Effect of Asparaginase Treatment on Biscuit Volatile Compounds

Featured Application

This study suggests that the use of asparaginase does not markedly alter sensory-related attributes of the final product, providing further confirmation of the validity of this strategy for mitigating the undesirable effects of the Maillard reaction.

Abstract

Among the strategies proposed to reduce the formation of acrylamide in bakery products, the use of asparaginase is considered one of the most promising, also due to its limited impact on the sensory quality of the final product. Asparagine, the key precursor of acrylamide that is selectively hydrolysed by the asparaginase treatment, is generally not considered a major contributor to the overall flavour and colour of baked foods. This study investigates the effect of three addition levels of the enzyme asparaginase (500, 750, 1000 ASNU compared to a not added control) and three different preparation conditions (without resting time and with 15 min resting time at 20 °C or 50 °C) on asparagine content in the dough and on the formation of Volatile Organic Compounds (VOCs) and colour of shortbread biscuits. Results showed that the addition of asparaginase, at all levels, markedly reduced the asparagine content in the dough, with limited effects on the VOC profile and colour. The examined preparation conditions significantly affected the VOC profile: the application of a resting time at 20 °C tended to promote the formation of VOCs through lipid oxidation while reducing the level of many MR-related VOCs. This last effect seemed to parallel the slight reduction of the browning index observed when the resting time was applied. Results suggest that the use of asparaginase does not markedly affect the VOC profile of shortbread biscuits, thus confirming its limited effects on sensory-related quality attributes of baked foods.

1. Introduction

Maillard reaction (MR) plays a key role in thermal treatments during the processing of cereal-based foods, being responsible for several attributes related to quality and safety [1,2,3]. Undesired effects of the MR have also been highlighted, such as heat damage of the proteins, assessable through the determination of furosine, and neo-formation of chemical contaminants, such as acrylamide and 5-hydroxymethylfurfural [4,5]. Thus, measures to mitigate the extent of these unintended effects of MR, while keeping the desired effects on food quality, need to be carefully considered. This study will investigate one of the mitigation strategies used for reducing acrylamide formation, focusing on the use of the enzyme asparaginase in the conventional production of shortbread biscuits. Asparaginase catalyses the conversion of asparagine into aspartic acid and ammonia by hydrolysing the amide group in asparagine’s side chain [4,6]. This enzyme selectively degrades asparagine, which is the primary precursor of acrylamide and, generally, is not regarded as a major determinant of flavour or colour in thermal treated foods. In cereal-based products, asparagine, rather than reducing sugars, is considered the limiting factor in acrylamide formation [6]. The free asparagine content in widely used cereal species has been reported to range from 426 to 1179 mg kg⁻¹ [7]. Commercial asparaginase preparations, derived from Aspergillus oryzae and Aspergillus niger, are widely used in industrial applications and have been deemed safe by regulatory bodies such as FAO/WHO and the FDA [8]. The use of asparaginase has been considered one of the most promising strategies to limit acrylamide formation in bakery products, even by virtue of the limited impact on the sensory quality. In particular, several investigations have demonstrated that the combination of asparaginase addition and controlled resting time can reduce acrylamide formation in biscuits, along with the formation of other undesired MR products, such as furfurals [9,10,11]. However, only a few studies explored in detail the quality and sensorial attributes of biscuits after the use of asparaginase, generally focusing on the effects on taste and colour, and with little information on the aroma of the final product [11,12,13].

