# Effect of Asparaginase Treatment on Biscuit Volatile Compounds
## 4 Discussion  
Results collected in the present study confirmed that the asparaginase treatment is effective in reducing the asparagine content in the dough prepared before the baking of shorthead biscuits, as previously observed [16]. Interestingly, the lowest addition level recommended by the enzyme manufacturer, 500 ASNU, appeared to be sufficient to achieve a substantial reduction in asparagine content, while higher doses of the enzyme did not result in a further decrease in asparagine in the dough. Therefore, for the preparation of shorthead biscuits of the type examined in this study, a dose of 500 ASNU would seem suitable to achieve the intended goal. On the other hand, this reduction only caused changes in the VOC profile and colour parameters that were limited when compared to the strong reduction of asparagine in the dough. Thus, the reduced concentration of asparagine in the dough has a limited effect on VOC formation. This seemed to suggest a minor contribution of asparagine, as an MR reagent, to the formation of VOCs in shorthead biscuits. Limited information is available on the effects of asparaginase use on the formation of volatile compounds in foods. To the best of our knowledge, only one study has examined this effect in detail in food, specifically a study on the application of asparaginase for reducing acrylamide formation in coffee [20]. In this study, asparaginase application was shown to be effective in reducing asparagine levels in coffee beans and lowering acrylamide levels in the final product (coffee powder), while significant but rarely consistent effects were observed on the formation of various volatile compounds. The study reported a tendency for increased levels of certain Maillard reaction products, such as furfural, some pyrazines, and certain ketones (3-hydroxy butanone, 1-hydroxy-2-propanone), along with a reduction in 2-furanmethanol levels in coffee samples treated with asparaginase, although the effect was not consistent across all examined samples. However, no possible explanation was provided for these effects. On the other hand, limited information is available on the contribution of asparagine to the formation of volatile compounds from the Maillard reaction in food matrices. In general, asparagine is not reported among the most important precursor amino acids responsible for the formation of volatile aroma compounds through the Maillard reaction [1]. Recently, in model systems based on hydrolys vegetable proteins (HVPs) and reducing sugars (glucose and fructose), the formation of volatile compounds from the Maillard reaction was studied as a function of the different amino acid compositions of various plant-based matrices (soy, wheat, corn) [21]. It was observed that asparagine, which accounts for approximately 5% of the total amino acid fraction in wheat, participates in the Maillard reaction, leading to the formation of certain pyrazines, including 2,5-dimethylpyrazine and 2-methylpyrazine. In our study, no significant reductions in these compounds were observed in biscuits made from doughs with drastically reduced free asparagine levels. However, differences in the conditions of the model used in the cited study [21] compared to those of the present study may result in certain reactions being predominant in one case but not in the other. Other studies on model systems of the Maillard reaction involving asparagine and reducing sugars have reported the formation of key compounds such as furfural, hydroxymethylfurfural and 5-methylfurfural [22], as well as the formation of volatile pyrazinones such as 3,5-dimethyl- and 3,6-dimethyl-2(1H)-pyrazinones [23], which, however, have neither been reported in biscuits, nor found in our study. Regarding furfural, a reduction was observed in biscuits treated with asparaginase at 750 ASNU, but this effect was not consistent across other asparaginase doses, whereas asparagine reduction in the dough was observed at all enzyme addition levels.