*3.4. Comparison between the Acrylamide Concentrations of the Eight FDOE Samples and the Reference Biscuits (RIB)*

Table 3 shows the results for the acrylamide concentrations obtained in the FDOE sample set as well as in the RIB samples. All of the samples belonging to the FDOE showed acrylamide concentrations lower than those of the RIB, demonstrating that the combination of using a lower baking temperature with the introduction of steam in the middle step along with decreasing the amount of ammonium bicarbonate in the recipe is a promising strategy to mitigate acrylamide in biscuits. The Kruskal–Wallis equality-of-population rank test was run to highlight any significant difference among the sample medians, in particular those belonging to the FDOE (samples 1 to 8) and the RIB. This non-parametric test showed a moderately significant difference across the samples (*p* = 0.0530). Although nonapplicable, the one-way ANOVA also showed significant differences (*p* ≤ 0.001) among the samples. Among the FDOE samples, a post hoc test showed significant differences only for samples 1 and 8, which exhibited the lowest and the highest acrylamide concentrations, respectively. The comparison of the conditions applied to the preparation of samples 1 and 8 indicated that all experimental factors (SRT, TP, and AB) were substantially different (Figure 1). The former showed an 87.2% reduction in acrylamide in comparison with the RIB, whereas the latter showed a 63.2% reduction.


**Table 3.** Results of measurements of size, CIELab color parameters, water activity, and acrylamide (expressed as mg/kgFM; FM: fresh matter) carried out on FDOE samples (samples 1–8) and reference samples (RIB).

The colors used to indicate the samples and cell backgrounds refer to the colors used in Figure 1.

In sample 1, the TP used and the amount of AB in the recipe were the lowest. At the same time, the amount of steam administered was the maximum in terms of time. As hypothesized, this combination of parameters resulted in the lowest acrylamide value among all of the samples, whereas the diametrically opposite conditions of TP, RST, and AB were applied to sample 8, in which the highest amount of acrylamide was found within the FDOE.

Samples 2 and 5 followed sample 1 in terms of acrylamide content. These samples were obtained with the lowest baking TP and only one of the other two factors most favorable to the mitigation of acrylamide (1.5 g of AB and 3 min of SRT for samples 2 and 5, respectively). Sample 3 also had only one factor out of three that differed from sample 1, namely the highest baking TP. However, TP caused the most intense effect on acrylamide formation, as widely demonstrated [8–10] and recommended [11]. It emerged that sample 3 showed contaminant concentrations higher than those in samples 2 and 5. Finally, samples 4, 6, and 7, all of which had higher acrylamide values than the previous samples, showed only one favorable mitigation factor out of three, i.e., the lowest AB amount, the lowest TP, and the highest SRT, respectively.

The obtained data seemed to demonstrate the formulated hypothesis, according to abundant scientific evidence derived from experimental works on biscuits and concerning the use of steam-cooking [32] or on different starchy matrices, such as bread and potatoes [10,32,33]. As far as the contribution to acrylamide formation by AB is concerned, the present data agree with data from the scientific literature and demonstrate that diminishing this leavening agent is an interesting approach: some authors [21] concluded that ammonium bicarbonate is the most effective ingredient in terms of causing the formation of acrylamide in biscuits and that its complete elimination from the recipe may be a solution. However, the complete replacement of a leavening agent such as AB would require the reformulation of the industrial process to obtain a biscuit appreciated by consumers.