It is known that the addition of asparaginase modifies the amino acid composition of the dough [4,6], that amino acids are key reagents in the MR, and that MR is one of the main pathways for the formation of volatile organic compounds (VOCs) in biscuits [1,14]. Thus, it is conceivable that this treatment may influence VOC formation. However, the literature does not clearly establish whether asparaginase treatment affects aroma quality or the significance of such an effect. On the other hand, it is known that VOCs are formed not only through MR but also from caramelisation reactions and lipid oxidation [1]. Varying baking times and temperatures have been shown to significantly impact the VOC formation in baked products and manipulation of these parameters can modify to a large extent the progress of the above-mentioned chemical pathways of VOC formation [14]. Key volatile compounds of cereal-based baked foods formed through these reactions belong to the chemical classes of alcohols, aldehydes, pyrazines, ketones, acids, furans, alkanes, sulphur compounds, pyrroles, and others [14]. Beyond aroma, MR plays a key role in determining also the colour of baked foods. Colour development has been attributed to the final stage of the MR [1]. In addition, a strong positive relationship between acrylamide concentration and colour development has been reported in the preparation of cereal-based baked products [15]. Moreover, no significant changes in colour associated with asparaginase treatment have been observed in baked foods [9,16]. Given the key role of sensory quality in determining consumers’ food choices, it is essential to evaluate the effects of asparaginase treatment on the sensory attributes of biscuits. In this study, the impact of asparaginase treatment at three different addition levels, combined with three dough preparation conditions, was investigated. The study focused on the free asparagine levels in the dough as well as on the VOC profile and the colour of the final shortbread biscuits.

2. Materials and Methods

2.1. Experimental Biscuits

Biscuits were prepared in our experimental baking laboratory using the following formulation: refined flour (cv Providence, 900 g), butter (270 g), sugar (225 g), whole milk (165 g), and baking powder (21 g), with or without the addition of asparaginase. The baking powder was a commercial preparation containing sodium diphosphate (E450i), sodium bicarbonate (E450ii), corn starch, and stabiliser sodium, potassium, and calcium salts of fatty acids (E470a) but no added flavourings. Asparaginase was added at levels of 500, 750, or 1000 ASNU per kilogram of flour (where ASNU is defined as the amount of enzyme that produces 1 μmol of ammonia per minute under assay conditions), with a control batch prepared without enzyme. The asparaginase used was Acrylaway^® L (Novozymes, Bagsvaerd, Denmark). The concentrations of added enzyme were chosen based on the manufacturer’s recommendations and previously published studies [9,11]. For biscuits, the optimal concentration range of asparaginase is between 500 and 1000 ASNU/kg of flour and may vary depending on the formulation and processing parameters. In the present study, at each level of asparaginase addition, the following conditions for the enzyme’s action were experimented: (A) no resting after dough mixing; (B) the dough was allowed to rest 15 min at ambient temperature (T = 20 ± 1 °C) before making the biscuits; (C) the dough was allowed to rest 15 min at 50 ± 1 °C before making the biscuits (

Table 1. Description of asparaginase addition level and conditions for preparation of experimental biscuits.

Sample Code	Asparaginase (ASNU/kg Flour)	Resting Time (min)	Dough T (°C)
B500a	500	0	-
B500b	500	15	20 °C
B500c	500	15	50 °C
B750a	750	0	-
B750b	750	15	20 °C
B750c	750	15	50 °C
B1000a	1000	0	-
B1000b	1000	15	20 °C
B1000c	1000	15	50 °C
BCtrlA	0	0	-
BCtrlB	0	15	20 °C

). A planetary mixer equipped with a 3 L bowl and C-shaped hook (KitchenAid, Whirlpool, Benton Harbor, MI, USA) was used to make the dough. First, the butter was placed in the mixer bowl and mixed for one minute to make it soft and smooth. Then, the sugar was added, and mixing was continued for another minute. Finally, the flour, the baking powder and the milk were added, and the mixture was mixed for six additional minutes. The enzyme, when used, was added to the milk just a moment before pouring the milk into the mixer bowl. After mixing, the dough was removed from the bowl and divided into 3 equal parts (A, B and C), which were processed as follows. Part A was immediately rolled out at 5 mm thickness with a rolling pin equipped with guides, then biscuits were cut by a round steel cookie-cutter, 5 cm in diameter, and baked in a steam-convection oven (Angelo Po, Carpi, Italia) at a temperature of 180 °C without steam for 16 min. At the same time, parts B and C were shaped into a loaf, wrapped in cling film and allowed to rest for 15 min, respectively, at 20 °C (B) and at 50 °C (C). As soon as the resting time was over, parts B and C were rolled out and biscuits were cut and cooked in the same way as it was conducted with A. The following characteristics were measured on all biscuit samples: length (5.19 ± 0.25 cm), width (4.39 ± 0.23 cm), height (1.18 ± 0.08 cm) and weight (10.0 ± 3.0 g).

