# Bread-Making Quality and Reduction of Acrylamide Content Using Weissella confusa Strain V20 from Desert Plant Stipagrostis pungens as Sourdough Additive

Imene Fhoula, Islem Dammak, Insaf Nachi, Imen Smida, Mnasser Hassouna, Hadda-Imene Ouzari

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Imene Fhoula, Islem Dammak, Insaf Nachi, Imen Smida, Mnasser Hassouna, et al.. Bread-Making Quality and Reduction of Acrylamide Content Using Weissella confusa Strain V20 from Desert Plant Stipagrostis pungens as Sourdough Additive. Fermentation, 2024, 10 (12), pp.647. 10.3390/fermentation10120647. hal-04874320

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Article

# Bread-Making Quality and Reduction of Acrylamide Content Using *Weissella confusa* Strain V20 from Desert Plant *Stipagrostis pungens* as Sourdough Additive

Imene Fhoula <fnref n="1" />*, Islem Dammak <fnref n="2" />, Insaf Nachi <fnref n="2" />, Imen Smida <fnref n="3" />, Mnasser Hassouna <fnref n="2" /> and Imene Hadda Ouzari <fnref n="4" />

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**Citation:** Fhoula, I.; Dammak, I.; Nachi, I.; Smida, I.; Hassouna, M.; Ouzari, I.H. Bread-Making Quality and Reduction of Acrylamide Content Using *Weissella confusa* Strain V20 from Desert Plant *Stipagrostis pungens* as Sourdough Additive. *Fermentation* **2024**, *10*, 647. https://doi.org/10.3390/fermentation10120647

Academic Editor: Patrizia Romano

Received: 19 November 2024

Revised: 9 December 2024

Accepted: 14 December 2024

Published: 16 December 2024

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**Copyright:** © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
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4.0/).

**Abstract:** The impact of wheat sourdough inoculated with a promising lactic acid bacterium *Weissella confusa* V20, isolated from a Tunisian desert plant, on the quality characteristics of sourdough and bread as well as on acrylamide formation was evaluated. *W. confusa* V20 sourdough (SWc) did not provide high final acidity and could effectively metabolize sucrose, resulting in significant (p < 0.05) increases in glucose (44.5%) and fructose (84.2%) levels. A substantial decrease (p < 0.05) in free asparagine was observed upon fermentation, reducing from 16 ± 0.9 mg/100 g dry weight (d.w.) in the control dough with baker's yeast (DBB) to 9.9 ± 0.4 mg/100 g d.w. in the dough inoculated with *W. confusa* V20 sourdough (DWc) and to 14.2 ± 0.2 mg/100 g d.w. in the dough produced with spontaneous sourdough (DSS). To improve the texture and flavor of the bread, the sourdough process should be further optimized in a strain-specific approach. V20 sourdough was more effective in enhancing the quality of the dough and bread compared to sourdough fermented with yeast. *W. confusa* V20 proved to be highly effective in reducing acrylamide levels, achieving a significant 62% decrease compared to the control.

**Keywords:** *Weissella confusa*; bread; sensory properties; acrylamide

## 1. Introduction

Making bread is most likely one of humankind's earliest inventions. Bread has been a staple food in the human diet since ancient times. It is consumed across the world, in various varieties and patterns, affected by many cultures [1]. Much research has been conducted by utilizing empirical and scientific approaches to fulfill the perceived new generation of customer demands for natural foods, better health, and quality of life [2,3]. A variety of undesirable chemicals could be generated during bread baking via thermal degradation at temperatures above 120 °C. Acrylamide is one such chemical that has attracted the interest of researchers over recent years. It is considered to be a cumulative neurotoxicant and carcinogen in both humans and animals [4]. This compound is generated by chemical reactions in heat-treated carbohydrate-rich and protein-deficient staple foods. In particular, acrylamide is formed by Maillard reactions in heated foods containing reducing sugars and free amino acid asparagine [5]. Several studies have reported approaches for the reduction of acrylamide in foods, mainly in cereal products, by optimizing the effects of raw ingredients, additives, enzymes, antioxidants, processing conditions, pH, water activity,

<footnote n="1">¹ Laboratory of Biochemistry and Microbiology LR16SP01, Aziza Othmana Hospital, University of Tunis El Manar, Tunis 1008, Tunisia</footnote>

<footnote n="2">² Laboratory of Technological Innovation and Food Security LR22-AGR01, Higher School of Food Industries of Tunisia (ESIAT), University of Carthage, 58 Alain Savary Street, El Khadhra City, Tunis 1003, Tunisia; islem.dammak.a@gmail.com (I.D.); nachiinsaf@gmail.com (I.N.); mnasser.hassouna@isbb.rnu.tn (M.H.)</footnote>

<footnote n="3">³ Institut National de la Santé et de la Recherche Médicale (INSERM), University Rennes, Institut National de la Recherche Agronomique (INRA), Centre Hospitalier Universitaire (CHU) de Rennes, Nutrition Metabolisms and Cancer (NuMeCan), UMR-1241, Biosit, MRic/ISFR, 35000 Rennes, France; smida_imen@yahoo.fr</footnote>

<footnote n="4">⁴ LR03ES03 Laboratoire Microorganismes et Biomolecules Actives, Université de Tunis El Manar, Faculté des Science de Tunis, 2092, Campus Universitaire, Tunis 2092, Tunisia; imene.ouzari@fst.utm.tn</footnote>

