## 4 Discussion  
The traditional method of making sparkling wine consists of a second fermentation in a closed bottle, which places the yeast in a harsh and hostile environment, with high ethanol concentrations and a lack of nitrogen sources, to which it has to adapt metabolically. The nitrogen fraction of wine consists mainly of amino acids. In this study, ammonium chloride, biogenic amines, and amino acids were identified and quantified (Table S1). The consumption and production of nitrogen compounds was different according to the strain; it is known that certain amino acids may be related to the maintenance of the redox potential of the cell [1,16]. The protein expression differences observed between the G1 and N62 yeast strains highlight distinct metabolic adaptations related to nitrogen utilization and resource allocation during fermentation. These differences are consistent with the established nitrogen metabolism regulation mechanisms in _S. cerevisiae_ and shed light on strain-specific strategies for managing nitrogen [17-20]. In the case of _S. cerevisiae_ G1, there is higher cell wall activity (Figure 6a,b), showing significant differences in several related proteins. The upregulation of EXG1 (exoglucanase), UTH1, YGP1, and CIS3 suggests enhanced cell wall remodeling, potentially to optimize the nutrient uptake efficiency in nitrogen-limited conditions [21]. This aligns with nitrogen stress responses, where vacuolar proteolysis increases to recycle nitrogen [19,21]. In addition, PRC1 is involved in phytochemicalain synthesis and is a serine-type carboxypeptidase that degrades small peptides [22]. This protein, together with the vacuolar aminopeptidase, APE3 and vacuolar protease, PRB1, are related to each other, releasing peptides and the serine amino acid intracellularly [23]. In addition, significant differences have been found in lipid-related proteins. PLB1 hydrolyses the phospholipid [24] SCS2, which is related to the plasma membrane and phospholipid biosynthesis [25], and ERG8, which is involved in the ergosterol synthesis pathway [26], and according to the results of [PERSON] et al. [27], this biosynthesis favors nitrogen assimilation, as reflected in this strain (Figure 3a). The presence of LYS9 (lysine biosynthesis) and GLY1 (threonine cleavage) indicates prioritized nitrogen allocation to essential amino acid production [18,20]. NCA3's role in mitochondrial organization and KGD1's involvement in the TCA cycle highlight energy metabolism adjustments. In nitrogen limitations, mitochondrial proteome allocation often shifts to optimize ATP yield per unit of nitrogen. ERG8's role in ergosterol biosynthesis may stabilize membranes during nitrogen stress, a response linked to TOR pathway modulation [28]. In _S. cerevisiae_ N62, upregulated ENO1 (glycolysis) and MDH3 (glyoxylate cycle) suggest enhanced carbon flux toward anaplerotic pathways. This compensates for nitrogen scarcity by prioritizing acetyl-CoA production for biosynthesis, a strategy observed during carbon overflow metabolism [19]. TEF2 (the translation elongation factor) and ILV5 (branched-chain amino acid biosynthesis) indicate increased translational activity and nitrogen redistribution. Such responses are consistent with chromatin regulators like Ahclp/Eaf3p, which globally upregulate nitrogen utilization pathways [17]. MRPL3 (the mitochondrial ribosome component) and ICP55 (mitochondrial proteome stabilization) reflect mitochondrial proteome restructuring under nitrogen stress, balancing energy demands with nitrogen availability. PDC6 and PDA1 upregulation highlights pyruvate metabolism redirection, potentially modulating carbon overflow dynamics to conserve nitrogen [29]. When a comparative analysis is performed, G1 prioritizes nitrogen conservation via arginine and lysine biosynthesis and cell wall maintenance, while N62 invests in translational machinery and alternative carbon utilization. These strategies mirror the proteome reallocations observed under nitrogen limitations.