# Effect of Asparaginase Treatment on Biscuit Volatile Compounds
## 4 Discussion
It is likely that the reduction of a single amino acid, in this case, asparagine, which represents only a small fraction of the total amino acid content, leads to only minor changes in the volatile compounds for which it serves otherwise as an important precursor. Moreover, in previous experimental studies on the application of asparaginase in biscuit preparation,it has been reported that even minimal variations in processing conditions, particularly baking conditions, can lead to significant changes in the texture and surface colour of the biscuits, despite careful control of experimental variables [13]. Considering the extreme sensitivity of Maillard reaction processes to environmental conditions, it is plausible that some of the minor differences observed in the levels of volatile compounds in biscuits made with different doses of asparaginase may have been caused by such minimal variations in processing conditions. Even though the addition of asparaginase did not largely affect the VOC profile of experimental biscuits, the different preparation conditions had a consistent, though slight, impact on it. The PCA bi-plot showed that the dough preparation conditions had the opposite effect on the formation of compounds produced by the MR and those formed through lipid oxidation. One possible explanation could be the different influence of the tested dough handling conditions on the two pathways of VOC formation. The application of a resting time of 15 min at 20 \({}^{\circ}\)C before baking reduced the formation of most MR-related VOCS, whereas the products of lipid oxidation were enhanced. [PERSON] et al. [24] previously observed that lipid oxidation products were more abundant in dough compared to the individual ingredients used in its formulation. This suggests that oxidation may start during the preparation stages, including ingredient mixing and dough kneading. Factors such as the activity of enzymes like lipoxygenases, particularly in flour, along with air incorporation during mixing, could contribute to this early onset of oxidation. On the other hand, the application of a resting time at room temperature appeared to have a slight inhibitory effect on the formation of many VOCs generated by MR. It can be hypothesised that during the resting time, the action of asparaginase could further modify the composition of the free amino acid pool in such a way as to reduce the concentration of the most reactive amino acids in the MR. A slight reduction in asparagine seemed to occur as a result of the application of the resting time, as reported in Figure 1. Moreover, other effects on amino acid composition could be related to the enzyme's activity on other amino acids as well. It is known that some asparaginases exhibit, albeit to a lesser extent, hydrolytic activity toward glutamine [6], which could lead to secondary effects on the composition of free amino acids in the dough before baking. Overall, the observed effect of resting time on most MR-related VOCs suggests that the enzyme's activity during the resting time tends to reduce the concentration of active MR precursors. This minor effect aligns with what was observed regarding the browning index. Maillard reaction is, in fact, known to be responsible also for colour development during baking which is a key sensory attribute. During the baking of biscuits, oven temperature and water activity are major influencing factors for browning development [25]. However, other parameters, such as dough composition and dough resting time, also affect surface colour depending on the quantities of sugar and amino acid content [25]. The colour measured in the experimental biscuits in the present study was poorly affected by the addition of asparaginase. In agreement with this, both [PERSON] et al. [12] and [PERSON] et al. [11; 26] did not observe significant effects on the final colour of cookies as a result of asparaginase treatment.