2.2. Determination of Levels of Asparagine in the Dough

The extraction of asparagine was carried out as described by Gazi et al. [17]. One gram of dough was placed in a centrifugation tube with a cap and dissolved in 10 mL water/acetonitrile 1:1 with 0.1% (v/v) formic acid to stop the enzyme action. The tube content was mixed for 20 min by a rotary shaker, then centrifuged at 6080× g for 5 min and the supernatant was retained. The pellet was extracted two more times with 5 mL of extracting solution and 5 min mixing, followed by centrifugation as before. Aliquots of the combined supernatants were placed in Eppendorf tubes and centrifuged at 10,000× g for 5 min. Then the supernatants were stored at −18 °C until chromatography. The extraction was carried out in duplicate. On the day of the analysis, the extracts were thawed; one ml was diluted 1:1 with acetonitrile, centrifuged at 14,000× g for 5 min and transferred to a 2 mL vial. For technical reasons, it was not possible to analyse the sample BCtrlB.

The free asparagine content was determined using an AB Sciex 3200 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA) coupled with a PerkinElmer 200 Series liquid chromatograph (PerkinElmer, Shelton, CT, USA). The chromatograph setup included two PE 200 Series micropumps, a column oven, and a thermostatic autosampler. Separation of asparagine from other matrix components was achieved through Hydrophilic Interaction Liquid Chromatography (HILIC), with a 5 µL injection onto a Luna Polar Pesticides column (100 × 2.1 mm, 3 µm, Phenomenex, Vaerloese, Denmark).

The mobile phase consisted of 0.1% formic acid in water (Phase A) and 0.1% formic acid in acetonitrile (Phase B). Chromatographic runs were conducted at a flow rate of 0.4 mL/min with the following gradient: 0–1 min: 95% B, 1–7 min: 95% to 45% B, 7–7.1 min: return to 95% B, 7.1–14 min: maintained at 95% B. The column temperature was held constant at 25 °C. Detection of the analyte was performed using electrospray ionisation (ESI) in positive polarity, with data acquisition in multiple reaction monitoring (MRM) mode. The monitored transitions were 133.0 → 74.0 (quantifier ion) and 133.0 → 87.0 (qualifier ion), each with a dwell time of 300 ms. Mass spectrometer settings included the following: curtain gas: 10 psi, ion spray voltage: 4000 V, source temperature: 200 °C, nebuliser gas: 5 psi, auxiliary gas: 10 psi, declustering potential: 30 V, entrance potential: 8 V, collision energy: 15 V, collision cell exit potential: 2 V. Asparagine quantification in the extracts was performed using the external standard method. The calibration curve consisted of seven levels (0.1, 0.2, 0.5, 1, 2, 5, and 10 µg/mL) of pure asparagine. To ensure optimal accuracy across all calibration levels, a linear regression model was applied, with the origin omitted and a 1/x² weighting factor.

2.3. Measurement of Quality Attributes of Biscuits

2.3.1. Colour

The colour of the biscuits was determined using a CR 400 chroma-meter (Konica-Minolta, Milan, Italy) in the absolute chromatic space CIE1976 (L* a* b*) with illuminant D65. Measurements were taken twice on the top side and twice on the bottom side of each piece. Ten pieces were analysed for each kind of biscuit (n = 40). The Browning index (BI) was then calculated based on the CIE values of L* a* b* according to the following formulas [18]:

BI = 100 × [(X − 0.31)/0.17], where, X = (a* + 1.75L*)/(5.645L* + a* − 3.012b*)

2.3.2. Volatile Organic Compounds (VOCs)

Biscuit samples were frozen with liquid nitrogen and ground by a laboratory grinding device (Ika, Staufen, Germany) in order to obtain a powder that was stored for each sample at −70 °C until analysis. Analysis was carried out within three weeks of grinding. For VOC analysis, the approach developed in our laboratory and previously described [19] was adopted with some changes. Volatile compounds were determined on each sample by a semi-quantitative HS-SPME/GC-MS method. The same amount (0.5 g) of biscuit powder was placed in a 15 mL vial for SPME extraction. The vial containing the sample was immersed in a water bath kept at 60 °C. Then HS-SPME extraction was carried out by exposing a 50/30 µm DVB/CAR/PDMS fibre (Supelco, Sigma-Aldrich, Milano, Italy) to the headspace of the biscuit powder for 45 min.

