Impact of Sourdoughs, Enzymes, and Their Combinations on Gluten-Based Bread Quality

Abstract

The study investigates the impact of sourdoughs made with different flours (white wheat, wholemeal wheat, and barley) and specific enzymes (laccase, lipase, and hemicellulase) on the technological properties of gluten-based wheat breads, thereby exploring the combined role of sourdough and enzymes. Three levels of each sourdough (20, 30, and 40%) were tested, and the optimal level was then used to evaluate the impact of individual and combined enzyme treatment. Pasting properties and FT-IR analyses of the flours were evaluated. White wheat flour displayed the highest peak viscosity (353.50 mPas) and final viscosity (526.50 mPas). β-sheet structures predominated in all samples, although they were most prevalent in wholemeal wheat flour (51%) as opposed to white wheat flour (47%) and barley (47%). Sourdough breads exhibited better texture and moisture retention at 40% inclusion than at other levels. After 72 h, white wheat sourdough maintained the highest specific volume (3.71 cm³/g), while barley sourdough retained the most moisture (38.83%) and the lowest chewiness and hardness results, suggesting better softness and crumb retention. Whereas for enzyme treatment, they had different effects. White wheat and wholemeal wheat sourdough breads treated with enzymes had decreased hardness, chewiness, and gumminess; barley sourdough breads with enzymes were negatively affected by the texture. Correlations and multivariate analysis reveal that bread texture is strongly influenced by the type of sourdough and enzymatic treatment. Higher doses of laccase or hemicellulase improve softness in wholemeal-based sourdough bread, while excessive lipase leads to a firmer and less pleasant crumb.

1. Introduction

Bread is a widely consumed basic food in many cultures. It is commonly prepared from wheat flour, water, (including or not yeast), and delivers a significant amount of energy as well as carbohydrates, lipids, proteins, dietary fiber, minerals, and vitamins [1]. In contrast, bread’s limited shelf life stands as a tough challenge for bakery products, mostly due to microbial deterioration and moisture loss. This process, known as staling, is the core factor of bread waste, resulting in a loss of softness, freshness, flavor, and aroma [2,3,4]. Staling occurs shortly after baking and involves complicated physical and chemical changes, most notably starch retrogradation and moisture migration from the crumb to the crust [5,6,7]. Despite profound investigation, the precise mechanics of stalling are still not entirely identified.

Sourdough fermentation is a promising strategy for improving the quality of bread and reducing its staling. It relies on a fermented mixture of flour and water, naturally colonized by wild yeasts (including but not limited to Saccharomyces candida) and lactic acid bacteria (Lactobacillus). These microorganisms generate an acidic fermentation that gives the bread its characteristic aromas [8,9,10,11,12]. Additionally, the sourdough fermentation improves the nutritional profile of bread since the metabolism of lactobacilli produces new, beneficial compounds such as peptides and amino acid derivatives as well as increases the bioavailability of minerals and phytochemicals. Numerous studies have demonstrated that compared to yeast-based breads, the use of sourdough, particularly type 1 sourdough (spontaneous), results in higher specific volume and better organoleptic qualities [9,13,14,15,16,17]. Although sourdough fermentation clearly has multiple advantages, the application of enzymes could supply further enhancements. Enzymes are evidence-based strategies to increase shelf life and quality of bread by modulating moisture retention [18]. They can, in addition, break down non-starch polysaccharides, causing an improvement in water absorption and dough strength with better gas retention (during fermentation), higher volume, and better crumb structure [19,20]. Due to their more natural origin, enzymes have been increasingly used in recent years as substitutes for chemical additives such as emulsifiers and hydrocolloids. A variety of enzymes from different sources—such as amylases, oxidoreductases, hemicellulases, and lipases—have been employed in bread making to improve dough properties and enhance the final loaf volume [21,22,23].

This study focused on three specific enzymes: laccase, lipase, and hemicellulase. These enzymes use different but complementary ways to target specific parts of wheat-based bread dough. They were selected for their ability to target proteins, lipids, and fibers, offering a comprehensive approach to enhancing the texture, volume, and shelf life of bread. Laccase enhances the gluten network and increases dough elasticity by catalyzing the cross-linking of phenolic compounds and the oxidation of free sulfhydryl groups in gluten proteins [23]. Whereas lipase creates polar lysolipids that serve as natural emulsifiers and help stabilize gas cells while enhancing crumb softness through hydrolyzing the lipids in wheat flour [21]. Hemicellulase facilitates gluten hydration and gas retention by changing water-unextractable arabinoxylans (WUAX) into water-extractable forms [22]. Despite the fact that each enzyme has already been the subject of scientific studies, the effect of their combination in leavened doughs with different types of natural sourdoughs has not yet been studied and remains poorly understood.

