*2.1. Biscuit Preparation*

The ingredients: soft wheat flour (Italian type "0"), sugar syrup, fats, flavoring agents, and leavening agents (sodium bicarbonate and ammonium bicarbonate) were bought at a local market. The formulation for the biscuits was obtained using the creaming method, which consisted of adding the ingredients into a kneading machine equipped with a leafshape beater (PastaMatic Gourmet 1950 Edition, De Longhi Appliances Srl, Campi Bisenzio, Italy), following two steps (Figure 1): (i) mixing the liquid ingredients (i.e., sucrose syrup and butter) for 10 min; and (ii) adding the powders (i.e., soft wheat flour and leavening agents) and thoroughly homogenizing them for 15 min.

**Figure 1.** Diagram of the whole process and of the sample set obtained through the FDOE. AB, ammonium bicarbonate; TP, baking temperature program; SRT, steam release time.

Once the doughs were obtained, biscuits were molded using stainless steel cookie cutters according to the following dimensions: thickness 2 mm, width 2.5 mm, and length 6 mm. The samples were baked in an electric oven (EKF 616 E UD, Tecnoeka S.r.l., Borgoricco, Italy) for 8 min. For each batch, 12 biscuit doughs positioned in the same arrangement on a perforated stainless-steel pan were baked. The pan was placed in the oven chamber on the third shelf (out of six) from the top.

Preliminary tests were carried out to optimize the temperature program to emulate as closely as possible the physical and sensory characteristics of a standard industrial biscuit (SIB). A reference biscuit (RIB) was prepared using a standard recipe and without the introduction of steam during baking. Aside from the use as a control sample, one RIB batch per week (for a total of three batches) was prepared to rule out every possible influence coming from changing environmental conditions.

#### *2.2. Factorial Design of Experiment (FDOE)*

An FDOE was created to optimize the number of experiments. The choice fell on a 2<sup>3</sup> FDOE to assess the three most critical factors: the steam release time (SRT), the baking temperature program (TP), and the amount of leavening agent (ammonium bicarbonate, AB). For each of the eight experiments, Figure 1 shows the levels of the three factors selected for the FDOE application, which were established after accurate tests on the TP to obtain experimental samples as similar as possible to the SIB. The oven temperature was controlled using the thermocouple system with an indication on the oven display and by introducing a certified thermometric probe with an accuracy of ±1 ◦C.

The steam was produced using tap water connected to the oven hose nozzle through a plastic pipeline. The release was regulated to achieve a relative humidity of 40% inside the oven.

Different relative quantities of leavening agents were used. For the preparation of 500 g of dough, the RIB contained ammonium bicarbonate 9.00 g and sodium bicarbonate 4.50 g, whereas the samples called "4.5 g AB" contained ammonium bicarbonate 4.50 g and sodium bicarbonate 9.20 g, and, finally, the samples called "1.5 g AB" contained ammonium bicarbonate 1.50 g and sodium bicarbonate 12.48 g (Figure 1).

The eight batches of biscuits from FDOE were cooked according to a balanced Latin square to compose an arrangement of eight rows and eight columns and considering only the first three columns as replicates (Table 1). The replicates were prepared in 3 days, once a week. The reference biscuits were also cooked on the same day.

**Table 1.** Latin square design used to randomize the cooking of the eight batches of biscuits, according to the FDOE design.


After baking, biscuits were left to sit for 60 min until complete cooling was achieved. For each of the eight FDOE points, eight biscuits were wrapped and sealed in oriented polypropylene bags using a FoodSaver sealing bar (IFS001X, JCS Europe Ltd., Cheadle, UK) and kept until analyses that were scheduled 7 days later.