At the end of the extraction, the fibre was immediately inserted into the GC split-spitless injection port, for the desorption step, and the GC run was started. GC/MS analyses were performed on an Agilent 6890GC 5973N MS system equipped with a quadrupole mass filter for mass spectrometric detection (Agilent Technologies, Palo Alto, CA, USA). The desorption was carried out by the split mode (split-less) at 240 °C for 8 min. Then GC separation was achieved on a DB-WAX column (0.25 mm i.e., 60, 0.5 µm film thickness); J&W, Agilent Technologies, Palo Alto, CA, USA) by setting the following conditions: inlet temperature 240 °C; oven temperature programmed from 40 °C (10 min) to 210 °C at 4 °C min⁻¹, and then to 220 °C (5 min) at 30 °C min⁻¹ (the total run time of 57.83 min); constant flow of the carrier gas was 2 mL min⁻¹ corresponding to a linear velocity of 36 cm s⁻¹. The MS detector operated by the electron ionisation mode at 70 eV; transfer line, source, and quadrupole temperatures were set, respectively, at 220, 230, and 150 °C. Detection was performed by full scan and single ion monitoring (SIM) mode for, respectively, identification and quantification purposes. Identification of the sample volatiles was done by comparison of linear retention indices (LRI) and mass spectra of chromatographic peaks with those reported in the literature and in the NIST/EPA/NIH Mass Spectra Library 2005. The list of the 36 individual VOCs determined, along with their retention index and m/z ion signal selected for SIM detection was reported in Table S1. For the semi-quantitative determination of each volatile compound, the peak area of the target analyte obtained by SIM detection was used. A triplicate analysis was performed on the biscuit powder obtained from each of the biscuit samples. The average coefficient of variations of triplicate determination of individual VOCs, as determined on all samples, was below 10% (9.8%).

2.4. Statistical Analysis

Statistical significance of differences in VOC levels and browning index measurements among the different biscuit samples was evaluated using ANOVA, followed by Tukey’s HSD test for post-hoc comparisons (p < 0.05). A Heat Map analysis was conducted on the VOCs dataset, applying hierarchical clustering with Euclidean distances to independently group both VOC composition variables and biscuit samples. The dataset’s rows (volatile compounds) and columns (biscuit samples) were reordered according to the clustering results, bringing similar columns and rows closer together. The resulting heat map visually represents the permuted data matrix, where values are encoded as colour intensities. Additionally, Principal Component Analysis (PCA) was performed on the VOCs dataset. Prior to Heat Map and PCA analyses, datasets were preprocessed by averaging replicate measurements and applying autoscaling. All statistical analyses were carried out using XLStat software (version 2020.01.01; Addinsoft, New York, NY, USA).

3. Results

3.1. Asparagine Content in the Dough

Figure 1 shows the strong effect on dough-free asparagine levels caused by the asparaginase treatment. Results confirmed that the use of asparaginase consistently reduced the free asparagine level, from 344 mg Kg⁻¹ in the untreated dough (without enzyme and resting time) to a range of 29–56 mg Kg⁻¹ in all the treated samples.

It is interesting to note that only minor differences were found between the three asparaginase addition levels. Moreover, dough subjected to resting time at 20 °C tended to show slightly lower asparagine content with respect to dough prepared without a resting time, even though differences were not significant according to Tukey’s test.

3.2. 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 °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 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.

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 biscuits (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 °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. As regards pyrazines, the two odour-active compounds contributing to roast notes, 2-ethyl pyrazine and 2,3-dimethyl pyrazine, were observed at a higher level when a resting time was not applied (a condition) and the same trend was observed for furfural, one of the key product of the intermediate stage of the MR [11]. Similarly, important furan compounds, such as 2-Furan-methanol, which contributes to the caramel flavour, and 2-acetyl furan, showed a higher value in condition as compared with the b, regardless of asparaginase addition level.

