# Effect of Asparaginase Treatment on Biscuit Volatile Compounds
## 3 Results
### VOCs  
The VOC profile was analysed by determining the level of 36 identified compounds. A 1-way ANOVA performed on the dataset of all treated biscuits showed significant differences between the experimental samples for 32 out of 36 determined compounds (Table S2). In Figure 2 the Heat map analysis showed that the biscuit samples were clustered (dendrogram of columns) based on the level of asparaginase addition rather than on the preparation conditions. Moreover, biscuits obtained with the addition of 750 ASNU differed from all the others, being assigned to a distinct cluster. The VOCs were clustered (dendrogram of rows) based on the chemical group and the formation pathway: the upper cluster included VOCs mainly formed through the MR (pyrazines, products of Strecker degradation, furanic compounds), the lowest cluster included VOCs mainly formed by lipid oxidation (hexanal, nonanal, E-2-nonenal, 1 hexanol, 2-pentylfuran). Pyrazines were highest in the B500 biscuits, followed by B1000 samples, whereas Strecker aldehydes (2- and 3-methylbutanal) and furans were highest in B1000, followed by B500. On the contrary, the addition of 750 ASNU was associated with higher levels of lipid oxidation products. However, these differences observed in the VOC profile do not seem to be related to the asparagine level in the dough. In fact, the strong reduction in its level does not correspond to marked differences in the VOC profile of the asparaginase-treated samples when compared to control biscuits (the cluster on the right in the dendrogram of columns). The PCA analysis (Figure 3) confirmed that the control biscuits had a VOC profile similar to the profile of biscuits obtained with the asparaginase addition at 500 and 1000 ASNU levels, whereas the profile of the 750 ASNU biscuits tended to differ from the others. In addition, the bi-plot obtained from the PCA allowed to better observe the effects of the preparation conditions: a resting time of 15 min at 20 \({}^{\circ}\)C (condition b) promoted the formation of VOC derived from lipid oxidation (E-2-nonenal, nonanal, 2-pentylfuran, hexanal, compounds associated with the positive axis of PC1) while lowering the level of pyrazines and furans formed through the MR (compounds associated with the negative axis of PC1). On the contrary, the formation of these last compounds was enhanced when a dough resting time was not applied (condition a). Thus, an inverse correlation between MR-related VOCs and VOCs formed by lipid oxidation was observed. Interestingly, the preparation conditions (a vs. b vs. c) had a similar impact on the VOC profile at all addition levels of the enzyme. As regards differences between asparaginase addition levels, in all control  
Figure 1: Asparagine level (mg Kg\({}^{-1}\)) in the dough experimental samples. Mean values, standard deviations (\(n=2\)). biscuits and those obtained with 500 and 1000 ASNU addition, the level of VOCs formed by MR tended to be higher than the 750 ASNU samples. Figure 3: PCA analysis of VOC profile instead of the preparation conditions (a, b, c) and level of enzyme addition (500, 750, 1000 ASNU). Figure 2: Heat maps analysis of VOCs: cluster based on the level of asparaginase addition (Crtl/500/750/1000) instead of the preparation condition (resting time and dough temperature). Results of the ANOVA also allowed us to consider the effects of the asparaginase addition and preparation conditions on important known key odorants of the bscuits (Table S2). For example, the level of 2,3-butanedione, responsible for the butter-like odour note, tended to be reduced when a resting time at 50 \({}^{\circ}\)C was applied, and also when asparaginase was added at a level of 500 ASNU. The 2-methyl butanal and 3-methyl butanal (characterised by malty, nutty, chocolate odour notes) showed the same trend of 2,3-butanedione, with significantly reduced level at the same preparation condition (c), particularly at the enzyme addition level of 500 ASNU.