<footnote>* Correspondence: imene.fhoula@fst.utm.tn</footnote>
acids, yeast, cell-free extracts, and fermentation [5,6]. Lactic fermentation has also been
observed to suppress the formation of acrylamide in bread by using lactic acid bacteria
as an additive to sourdough [5]. Sourdough is a mixture of wheat flour and water that
has been fermented spontaneously or with the addition of lactic acid bacteria (LAB) and
yeasts and has been used as a leavening ingredient in bread manufacturing for centuries [7].
In recent years, sourdough bread production has received much attention in terms of
possible improvements to bread quality [8]. Furthermore, the use of sourdough in wheat
bread significantly improves dough and bread characteristics such as taste, texture, and
preservability [9,10]. LAB can produce a variety of secondary metabolites, offering benefits
in terms of flavor, texture, shelf life, and nutritional qualities [3]. The metabolic activity of
LAB in sourdough has been reported to have a beneficial impact on numerous features
during the bread-making process, enhancing bread quality via multiple metabolites, such
as organic acids; prolonging shelf life; and obviating the need for preservatives [10,11].
LAB may produce a variety of secondary metabolites that contribute to the sourdough
flavor. In addition, the activity of peptide hydrolysis, exopolysaccharide synthesis, and the
antibacterial ability of sourdough LAB might impact bread quality [3]. We hypothesize
that using a LAB strain from a desert plant as a starter for fermentation in bread making
can produce bread with distinctive organoleptic qualities. As a result, this study aimed to
determine the effect of using an exopolysaccharide-producing *Weissella confusa* strain V20
from the phyllosphere of the desert plant as a sourdough starter for dough making and its
impact on bread quality in terms of technological characteristics and acrylamide reduction.

**2. Materials and Methods**

2.1. *Microbial Strains and Culture Conditions*

The *Weissella confusa* strain V20 applied in this work was isolated from the leaves of Stipagrostis pungens, collected from the arid land of southern Tunisia. It was selected based on the production of exopolysaccharides and its rapid acidification activity in sterile flour extract SFE according to the method described by Alfonzo et al. [12]. The LAB strains were isolated from leaves using De Man, Rogosa, and Sharpe (MRS) agar medium pH 6.6 supplemented with cycloheximide (0.01%) to inhibit fungal growth after incubation for 48 h at 30 °C. Distinct colonies of acid-producing bacteria were chosen, as indicated by a yellow zone in the medium around each colony, and purified on MRS agar. The screening for exopolysaccharide-producing phenotype was performed on solid basal media derived from the MRS agar and supplemented with 2% (w/v) sucrose instead of glucose [11]. The total sugar content of the exopolysaccharides was determined in liquid medium (MRS) using the phenol-sulfuric acid procedure, with glucose as the reference [13]. The V20 strain was selected for further studies thanks to its exopolysaccharide-production, ability to grow in up to 8% NaCl, and the rapid acidification produced in sterile flour extract according to the method described by Alfonzo et al. [12]. Based on a partial sequencing of the 16S rRNA gene, this bacterium was categorized as a *Weissella confusa* strain (Genbank ID: MT106831). The above-mentioned strain was maintained at -80 °C in 50% glycerol as a stock. Fresh yeast was purchased at bakeries and kept in a refrigerator to keep it alive for as long as possible.

## 2.2. Wheat Sourdough Preparation

Wheat flour (product type PS-7 according to the manufacturer, 14.4% moisture content, 0.45% ash, 10.4% protein, and 8.5% wet gluten) was obtained from Tunisian mill industry. The selected *W. confusa* strain V20 was used to make sourdough bread. The strain was initially grown at 30 °C for 18 h in a modified MRS broth (1% lactose *w/v*, 1% maltose *w/v* at pH 5.6). The sourdough preparation and cell suspensions were made as shown by Nionelli et al. [14]. The selected *W. confusa* strain V20 inoculum was evenly disseminated in tap water (37.5 mL) at a cell density estimated at about log 9.0 cfu/g, then mixed with wheat flour (62.5 g) to make 100 g of sourdough (SWc). As a control, we used a spontaneous sourdough (SS) that had not been treated with the lactic bacterium strain. The sourdoughs were fermented at 30 °C for 16 h.
## 2.3. Bread Production

The dough was prepared according to a modified recipe of Nionelli et al. [14]. The sourdoughs were added at the level of 20% (w/w), and baker's yeast was added at the level of 2% (w/w) to all doughs. The 20% sourdough content was chosen based on the study by Nionelli et al. [14], which demonstrated optimal bread quality and characteristics at this proportion. Baker's yeast bread with no sourdough added (reference bread) served as the control (BBB). Bread doughs with a dough yield of 160 were fermented at 30 °C for 1.5 h before baking at 220 °C for 20 min. Dough yield was calculated according to Formula (1):

$$ \text{Dough yield} = \frac{\text{Dough weight}}{\text{Flour weight}} \times 100 \qquad (1) $$

The fermentation experiments were performed in triplicate on the same day.

## 2.4. Physicochemical Analysis

### 2.4.1. pH and the Total Titratable Acidity

The pH values and total titratable acidity (TTA) were determined for sourdoughs or doughs. The samples (10 g) were homogeneously mixed with distilled water (90 mL) for 3 min in a Stomacher mixer. The acidification capability was measured with a pH meter and expressed as the change between the pH after and before fermentation. TTA was determined by titrating on aliquots of homogenates with NaOH solution (0.1 N) to attain a final pH of 8.3 [7]. The TTA was expressed in mL NaOH (0.1 N)/10 g. All tests were performed in triplicate.