In this context, the objective of this study is, first, to examine the impact of three sourdoughs—made from white wheat, barley, and wholemeal wheat flour—on the technological characteristics and shelf life of wheat-based breads. In a second phase, the effect of enzymes (laccase, lipase, and hemicellulase) as well as their combinations on the textural properties of breads made with these sourdoughs was studied in order to better understand potential interactions, whether synergistic or antagonistic, between these enzymes, which could improve the technological quality of the bread.

2. Materials and Methods

2.1. Raw Materials

White wheat flour, wholemeal wheat flour, barley flour (Polskie Młyny Sp. Z o.o., Warszawa, Poland), instant yeast (Saccharomyces cerevisiae, Dr. Oetker Polska Sp. z o.o., ul. Adm. Dickmana 14/15, PL-80-339 Gdańsk, Poland), sunflower oil, salt, and sugar were all purchased from a local market in Poland. White wheat flour (type 750) contained 12.00% protein, 1.80% fat, and 0.75% ash. Wholemeal wheat flour (type 1850) contained 13.00% protein, 2.10% fat, and 1.80% ash. Barley flour (type 1850) contained 8.80% protein, 2.40% fat, and 1.80% ash (producers’ data).

2.2. Enzymes

Lipase used in this study was provided by VERON Pasta L, from AB Enzymes GmbH (Darmstadt, Germany). A lipase (Trichoderma reesei), with an activity of IUB 3.1.1.3, CAS 9001-62-1. The recommended dosage is 3–9 g/100 kg flour (30–90 ppm). Hemicellulase was provided by Bakezyme ARA 10,000 BG, from DSM Food Specialties (Heerlen, The Netherlands). An arabinofuranosidase (Aspergillus niger), with an activity of 10,000 ARF/g (±5%). The recommended dosage is 1–10 ppm (0.1–1 g/100 kg flour). Laccase was provided by Novozym 51003, from Novozymes (Madrid, Spain). It is a laccase enzyme preparation derived from Myceliophthora thermophila, with a protein concentration of 39.86 mg/mL.

2.3. Determination of Pasting Properties of Flours

The pasting properties were carried out in 2 replications and evaluated using a Micro Visco-Amylo-Graph Brabender (Brabender, Duisburg, Germany) as described by Kupryaniuk et al. [24]. The procedure was initiated by mixing 10 g of each flour sample in 100 mL of distilled water. Thereafter, using a sensitivity of 235 cmg, the characteristics were assessed at a steady rotation speed of 250 rpm. Regarding the temperature profile, it included heating at a rate of 7.5 °C/min from 30 to 93 °C, holding at 93 °C for 5 min, cooling at a rate of 7.5 °C/min from 93 to 50 °C, and holding at 50 °C for 1 min. The following properties were assessed using Brabender Viscograph software (version 4.1.1): final viscosity (FV), peak viscosity temperature (PVT), onset gelatinization viscosity (OGV), peak viscosity (PV), and onset gelatinization temperature (OGT).

2.4. Fourier Transformed Infrared (FT-IR) Analysis of Flours

The flours from white wheat, wholemeal wheat, and barley were analyzed by Fourier-transform infrared (FT-IR) spectroscopy. The spectra were acquired with a SHI-MADZU IRAffinity-1S FT-IR spectrometer (SHI-MADZU, Burladingen, Germany) with a diamond attenuated total reflectance (ATR) accessory. They were collected between 4000 and 400 cm⁻¹, with a resolution of 4 cm⁻¹. Each obtained spectrum was baseline corrected using ORIGIN software (version 8.0724 PRO, Origin Lab Corporation, Northampton, MA, USA). The analyzed spectra were averaged over five separate analyses of registered spectra series. To investigate the structural characteristics of the protein network of the studied flours, the secondary structure analysis of proteins was conducted in the amide I region (1590–1710 cm⁻¹). In order to differentiate the overlapping bands in the amide I region and to precisely identify the underlying protein secondary structure, the second derivative of the amide I band was calculated using a five-point, two-degree polynomial function. The second derivative spectra were then smoothed with a 13-point, two-degree polynomial Savitsky-Goly function following the methods of Susi et al. [25] and Seabourn et al. [26]. To estimate quantitatively the amount of each type of secondary structure component indicated by the second derivative, the amide I band (1590–1710 cm⁻¹) was deconvoluted with Gaussian curves using ORIGIN software (version 8.0724 PRO, Origin Lab Corporation, Northampton, MA, USA). Following parameters: R² > 0.99, solution converged and χ² < 0.001 was used to estimate the quality of the band deconvolution. Differential spectra in the amide I region were calculated in order to confirm deconvolution results [27]. The relative composition of secondary protein structures composing the amide I band was expressed as the percentage of the area of the fitted region expressed as a relative area of components centered at specific wavenumbers [28]. Secondary structure assignment was based on absorption wavenumbers according to the previous studies [28,29,30,31].