On the contrary, the VOCs formed by lipid oxidation tended to show higher levels in the preparation with 15′ resting time at 20 °C more than the condition a and c, even though in many cases (2-pentylfuran, E-2-nonenal, nonanal, hexanal) differences were not statistically significant. It is important to note that, in most cases, these differences are not statistically significant or are only significant for certain levels of asparaginase addition. However, despite the lack of statistical significance in the differences for individual volatile compounds, the overall data, as indicated in the description of the Heat Map and PCA results, suggested an influence of the different conditions (asparaginase levels and preparation methods) on the formation of classes of compounds associated with different reactions (Maillard reaction and lipid oxidation).

3.3. Colour

The differences in the browning index (BI) between treated samples are reported in Figure 4. As regards the effect of asparaginase, biscuits with 750 ASNU addition level showed a slight, though significant, reduction of browning when a resting time was applied (both b and c condition). Regarding the preparation conditions, at all levels of asparaginase addition, the use of a resting time, at both temperatures, tended to lower the browning index with respect to the preparation without any resting time.

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 shortbread 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 shortbread 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 shortbread 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 hydrolysed 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. 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 °C before baking reduced the formation of most MR-related VOCS, whereas the products of lipid oxidation were enhanced. Maire 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 Kukurova et al. [12] and Anese et al. [11,26] did not observe significant effects on the final colour of cookies as a result of asparaginase treatment. More recently, significant but minor effects on chromatic coordinates have been reported in the preparation of wheat flour biscuits treated with asparaginase [13]. In this case, at higher doses of asparaginase, an increase in L* values and a reduction in a* and b* values were observed, but the Browning Index was not calculated in that study. The article did not provide a possible explanation for these variations, while sensory analyses of the biscuits revealed no significant differences in their perceived colour. Similarly, in a related study on biscuits made with oat, corn, and rice flour, the application of asparaginase led to significant variations in chromatic coordinate values, although these variations did not follow a consistent pattern [27]. In this case, as well, the authors did not report any visually perceptible changes in biscuit colour, although this observation was not supported by sensory analysis. The variations in chromatic coordinates were attributed to minor changes in processing conditions, such as slight variations in baking temperature, rather than to the addition of asparaginase. On the other hand, as commented above, in the present study the application of a resting time at 20 °C seemed to mildly inhibit the browning process, and this minor effect appeared to correspond to the lowered rate of MR occurring when a resting time was applied, as shown by the dynamics of formation of MR-related VOCs. Moreover, the variation in incubation temperature during the resting time, from 20 to 50 °C, did not appear to have any effect on the browning index. The same result was observed in a previous study [26], which explored the effect of asparaginase within a temperature range of 20 to 54 °C. The authors of that study suggested that increasing incubation temperatures could promote the progression of non-enzymatic browning reactions. However, within the temperature range in which asparaginase activity is not compromised (20–60 °C), this effect seems to be relatively limited.

5. Conclusions

The application of asparaginase in biscuit preparation represents a highly effective approach to significantly reducing asparagine levels in the dough, thereby limiting its role as a precursor of acrylamide. The experimental study presented here established that the minimum dose recommended by the manufacturer resulted in a reduction of the asparagine content in the dough, which was not further reduced by using higher doses of the enzyme. For the first time, this study investigated the effect of using asparaginase in the preparation of shortbread biscuits on the formation of volatile compounds responsible for their aroma. At all asparaginase addition levels, limited effects on sensory-related quality attributes of the biscuits were observed, suggesting a minor role of free asparagine in the dough as a precursor of VOCs and compounds responsible for browning. These results suggest that while the formation of acrylamide is considered to be inextricably linked to the development of colour and aroma, the application of asparaginase allows for the decoupling of asparagine content, and, consequently, acrylamide formation, from the formation of the compounds responsible for aroma and browning. Moreover, significant effects have been observed regarding the tested preparation conditions, and in particular, the application of a resting time at 20 °C, which seemed able to modify to a limited, though significant, extent the relative content of some known odorants of biscuits. Further investigations, including sensory analysis, could determine whether such variations are capable of causing perceptible changes in the aroma of the biscuits and significantly influencing their sensory quality.