### 2.4.2. Determination of Reducing Sugars

A high-performance liquid chromatography (HPLC) system was used for the analysis of reducing sugars in freeze-dried sourdough as well as in dough and bread samples according to the method of Bartkiene et al. [15]. Aliquots of the filtrates were subjected to HPLC analysis (Waters 2695 Separations Module) after purification with an ÄKTA system. The sugar separation was performed using a Luna-NH2 column (5 µm, 150 × 4.6 mm; Phenomenex, Torrance, CA, USA) and a Waters 2414 Refractive Index Detector (Waters Corporation, Milford, MA, USA). The reduced sugars were quantified according to a calibration curve of the corresponding standards (50 to 500 mg/mL). The three sugar standards (glucose, sucrose, and fructose) were acquired from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

### 2.4.3. Analysis of Asparagine

The procedure of asparagine extraction was performed as described by Nachi et al. [7]. Each sample (2 µL) was introduced into an HPLC system coupled with a Shimadzu LC-20AD evaporative light scattering detector (ELSD). The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in 98% acetonitrile) with a flow rate of 0.7 mL/min at 30 °C and an injection volume of 10 µL. The gradient started with 0% B for 5 min, increased to 15% B for 7 min and then to 30% B for 5 min, and finally was maintained at 0% B for 18 min. Asparagine was quantified using a calibration curve prepared with L-asparagine (Sigma-Aldrich).

### 2.4.4. Determination of Acrylamide by LC-MS/MS

Acrylamide was extracted from bread samples and analyzed using LC-MS/MS according to the procedure provided by Bartkiene et al. [15]. Analysis of the prepared samples and standards was accomplished with a QTRAP® 5500 LC-MS/MS system (AB Sciex, Framingham, MA, USA) linked with an Acquity LC system (Waters Corporation, Milford, MA, USA). The HPLC method employed a 100 × 3 mm HILIC column with a 3 µm particle size (Phenomenex, Torrance, CA, USA) for the examination of the prepared samples and standards. The mobile phase consisted of components A (0.1% formic acid in water) and B
(0.1% formic acid in methanol), the flow rate was 0.3 mL/min, the injection volume was 10 µL, and the column temperature was 30 °C. The determination of the acrylamide content was performed using a calibration curve with an internal standard (acrylamide-d3).

### 2.4.5. Bread Quality Characteristics

The hardness of bread crumb samples was determined using a Perten's TVT 6700 texture analyzer (North Ryde BC, NSW, Australia) as described by Nachi et al. [7]. For each treatment, three portions of bread were compressed with a metal cylinder probe (75 mm diameter). The following conditions were selected: 5 mm/s test speed, 50% sample deformation, and two compression cycles with a 12 s interruption. The TexCal program was used to examine the data. Each batch was subjected to three measurements.

The color of the crust and crumb portions of bread samples was assessed using a CR-300 reflectance colorimeter (Minolta, Tokyo, Japan) based on the color parameters (lightness L*, redness a*, and yellowness b*). For each sample, the measurements were taken three times. After 24 h of storage at room temperature, the properties of the bread crumb were evaluated. Three pieces of bread were photographed using a Nikon D3100 digital camera (Nikon Corporation, Tokyo, Japan), featuring a 55 mm lens, shutter speed: 1/640s, and an ISO sensitivity setting of 1600).

During the sensory evaluation, 38 panelists previously trained (19 male and 19 female, aged 28 to 33 years) were recruited among students of the Higher School of Food Industries of Tunisia to assess the baked bread samples that had cooled after one hour. The bread samples were evaluated at room temperature. Breads were cut into 1.5 cm thick slices and then served in a random order (including crumb and crust). Each sample was coded with a 3-digit random number. Between tastings, the panelists cleansed their palates by rinsing with tap water. The panelists used a scoring procedure to rate the sensory features of the bread samples on a structured 8-point hedonic scale (1—extremely unpleasant, 2—very unpleasant, 3—unpleasant, 4—quite unpleasant, 5—neither pleasant nor unpleasant, 6—quite pleasant, 7—pleasant, and 8—extremely pleasant). Elasticity, acidic and salty flavor, crust and crumb colors, texture, volume, and crumb aeration were among the sensory attributes. These attributes were evaluated for the optimized and control bread samples. These samples were considered acceptable when their mean scores for overall acceptability were above 4.0. All sensory assessment tests followed the ethical and professional norms outlined by the Institute of Food Science and Technology [16].

### 2.4.6. Scanning Electron Microscopy (SEM) Imaging of Bread

The pieces of bread were glued to a sample holder and covered with a layer of carbon. The samples were then placed into a cathode sputtering apparatus to ensure their metallization. This operation was important to minimize the accumulation of negative parasitic charges on the sample surface during its observation with the SEM, thus enabling good visualization. The microstructure of the bread crumbs was observed with a JSM-5400LV scanning electron microscope with LaB6 filament (JEOL, Tokyo, Japan). The SEM images were obtained at 50× magnification and an electron acceleration voltage of 5000 V.

### 2.5. Statistical Analysis

All of the tests were carried out three times, and the results were presented as the mean values with standard deviations. The IBM SPSS Statistics software version 23.0 was used to conduct the statistical analysis. In order to determine the significance of differences between the samples, one-way analysis of variance (ANOVA) was used, preceded by Duncan's test with a significance threshold of *p* < 0.05. In addition, a Pearson correlation analysis was carried out.

