**1. Introduction**

Acrylamide is a neo-formed contaminant (NFC) that has been found in different kinds of foods that contain a high quantity of carbohydrates and are cooked by baking, roasting, or frying at temperatures higher than 100 ◦C [1]. In 1994, acrylamide was classified by IARC as Group 2A (potential carcinogen and neurotoxic to humans). This contaminant has genotoxic properties and causes carcinogenesis since it bonds strongly with DNA [2]. For these reasons, acrylamide levels in food are strictly controlled by European Food Safety Authority (EFSA) [3].

The highest levels of contamination, among the foods investigated, have been found in french fries, coffee, and biscuits. The European authorities have established benchmark levels for food classes in Regulation (EU) n. 2017/2158 (Attachment IV). The critical acrylamide threshold for biscuits and waffles is 350 μg/kgFW, while remarkable attention has been put on infant biscuits, setting a very low threshold of 150 μg/kgFW. Because baby foods can pose significantly higher health risks due to the lower body weight of infants, baby food manufacturers have to abide by stringent parameters and undergo careful scrutiny.

Most acrylamide is formed primarily through the Maillard reaction, which specifically involves the amino group of asparagine, the carbonyl group of reducing sugars, and intermediate molecules of the Maillard reaction [4]. Another way acrylamide forms is through a reaction that involves acrolein [5]. Both chemical reactions are strongly dependent

**Citation:** Lo Faro, E.; Salerno, T.; Montevecchi, G.; Fava, P. Mitigation of Acrylamide Content in Biscuits through Combined Physical and Chemical Strategies. *Foods* **2022**, *11*, 2343. https://doi.org/10.3390/ foods11152343

Academic Editors: Dapeng Peng and Yongzhong Qian

Received: 8 July 2022 Accepted: 3 August 2022 Published: 5 August 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

on the time–temperature factor, which means that the final result is similar when high temperatures and a short processing time or the reverse are applied [6].

Currently, there are no technological strategies to completely prevent acrylamide formation, although there are some ways to mitigate its concentration in food [7–10]. The acrylamide Toolbox lists the intervention steps that may be applied to reduce formation of acrylamide in specific manufacturing processes and products. The "ALARA" principle (As Low As Reasonably Achievable), which the Toolbox relies upon, states that the acrylamide presence needs to be reduced to the minimum, taking into account the risk presented, other legitimate considerations, such as potential risks from other contaminants, sensory properties and quality of the final product, and the feasibility and effectiveness of controls [11]. Moreover, the most effective methods applied so far have involved: (i) use of the asparaginase enzyme [12,13], (ii) use of different kinds of flours with low asparagine levels [14], (iii) replacement of ammonium bicarbonate (through the release of NH3, which supports the presence of Maillard reaction intermediates such as glyoxal and methylglyoxal) with non-ammoniagenic leavening agents [15,16], and (iv) assessment of different methods of baking, such as steaming (40% relative humidity), vacuum baking, and time–temperature optimization [11,17]. In fact, steam application helps to move the water activity (*aw*) value from the Maillard reaction's optimum, hence takes place more slowly [11].

While reducing the quantity of acrylamide in biscuits is paramount, keeping the biscuits' sensory characteristics, such as color and size, without losing their nutritional value is also essential. However, asparaginase poses several technological challenges for which the main objective of studies on food technologies aims to find suitable alternative strategies. These challenges include: asparaginase, which is currently and effectively employed as an acrylamide-mitigating agent [13], but makes the confectionery production process less cost-effective; the commercial preparation of the enzyme comes from the microorganism *Aspergillus niger*, a classified self-cloned micro-organism thus considered a genetically modified organism (GMO) and recently banned from the "organic" regime regulations.

Many researchers have focused their attention on the recipe modification, considering the influence of the substitution of inverted sugar with sucrose [18] and the replacement of wheat with other grains [14]. Good results have been obtained with the introduction of legume flours [19,20], even if sometimes the sensory properties are not always accepted by consumers [19]. The effect of cookie ingredients and cookie formulation has also been studied [21]. Of course, processing, i.e., the time–temperature condition of baking, plays an important role in acrylamide formation [8]. Oven-type baking processes affect acrylamide formation [18]. In particular, baking in ventilated mode promotes acrylamide formation, unlike baking in static mode [22]. Conversely, microwave baking has been studied for its capability to reduce thermal processing hazards and, consequently, acrylamide concentrations [23]. Vacuum-combined baking possesses these same capabilities and seems to be able to reduce acrylamide by about 30% in comparison with conventional baking [24].

Despite the possibly infinite combinations of recipes and baking conditions and the feasibility of such solutions in an industrial environment, this work considered only a slight modification of the recipe (i.e., the ratio of two different leavening agents) and the introduction of steam into a phase of the time–temperature program with a fixed total baking time, emulating the industrial conditions. Therefore, the present work aimed to investigate the influence, on the acrylamide amount in biscuits, of four baking conditions that differ in time/temperature cooking programs and use two different times of steam release to prepare biscuits from two recipes in which the ratios of the leavening agents ammonium bicarbonate/sodium bicarbonate were equal to 1:8 and 1:2. To achieve this goal, a factorial design of experiment (FDOE) 2<sup>3</sup> was applied to produce eight batches of experimental biscuits that were compared with reference biscuits obtained from standard recipes and baking conditions. As a secondary goal, size parameters (thickness, length, and width), color coordinates in the CIELab space (L\*, a\*, and b\*), and water activity of the sample biscuits were measured to assess the magnitude of their correlation with the acrylamide content.
