## 3 Results and Discussion
### Final Discussion of Release and Bound State Aldehydes  
Based on both these release methods, we finally investigated whether all the aging-related aldehydes are released during aging. Therefore, the sums of all investigated free and bound state aldehydes were calculated. Figure 5 shows the equilibrium between the sum of free aldehydes and the sum of bound state aldehydes assessed by the two applied release methods. We observed that the equilibrium shifted toward the free form during aging. After M9 versus M6, the equilibrium was fully toward the free form, indicating either hydrolysis or a different degradation of an important bound state fraction. Further possibilities could be the irreversible binding on proteins [10], proline-catalyzed aldol condensation [34], or further oxidation reactions, which will be discussed in part 2 of this study.  
Furthermore, the equilibrium was influenced by the amount of soluble N in the sample. In fresh samples, the ratio of bound state and free aldehydes increased with the amount of soluble N for 4 VP, and \(\sim\)82% of aldehydes were free in P3, \(\sim\)73% in P2, and 51% in P1 (R = 0.69). The same result was observed for ACA but to a lower extent (\(\sim\)57% in P3, 51% in P2, and 34% in P1; R = 0.26). A higher proteolytic malt modification level resulted in elevated concentrations of all amino acids except proline (see part 2). Therefore, amino acids are important binding agents for aldehydes. It was assumed that cysteinylated aldehydes have a major influence on beer flavor instability. In fact, they do exist in the beer matrix but only at negligible concentrations and increase during aging since they are in equilibrium with free aldehydes [12]. Thus, the N adducts that we observed in this study are more likely to be in the form of mines, either with amino acids or with larger peptides and proteins. These compounds would be hydrolyzed slowly during beer aging and thus would release free aldehydes. The degradation is favored at pH 4.0 because of the presence of a zwitterionic hemiaminal. Therefore, the targeted analysis of mines after reduction, as described in the literature, is necessary in future research [35]. Furthermore, untargeted analysis of protein-bound aldehydes seems highly promising for further elucidation of bound state species.  
In summary, 4 VP can release more bound state aldehydes compared with ACA. 4 VP acts via a competitive (for nucleophiles) mechanism and also elevates the pH of the sample.  
ACA only acts competitively. Using ACA is the softer, less invasive way of releasing bound state aldehydes. Ultimately, beer is a dynamic equilibrium system, and each method can only shift the equilibrium to a certain extent. Both presented methods, 4 VP- and ACA-induced release, are promising tools for the early-stage assessment of beer flavor instability regardless of the maximum degree of release. Which method comes closest to reality is highly influenced by the samples, its way of production, and, ultimately, storage conditions.  
Contrary to the literature, 4 VP does not mostly release SO2 adducts, as observed in beers with up to 10 mg/L of SO2 [17]. 4 VP and ACA can also release N-related adducts.