2.5. Sourdough Preparation, Incorporation Levels, and Shelf Life Evaluation

2.5.1. Sourdough Preparation Method

Three types of sourdough were prepared using the same ratio of flour to water (40 g/40 g, flour/water each), but with different flours: sourdough 1 (white wheat flour), sourdough 2 (wholemeal wheat flour), and sourdough 3 (barley flour). Each sourdough was refreshed every 12 h during the first five days, with half of the mixture discarded at each feeding. The cultures were placed in sterilized glass jars (pre-treated with boiling water) and stored at room temperature (28 °C ± 2 °C) in a dry location. After the initial five days, feedings were reduced to once every 24 h. The sourdough was considered mature and ready for baking when it consistently doubled in volume after each feeding.

2.5.2. Incorporation Levels

For each sourdough prepared, three levels were used (20%, 30%, and 40%) in bread making to determine which sourdough level yields the best bread characteristics.

The incorporation levels were selected based on preliminary laboratory trials and relevant literature. In our experiments, sourdough additions below 20% did not produce a noticeable improvement in loaf volume, whereas levels above 40% resulted in a pronounced acidic taste without further enhancing bread quality compared to 40% incorporation. These findings align with the results of Naji-Tabasi et al. (2022) [32], who reported that the optimal fermentation condition for whole barley–wheat flour Barbari bread was achieved with 30% sourdough. Therefore, based on both our preliminary experiments and published evidence, incorporation levels of 20%, 30%, and 40% were selected for evaluation in this study. For each bread produced, the specific volume, texture, and moisture content were determined. The sourdough level that provides the highest specific volume, lowest moisture content, and most desirable textural attributes will be considered optimal and used for the remainder of the analysis.

2.5.3. Shelf Life Monitoring

Breads produced with the three types of sourdough with optimized levels were used to investigate the effect of storage on the bread’s shelf life. The bread samples were designated as follows:

• CB: Control Bread (made with yeast),
• GBBS1: gluten-based bread with S1 (white wheat flour sourdough),
• GBBS2: gluten-based bread with S2 (wholemeal wheat flour sourdough),
• GBBS3: gluten-based bread with S3 (barley flour sourdough).
• Textural properties, specific volume, and moisture content at intervals of 1 h, 24 h, 48 h, and 72 h post-baking were determined. The samples were stored in airtight plastic bags at room temperature (20 °C ± 2 °C).

2.6. Bread Making Procedure

The bread making process used in this study was adopted in the laboratory from Bourekoua et al. [33] with modifications. Sourdough bread samples were prepared using the following formulation: 100 g of white wheat flour, 5 g of sunflower oil, 3 g of sugar, 2 g of salt, sourdough (tested at various levels as described below), and 63 g of water. The amount of water was determined based on preliminary experimental trials conducted in the laboratory. A bread using 3 g of instant yeast was prepared as control bread. The preparation process began by manually mixing the sourdough with water and sunflower oil. The dry ingredients—flour, sugar, and salt—were previously combined and then added to the wet mixture. Mixing was performed using a Philips Daily Collection hand mixer (300 W, model with 5+ turbo speed settings, Eindhoven, The Netherlands). The dough was mixed for 1 min at low speed followed by 3 min at high speed. Following mixing, the dough was subjected to a first fermentation (bulk fermentation) at 40 °C for 60 min. It was then transferred to a refrigerator and stored at 4 °C for 14 h (overnight cold fermentation). On the following day, the dough was divided into 80 g portions and placed in molds. A second fermentation was carried out at 40 °C for 90 min. The dough was then baked at 220 °C for 25 min. After baking, the loaves were allowed to cool at room temperature before undergoing further analysis.