## 3. Results and Discussion

### 3.1. Physicochemical Analysis of Sourdough

It was assumed that the fermentation of sourdough with a LAB strain isolated from plants growing under adverse conditions may enhance the characteristics of dough
and bread. In this work, wheat flour fermented with or without an exopolysaccharide-producing *W. confusa* strain V20 from *Stipagrostis pungens* was investigated. *W. confusa* species is an indigenous LAB in plant cereal raw materials and sourdough [11]. The inoculated fermented sourdough was characterized with regard to the pH, TTA, sugar content (glucose, sucrose, and fructose), and free asparagine, as shown in Table 1. After 16 h of fermentation at 30 °C, sourdough treated with *W. confusa* strain V20 (SWc) exhibited a significant pH drop from 6.36 to 3.84 (ΔpH = 2.52; *p* < 0.05) in comparison with spontaneous sourdough (SS), (ΔpH = 2.33; *p* < 0.05). As expected, the pH values decreased, and the TTA increased. This finding was in agreement with Kajala et al. [17], where more acidic pH values were obtained in LAB sourdough than in fermentation without LAB. Indeed, LAB has been shown to lower the pH of sourdoughs considerably. As observed in several studies, the pH values in sourdoughs using the classic LAB starters (between pH 3.5 and pH 4.2) were quite similar to our results, depending on the variety of flour, the fermentation procedure, and the bacterial strains used [8,17]. Once organic acids were produced, especially acetic acid for heterofermentative LAB, lowering the pH favored the undissociated form of acids and promoted the main antimicrobial activity of the LAB [18]. It was supposed that the lower pH driven by LAB such as *W. confusa* strain V20 may suppress harmful bacteria in the sourdough microbiota. As stated by Cizeikiene et al. [2], the introduction of LAB sourdough into bread may prevent the growth of rope spoilage microorganisms by decreasing the pH during fermentation.

<table><caption>**Table 1.** Physicochemical parameters and composition of sourdoughs with or without *Weissella confusa* strain V20 after fermentation for 16 h.</caption><thead><tr><th>Samples</th><th></th><th>SS</th><th>SWc</th></tr></thead><tbody><tr><td rowspan="2">pH</td><td>T<sub>16</sub></td><td>4.03 ± 0.06 <sup>a</sup></td><td>3.84 ± 0.08 <sup>b</sup></td></tr><tr><td>ΔpH</td><td>2.33 ± 0</td><td>2.52 ± 0.02</td></tr><tr><td>Total titrable acidity (mL NaOH/10 g d.w.)</td><td>T<sub>16</sub></td><td>5.2 ± 0.3 <sup>a</sup></td><td>10.7 ± 0.8 <sup>b</sup></td></tr><tr><td rowspan="2">Glucose (mg/100 g d.w.)</td><td>T<sub>0</sub></td><td>612 ± 38</td><td>612 ± 38</td></tr><tr><td>T<sub>16</sub></td><td>529 ± 18 <sup>b</sup></td><td>884 ± 21 <sup>a</sup></td></tr><tr><td></td><td>%Δ Glucose</td><td>-13.6</td><td>+44.5</td></tr><tr><td rowspan="2">Fructose (mg/100 g d.w.)</td><td>T<sub>0</sub></td><td>266 ± 48</td><td>266 ± 48</td></tr><tr><td>T<sub>16</sub></td><td>250 ± 3 <sup>b</sup></td><td>490 ± 34 <sup>a</sup></td></tr><tr><td></td><td>%Δ Fructose</td><td>-6</td><td>+84.2</td></tr><tr><td rowspan="2">Sucrose (mg/100 g d.w.)</td><td>T<sub>0</sub></td><td>460 ± 71</td><td>460 ± 71</td></tr><tr><td>T<sub>16</sub></td><td>413 ± 22 <sup>a</sup></td><td>160 ± 22 <sup>b</sup></td></tr><tr><td></td><td>%Δ Sucrose</td><td>-10.2</td><td>-65.2</td></tr><tr><td>Total reducing sugars (mg/100 g d.w.)</td><td>T<sub>16</sub></td><td>1192 ± 43 <sup>b</sup></td><td>1534 ± 77 <sup>a</sup></td></tr><tr><td rowspan="2">Asparagine (mg/100 g d.w.)</td><td>T<sub>0</sub></td><td>16.1 ± 0.6</td><td>16.1 ± 0.6</td></tr><tr><td>T<sub>16</sub></td><td>18.0 ± 1 <sup>b</sup></td><td>19.6 ± 0.2 <sup>a</sup></td></tr></tbody></table>

The information is provided as mean values ± standard errors (n = 3). The mean values with different superscript letters in the same row indicate a significant difference (p < 0.05); SWc: *W. confusa* V20 sourdough. SS: Spontaneous sourdough; d.w.: dry weight; T₀: initial mixture of sourdough. T₁₆: Final mixture of sourdough has been fermented for 16 h.

Moreover, following fermentation, the TTA value increased considerably (p < 0.05) from 2.5 mL to 5.2 mL (SS) and 10.7 mL (SWc). In sourdough fermented with *W. confusa* V20, the TTA was significantly (p < 0.05) higher than determined for SS. The TTA values were similar to those found by Şimsek et al. [19], who reported TTA levels ranging from 7.6 mL to 19.3 mL for sourdough samples.

Regarding the analysis of sugar content, it should be noted that the concentrations of glucose and fructose were significantly (p < 0.05) increased across sourdough samples
treated with *W. confusa* strain V20 by 44.5% and 84.2% (mg/100 g dry weight (d.w.)), respectively, while sucrose level was decreased (−65.2%) (Table 1). However, in spontaneous sourdough, the concentrations of glucose, fructose, and sucrose were reduced by 13.6%, 6%, and 10.6%, respectively. The sucrose levels in sourdoughs dropped significantly (*p* < 0.05) after 16 h of fermentation, ranging from 413 ± 22 mg/100 g d.w. in SS to 160 ± 22 mg/100 g d.w. in *W. confusa* sourdough, compared to the initial amount of sucrose at 460 ± 71 mg/100 g d.w. Apparently, the *W. confusa* strain V20 could metabolize sucrose in wheat flour sourdough, transforming it into glucose and fructose. It has been reported that glucose and fructose are produced in higher quantities from sucrose by secreted glucansucrase and fructansucrase, which convert sucrose into glucans or fructans, respectively, with the release of fructose and glucose [20].

