles showed variations that correspond to the breakdown or transformation of plant materials over time. For example, various metabolites present at the beginning of the fermentation process, including hexadecanoic acid, heptadecane, tetradecane, and heneicosane, are components derived from plant material [41; 42; 43]. Other plant-associated metabolites formed during fermentation may result from plant-aided enzymatic reactions (e.g., geranyl acetone, isopropyl palmitate, pentadecane, and eicosane) or even through the leaching process from the grain to the exterior, like hexadecanoic acid (methyl and ethyl ester), which is one of the main fatty acids found in coffee beans [41; 44; 49]. It is speculated that the leaching of these compounds may result in a lower integrity of the cell membrane system of the coffee beans. Thus, this cellular damage could affect both the diffusion of volatile compounds from the liquid fraction into the beans and allow chemical components previously compartmentalized in the endosperm to encounter oxidative and hydrolytic enzymes, affecting the quality of the final beverage [50]. However, more studies are needed to analyze these phenomena.