# Effect of Production Technique on Pilsner-Style Non-Alcoholic Beer (NAB) Chemistry and Flavor
## 3 Results and Discussion
### Evaluating the Impact of Production Technique on the Volatile Chemistry of the Different NABs  
In this study, a broad spectrum of volatile compounds (i.e., aldehydes, hop-derived terpen/terpenoids, esters, and fermentation by-products) was analyzed. Overall large concentration ranges were observed for most of the volatiles amongst the NABs made from different production techniques. The concentration ranges of the volatile analytes measured were in line with what has been previously observed in NAB [15; 16]. The concentrations of fermentation by-products (i.e., acetaldehyde, acetate esters, higher alcohols, and ethyl esters) were low in a majority of the NABs tested and for most of the analytes the results were in the range or below 1 mg/L. Exceptions from this were 3-methyl-1-butanol and phenyl ethanol, which were the two-primary fermentation-derived alcohols observed in the NABs tested (Table S2). The volatile profile of C1 is particularly unique because, as previously mentioned, this NAB had a natural aroma extract added to it. Interestingly, the fermentation by-product spectrum of C1 is more similar to what one would generally expect in an alcoholic pilsner type beer. For instance, C1 contained 4 mg/L ethyl acetate, 31 mg/L 3-methyl-1-butanol, and 19 mg/L of phenyl ethanol, whereas alcoholic pilsner beer has been shown to contain \(\sim\)8-32 mg/L ethyl acetate [29], \(\sim\)50 mg/L 3-methyl-1-butanol [30], and \(\sim\)18 mg/L of phenyl ethanol [30]. However, compared to its alcoholic counterpart (data not shown),it is clear that the aroma addition of C1 did not target to add equivalent concentrations of the fermentation by-products and is in the range of 50% or less for most volatiles. This is because it has been shown that the amount of ethanol has an impact on the release and perception of volatiles in NAB/alcoholic beer [31] and this should be taken into consideration when brewers choose to use aroma extracts. When considering the analytical precision (%RSD) and concentration range, the concentrations of ethyl esters detected in this study (Table S4) fall well below previously reported thresholds of these compounds [32]. Therefore, it is likely that these esters are playing a limited role in German NAB pilsner beer flavor. However, the concentrations of ethyl esters in C1 were also unique to the other NABs. In general, the concentrations of ethyl butyrate (15 \(\mu\)g/L), ethyl hexanoate (62 \(\mu\)g/L), and ethyl octanoate (14 \(\mu\)g/L), were all detected in levels of \(>\)5 \(\mu\)g/L in C1. However, this was not the case for the majority of the other NABs tested. One could assume that ethyl butyrate and ethyl hexanoate were included in the natural aroma added to this beer because C1 contained comparably high concentrations of ethyl butyrate and ethyl hexanoate, whereas the concentration of ethyl octanoate was in the range of most other NABs. The only other NABs that contained detectable levels of all three ethyl esters were M1 and V3, while for the majority of the other NABs, only ethyl octanoate was found. As reported previously [25], the concentrations of staling aldehydes in NAB are commonly higher than compared to the concentrations observed in beer. In this study, the concentrations of aldehydes varied significantly amongst the NABs and occurred in concentrations below previously reported sensory thresholds [33] (Table S3). The concentrations measured are also in agreement with previously reported values [15; 17]. Across the samples tested, furfural was the primary aldehyde and ranged from 31 \(\mu\)g/L (V4) to 250 \(\mu\)g/L (M1) (Table S3). The Strecker aldehydes 3-methylbutanal, 2-methylpropanal, phenylacetaldehyde, methional, and 2-methylpropanal ranged from \(\sim\)1 \(\mu\)g/L to \(\sim\)60 \(\mu\)g/L, while the (unsaturated) linear aldehydes were typically at concentrations \(<\)1 \(\mu\)g/L (Table S3).