With EPS-producing *W. confusa* strain V20, which produced 1.126 ± 0.013 g/L of exopolysaccharides, dextran production was observed in the presence of glucose or sucrose. The breakdown of sucrose and starch by cereal amylases and LAB may be linked to the observed increase in glucose concentration in sourdough [11,15]. Indeed, there are several mechanisms for sucrose metabolism by LAB. *Weissella* strains cleave sucrose by the action of dextransucrase [21].

According to Rizzello et al. [11], in the presence of sucrose and maltose, *Weissella* strains were involved in the production of gluco-oligosaccharides and exopolysaccharides. Hypothetically, during the exponential growth phase, sucrose was utilized for bacterial growth, and then, during the stationary phase, it was used to produce slimy exopolysaccharides. The activity of glucansucrase on sucrose resulted in the formation of glucan while releasing fructose. This was supported by the residual fructose detected in sourdough fermented with *W. confusa* strain V20 (Table 1). *Weissella cibaria* did not use the fructose moiety of sucrose for growth, exopolysaccharides, or mannitol production during fermentation, as Rizzello et al. [11] found for this species, which is closely related to *W. confusa*. As a result, fructose accumulated in sourdoughs in that case.

*W. confusa* is a heterofermentative species that can absorb glucose, sucrose, fructose, maltose, and certain pentose sugars, among other wheat flour carbohydrates [22]. A study by Kajala et al. [17], based on carbohydrate profile analyses, showed that *W. confusa* effectively utilized sucrose during sourdough fermentation, while some *Lactobacillus* spp. consumed only a portion of it. Only a few papers provide information on the glucose metabolism of *Weissella* strains [21].

## 3.2. Physicochemical Analysis of Dough

Table 2 shows the results of fermentation (with baker's yeast, SS, and SWc) of doughs prepared using various constituents for the purpose of exploring the pH and the concentrations of reducing sugars and asparagine. The control dough was made with baker's yeast dough (DBB) that did not include any LAB sourdough. Doughs prepared with spontaneous sourdough were noted as DSS, and those with *W. confusa* V20 sourdough were described as DWc.

<table><caption>**Table 2.** The pH values and content of glucose, fructose, sucrose, total reducing sugars, and asparagine in various doughs after fermentation for 1.5 h.</caption><thead><tr><th colspan="2">Samples</th><th>DBB</th><th>DSS</th><th>DWc</th></tr></thead><tbody><tr><td rowspan="2">pH</td><td>T<sub>0</sub></td><td>6.02 ± 0.11 <sup>a</sup></td><td>5.69 ± 0.15 <sup>b</sup></td><td>5.02 ± 0.03 <sup>c</sup></td></tr><tr><td>T<sub>90</sub></td><td>5.77 ± 0.09 <sup>a</sup></td><td>4.97 ± 0.01 <sup>b</sup></td><td>4.71 ± 0.01 <sup>c</sup></td></tr><tr><td rowspan="3">Glucose<br>(mg/100 g d.w.)</td><td>T<sub>0</sub></td><td>264 ± 11 <sup>c</sup></td><td>1287 ± 17 <sup>b</sup></td><td>1653 ± 20 <sup>a</sup></td></tr><tr><td>T<sub>90</sub></td><td>826 ± 23 <sup>b</sup></td><td>748 ± 31 <sup>c</sup></td><td>916 ± 4 <sup>a</sup></td></tr><tr><td>%Δ Glucose</td><td>+133.4</td><td>-35.8</td><td>-44.5</td></tr><tr><td rowspan="3">Fructose<br>(mg/100 g d.w.)</td><td>T<sub>0</sub></td><td>58 ± 4 <sup>c</sup></td><td>800 ± 9 <sup>a</sup></td><td>781 ± 6 <sup>b</sup></td></tr><tr><td>T<sub>90</sub></td><td>230 ± 7 <sup>b</sup></td><td>532 ± 1 <sup>a</sup></td><td>195 ± 1 <sup>c</sup></td></tr><tr><td>%Δ Fructose</td><td>+296.6</td><td>-33.5</td><td>-75%</td></tr></tbody></table>
<table><caption>**Table 2.** *Cont.*</caption><thead><tr><th>Samples</th><th></th><th>DBB</th><th>DSS</th><th>DWc</th></tr></thead><tbody><tr><td rowspan="2">Total reducing sugars<br>(mg/100 g d.w.)</td><td>T<sub>0</sub></td><td>321 ± 8<sup>c</sup></td><td>2087 ± 24<sup>b</sup></td><td>2434 ± 20<sup>a</sup></td></tr><tr><td>T<sub>90</sub></td><td>1281 ± 39<sup>a</sup></td><td>1057 ± 30<sup>c</sup></td><td>1111 ± 4<sup>b</sup></td></tr><tr><td rowspan="2">Sucrose<br>(mg/100 g d.w.)</td><td>T<sub>0</sub></td><td>ND</td><td>ND</td><td>172 ± 5</td></tr><tr><td>T<sub>90</sub></td><td>ND</td><td>ND</td><td>126 ± 9</td></tr><tr><td>Asparagine<br>(mg/100 g d.w.)</td><td>T<sub>90</sub></td><td>16 ± 0.9<sup>a</sup></td><td>14.2 ± 0.2<sup>b</sup></td><td>12.65 ± 0.2<sup>c</sup></td></tr></tbody></table>

The data are shown as the mean values ± standard errors (n = 3). The mean values with different superscript letters in the same row indicate a significant difference (p < 0.05); DBB: baker's yeast dough made without the addition of sourdough. DSS: Dough produced with spontaneous sourdough; DWc: dough inoculated with *W. confusa* V20 sourdough; ND: not detected due to complete hydrolysis or under the threshold of detection; d.w.: dry weight. T₀: Initial mixture of dough; T₉₀: final mixture of dough has been fermented for 90 min.

The dough prepared with *W. confusa* strain V20 showed a significantly (p < 0.05) more pronounced decrease in pH value to 4.71, compared with DSS (pH = 4.97) and the control sample DBB (pH = 5.77), demonstrating the influence of LAB cultures in reducing dough pH. Several studies found that using LAB sourdough for dough preparation increased the acidity of the resulting product [23], which is consistent with our observations during the present study. In dough fermentation, the obtained pH values reached similar results as in the study by Nachi et al. [7].

The fermentation of doughs made with spontaneous sourdough or *W. confusa* strain V20 was performed to investigate whether the content of free asparagine was reduced by this process (Table 2). The content of reducing sugars in the DBB (control) dough was considerably (p < 0.05) higher than in the other doughs after 1.5 h of fermentation. Glucose and fructose levels were measured at the same time (Table 2). A significant increase (p < 0.05) in fructose and glucose levels was observed in DBB after 1.5 h of fermentation, reaching 58–230 mg/100 g d.w. and 264–826 mg/100 g d.w., respectively. It is supposed that the original sucrose was converted to fructose and glucose. However, the amount of total reducing sugars was significantly (p < 0.05) lower for DWc (by 54.4%) than for DSS (by 49.4%). The amount of free fructose was greatly diminished by 75% and 71.3% with DWc and DSS, respectively, while glucose was less depleted by 44.5% and 35.8%, respectively. These findings could be supported by the notion that heterofermentative LAB utilizes fructose as an electron acceptor and hence converts it to mannitol [7].

Similarly to the report by Koschwanez et al. [24], sucrose was under the threshold of detection or only traces were detected in dough samples. This result can be explained by the presence of a highly active invertase enzyme in yeast, which promoted the rapid hydrolysis of sucrose to glucose and fructose. Contrary to the results of Katina et al. [25] for the closely related species *W. cibaria*, in the case of *W. confusa* strain V20, the presence of glucose did not suppress the utilization of fructose, and sucrose was also co-metabolized with glucose.

### 3.3. Analysis of Acrylamide

For the study of acrylamide formation during baking, the amount of free asparagine was measured in SS and V20-supplemented sourdough SWc before and after fermentation (Table 1) in order to determine if the content of this amino acid was lowered by fermentation. Indeed, free asparagine is a key precursor for acrylamide in cereal products [26]. Moreover, fermentation could reduce acrylamide content in bread via the reduction of the asparagine level [27]. The free asparagine concentration was reported to be 16 ± 1 mg/100 g d.w. prior to the fermentation, similar to the 14 to 17 mg/100 g d.w. in processed wheat flour indicated by Wang et al. [26]. However, *W. confusa* V20 was significantly (p < 0.05) capable of slightly increasing the asparagine content after 16 h of fermentation to 19.1 ± 0.6 mg/100 g d.w. Although the addition of *W. confusa* V20 initially resulted in a slight increase in free asparagine content, subsequent fermentation led to a significant reduction, indicating that the strain
eventually metabolized asparagine. This transient increase is likely due to strain-specific metabolic characteristics and the dynamic nature of microbial fermentation.

Table 2 shows the influence of various sourdoughs on the quantity of asparagine in dough samples. After fermentation, significantly lower levels of free asparagine were detected in both DV20 (9.9 ± 0.4 mg/100 g d.w.) and DSS doughs (14.2 ± 0.2 mg/100 g d.w.) (*p* < 0.05) than in the control dough DBB (16 ± 0.9 mg/100 g d.w.) (Table 2). These findings support previous research that found decreased amounts of free asparagine after wheat sourdough fermentation [7,26]. It has been observed that the amino acid asparagine was metabolized by yeast or LAB and utilized as a nitrogen source during bread dough fermentation. Initially, the dough made with wheat flour had a higher asparagine content (30 mg/100 g). Acrylamide concentration in various wheat bread samples was analyzed to further investigate the process of acrylamide formation and its association with dough fermentation. The findings are displayed in Figure 1. The acrylamide concentration in bread samples ranged from 7.54 µg/kg to 35.6 µg/kg. Both the use of spontaneous sourdough and lactic acid fermentation before baking considerably decreased acrylamide generation during wheat bread manufacturing compared to the control sample BBB that was fermented with yeast alone. The highest mean acrylamide content was detected in BBB (35.6 µg/kg).

<figure><img src="image_5.png" /><figcaption>**Figure 1.** Acrylamide levels in different bread crust samples prepared with fermented doughs. BBB: Baker's yeast bread (reference bread). BSS: Bread prepared using spontaneous sourdough without LAB strains. BWc: Bread made with *W. confusa* V20 sourdough. The results are given as the mean values ± SD (n = 3). Different letters above the bars mean a significant difference (*p* < 0.05).</figcaption></figure>

According to the European Commission Recommendation [28], a new benchmark value for the acrylamide content in soft bread obtained from wheat flour was set at 50 µg/kg. Bread is one of the major nutrition sources; therefore, acrylamide intake with bread may be a human health concern (EFSA, 2011). Furthermore, high levels of bread consumption in Tunisia (74 kg of bread/person/year) on average [7] make this study especially relevant to the region of North Africa.

Our results showed that the addition of *W. confusa* strain V20 to sourdough (SWc) considerably reduced (*p* < 0.05) the acrylamide content by 62% (to 13.53 µg/kg) relative to the control bread (35.6 µg/kg) for asparagine of 12.65 and 16 mg/100 g d.w. in dough, respectively. Although the bread treated with spontaneous sourdough (SS) had the lowest acrylamide concentration at 7.54 µg/kg d.w. (reduction by 78.8%) for asparagine of 14.2 mg/100 g d.w. in dough, it had an unattractive gray color and putrid flavor. Therefore, acrylamide reduction is influenced by the use of LAB-fermented sourdough through the metabolic utilization of nutrients in the presence of yeast [27]. Some lactic acid bacteria strains used for sourdough treatment were shown to be capable of lowering the acrylamide level in wheat bread [29]. In line with Nachi et al. [7], it was found that acrylamide gen-
eration during wheat bread manufacturing may be effectively suppressed by the lactic fermentation of wheat dough before baking. The concentrations of glucose and fructose in doughs were substantially (p < 0.05) and modestly connected with the production of acrylamide (r = 0.565 and r = -0.617, respectively). However, there was no significant relationship between asparagine concentration in sourdough and acrylamide content in baked bread. According to Pollien et al. [30], fructose is more effective than glucose in producing acrylamide from asparagine. The lowering of reducing sugar levels in dough appears to be the most critical factor influencing acrylamide production during baking rather than the elimination of available asparagine or other amino acids implicated in Maillard reactions [6]. The bread made with *Weissella confusa* strain V20 had a lower pH of 4.69 ± 0.02 and higher humidity (28.93%) compared to the other bread samples. Edna Hee et al. [31] reported that lowering the pH and water activity are the important factors for minimizing acrylamide formation in heat-processed foods because they prevent the Maillard reaction.

### 3.4. Physicochemical Quality of the Tested Breads

Table 3 lists the color characteristics (L*, a*, b*) of the crust and crumb determined for the tested bread samples. The crust and crumb of inoculated sourdough bread BWcV20 indicated a significant decrease (p < 0.05) in the lightness (L*) parameter compared to the control bread samples BBB and BSS. In fact, the crumbs of the bread made with *W. confusa* V20 sourdough BWc had a significantly (p < 0.05) lower value of lightness (L*) (68.80) compared to the BSS (70.11) and BBB (74.89) samples.

<table><caption>**Table 3.** Crust and crumb color attributes of bread samples.</caption><thead><tr><th rowspan="2">Samples</th><th colspan="3">Crust Color</th><th colspan="3">Crumb Color</th></tr><tr><th>L*</th><th>a*</th><th>b*</th><th>L*</th><th>a*</th><th>b*</th></tr></thead><tbody><tr><td>BBB</td><td>63.09 ± 0.07 <sup>a</sup></td><td>9.58 ± 0.38 <sup>b</sup></td><td>35.36 ± 0.11 <sup>a</sup></td><td>74.89 ± 0.30 <sup>a</sup></td><td>-1.53 ± 0.1 <sup>b</sup></td><td>14.63 ± 0.18 <sup>a</sup></td></tr><tr><td>BSS</td><td>63.77 ± 0.44 <sup>a</sup></td><td>9.56 ± 0.05 <sup>b</sup></td><td>34.91 ± 0.66 <sup>a</sup></td><td>70.11 ± 0.67 <sup>b</sup></td><td>-1.14 ± 0.2 <sup>ab</sup></td><td>13.85 ± 0.07 <sup>b</sup></td></tr><tr><td>BWc</td><td>61.94 ± 0.34 <sup>b</sup></td><td>10.56 ± 0.23 <sup>a</sup></td><td>35.16 ± 0.62 <sup>a</sup></td><td>68.80 ± 1.26 <sup>b</sup></td><td>0.25 ± 0.67 <sup>a</sup></td><td>14.32 ± 0.32 <sup>a</sup></td></tr></tbody></table>

L* = Lightness, a* = redness, b* = yellowness; Samples in the same column with the same letter are not substantially different (p < 0.05). BBB: Baker's yeast bread; BSS: bread made with spontaneous sourdough; BWc: bread made with *W. confusa* V20 sourdough.

Furthermore, the bread samples made with sourdough SWc had significant (p < 0.05) variations in the crust and redness (a*) values. A slight change was detected in the a* and b* parameters of the crust and crumb. Although the statistical analysis did not reveal significant differences, a trend towards increased redness (a) was observed in the crust of bread made with sourdough SWc. The BBB and BSS bread crumb samples indicated negative a* and positive b* values associated with greenish-yellow hues, whereas those bread crust samples showed positive a* and b* values, suggesting reddish-yellow hues. A moderate correlation was noted between the "L*" and "b*" parameters of crusts and the acrylamide levels (r = 0.486 and r = 0.510, respectively, p < 0.05) of the bread samples. These results agreed with those of Bartkiene et al. [29].

Hardness measurements revealed that bread prepared with BBB (yeast only) had a significantly higher value (5149 ± 119 g) than bread made with spontaneous sourdough BSS (4060 ± 57 g) (Table 4). The softest bread texture was obtained using SWc sourdough (2647 ± 7 g). Additionally, as described by Dastmalchi et al. [27], the usage of sourdoughs lowered the bread hardness, which was typically dependent on the reduction in pH associated with acid generation in sourdough. In accordance with Novotni et al. [9], we suggested that crumb softening after using sourdough with V20 (SWc) could be ascribed to an increase in the protease and amylase activity as well as the formation of EPS. Indeed, EPS can enhance the viscoelastic characteristics of dough, increase the loaf volume, and decrease crumb hardness [32].
<table><caption>**Table 4.** Baking characteristics of bread.</caption><thead><tr><th>Samples</th><th>Hardness (g)</th><th>Void Fraction (%)</th><th>Cell Density (cells/mm²)</th><th>Average Diameter of Holes (μm) at 50× Magnification</th></tr></thead><tbody><tr><td>BBB</td><td>5149 ± 119 <sup>a</sup></td><td>39.57 ± 0.87 <sup>b</sup></td><td>3831.8 ± 659.82 <sup>b</sup></td><td>390.00 ± 135 <sup>b</sup></td></tr><tr><td>BSS</td><td>4060 ± 57 <sup>b</sup></td><td>31.15 ± 0.90 <sup>c</sup></td><td>5256.74 ± 27.67 <sup>a</sup></td><td>513.67 ± 89 <sup>ab</sup></td></tr><tr><td>BWc</td><td>2647 ± 7 <sup>c</sup></td><td>46.65 ± 0.06 <sup>a</sup></td><td>2687.25 ± 107 <sup>c</sup></td><td>605.25 ± 24 <sup>a</sup></td></tr></tbody></table>

Data are displayed as the mean value ± standard error (n = 3). Different letters in the same column indicate significant differences (p ≤ 0.05). BBB: Baker's yeast bread. BSS: Bread made with spontaneous sourdough. BWc: Bread made with *W. confusa* V20 sourdough.

The digital image analysis of the bread crumb structure showed visual variance amongst the tested formulations (Figure 2). As stated in Table 4, bread loaves (BWc) exhibited a significantly higher void fraction (46.65 ± 0.06%) and a significantly lower cell density (2687 ± 107 cells/mm²) compared to both BSS (31.15%; 5257 cells/mm²) and BBB (39.57%; 3832 cells/mm²) (Table 4 and Figure 2).

<figure><img src="image_6.png" /><figcaption>**Figure 2.** The bread structure using scanning electron microscopy at 50× magnification. BBB: Baker's yeast bread (reference bread). BSS: Bread made with the spontaneous sourdough. BWc: Bread prepared with *W. confusa* V20 sourdough.</figcaption></figure>

Examining the microtexture of bread by scanning electron microscopy (SEM) has been useful for determining the changes in the physical structure of bread following the addition of the sourdoughs. SEM images of experimental bread at 50× magnification are
shown in Figure 2. The examination of the mean gas bubble diameters (holes) measured from SEM images at 50× magnification (Table 4 and Figure 2) showed that the size of the bubbles depended on the starter used. Bread fermented with baker's yeast (Saccharomyces cerevisiae) alone (reference bread) (BBB) had an average hole diameter of 390 ± 135 µm. This leavening agent is known for its CO₂-producing capacity, resulting in the formation of gas bubbles [33]. Bread fermented by *S. cerevisiae* in combination with sourdoughs exhibited larger gas bubbles. Bread (BWc) prepared with *W. confusa* V20 sourdough exhibited a significantly larger average hole diameter (605 ± 24 µm). This finding was expected when taking into account that lactic acid bacteria are able to produce carbon dioxide. In fact, *Weissella confusa* is a heterofermentative bacteria that produces carbon dioxide. By releasing carbon dioxide during fermentation, the lactic starter added to the dough helped to increase the volume of the crumb, thus reinforcing the rheological qualities and making the bread airier and more flavorful [33].

### 3.5. *Sensory Attributes of Bread*

A sensory assessment is an important technique with a large variety of applications in the bakery industry. Figure 3 shows the findings of the sensory study of the experimental bread. The majority of physicochemical properties tested were substantially influenced (p < 0.05) by the addition of SWc sourdough to bread dough, including crumb aeration, volume, elasticity, acidic taste, and salty taste. However, the color of the crust and crumb was minimally affected. The BWc bread sample displayed a higher attribute score of 6.27 in comparison to BSS and BBB (6.03 and 5.41, respectively). According to several studies, it has been well recognized that microbial fermentation is linked to improved taste characteristics. In fact, LAB could generate a variety of metabolites, which may be related to technological improvements in flavor, texture, shelf life, and nutritional value [10].

<figure><img src="image_7.png" /><figcaption>**Figure 3.** A spider web chart depicting the sensory analysis findings for the tested breads. BBB: Baker's yeast bread (reference bread); BSS: bread prepared using spontaneous sourdough without LAB strains; BWc: bread made with *W. confusa* V20 sourdough.</figcaption></figure>

## 4. Conclusions

The efforts aimed at controlling acrylamide production in wheat flour bread by lactic acid bacteria sourdough demonstrated an intriguing strategy for enhancing bread quality characteristics. In comparison to the control, the inclusion of *Weissella confusa* strain V20 sourdough reduced the acrylamide levels considerably (62%). Moreover, lactic acid fermentation significantly influenced the physical and sensory features of the experimental bread. This study confirmed that LAB sourdough had a significant influence on improving the texture, flavor, and sensory properties of bread throughout the baking process. The
selected *W. confusa* strain as well as other LAB strains should be characterized in more detail regarding the specific metabolites involved in the formation of qualitative attributes of sourdoughs and baked bread.

**Author Contributions:** Conceptualization, I.F., I.D. and I.N.; methodology, I.F., I.D. and I.N.; software, I.S.; validation, M.H. and I.H.O.; formal analysis, I.F.; investigation, I.F. and I.N.; writing—original draft preparation, I.F.; data curation, I.D.; visualization, M.H. and I.H.O.; supervision, M.H. and I.H.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article.

**Acknowledgments:** The authors acknowledge the financial support of the Tunisian Ministry of Higher Education and Scientific Research.

**Conflicts of Interest:** The authors declare no conflicts of interest.

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