2.7. Enzyme Incorporation into Optimized Sourdough Bread

Three selected enzymes (laccase, lipase, and hemicellulose) were incorporated into wheat bread formulations, each previously optimized with its corresponding level of sourdough. The enzymes were added individually at two concentration levels, as well as in defined combinations, as follows:

• Individual Enzyme Incorporation:

  -

  Laccase (lacc): 10 ppm and 20 ppm,

  -

  Lipase (lipa): 30 ppm and 50 ppm,

  -

  Hemicellulase (hemi): 5 ppm and 10 ppm;

• Combination Enzyme Incorporation:

  -

  Enzyme combination (EnzComb): Laccase 10 ppm + Lipase 30 ppm + Hemicellulase 5 ppm.

Only this combination was selected for an in-depth texture analysis for each type of sourdough bread; the choice was based on preliminary tests with high levels in combination. Indeed, higher doses of enzymes have resulted in undesirable textural properties, such as excessive softening or structural weakening. Moreover, such high concentrations of enzymes added simultaneously can exceed the acceptable limits of food additives in bread formulations, thereby raising concerns regarding consumer acceptability.

2.8. Bread Quality Evaluation

An hour after baking, the bread was characterized through taking measurements of its specific volume, analyzing its crumb texture profile, and determining its moisture content.

2.8.1. Specific Volume

The rapeseed displacement method was used to calculate volume in accordance with AACC Approved Method 10.05 [34]. By dividing the volume by the weight of each loaf, the specific volume (cm³/g) was determined. The resulting data were used to create the graphs with Excel software.

2.8.2. Moisture Content

The ICC Method 110/1 was used to calculate the moisture content [35].

2.8.3. Texture Profile Analysis (TPA)

The adapted protocols are those described by Różyło et al. [36] and Bourekoua [37], using a texture analyzer (ZWICK Z020/TN2S by ZwickRoell, Ulm, Germany). Bread crumb samples (30 × 30 × 20 mm) were double compressed using a plunger equipped with a 30 mm probe until a depth of 50% was reached, at a crosshead speed of 1 mm/s. The following characteristics were recorded: chewiness, gumminess, springiness, hardness, and cohesiveness.

2.9. Statistical Analysis

All results are presented as the mean of three independent replicates, except for pasting properties (2 replicates) and texture analysis, which was performed in quadruplicate, and five distinct FT-IR analyses were conducted. Data are expressed as means ± standard deviations (Excel 2016). Statistical analyses were conducted using one-way ANOVA, followed by Fisher’s post hoc test for multiple comparisons, with a significance level set at p < 0.05. Analyses were performed using STATISTICA software version 7.0 (StatSoft, Inc., Tulsa, OK, USA). Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were utilized to classify different attributes and bread samples, as well as to evaluate the correlation between textural parameters. The HCA employed the Ward approach utilizing Euclidean distances to elucidate the similarities and disparities among the bread samples. The results of both PCA and HCA were visually depicted using JMP Pro 17 version 17.0.0 software (Statistical Discovery LLC, Cary, NC, USA). Different indicate statistically significant differences between groups at 0.05.

3. Results and Discussion

3.1. Pasting Properties of Flours

The pasting properties of white wheat flour (WF), wholemeal wheat flour (WMF), and barley flour (BF) show how starch gelatinization and paste behavior are influenced by flour composition under heating and cooling. These parameters are critical in order to understand the functional behavior in baking and processing.

Table 1. Pasting properties of flours.

Flour Type	OGT (°C)	PVT (°C)	OGV (mPas)	PV (mPas)	FV (mPas)
WF	63.85 ± 0.07 a	89.60 ± 0.14 b	18.50 ± 0.71 a	353.50 ± 2.12 a	526.50 ± 3.54 a
WMF	66.15 ± 1.63 a	92.05 ± 0.78 a	19.00 ± 0.00 a	234.00 ± 7.07 b	415.00 ± 0.00 b
BF	66.20 ± 0.14 a	81.70 ± 0.00 c	19.00 ± 0.00 a	120.50 ± 0.71 c	91.00 ± 1.41 c

WF—white wheat flour, WMF—wholemeal wheat flour, BF—barley flour, OGT—onset gelatinization temperature, PVT—peak viscosity temperature, OGV—onset gelatinization viscosities, PV—peak viscosity, and FV for final viscosity. Data are expressed as mean values ± standard deviation; ^a–c letters show statistically significant differences between groups at the p < 0.05 level.

demonstrates the onset gelatinization temperature (OGT), peak viscosity temperature (PVT), onset gelatinization viscosities (OGV), peak viscosity (PV), and final viscosity (FV) of white and wholemeal wheat flours and barley flour.

According to Table 1, wholemeal wheat and barley flours exhibited the maximum values (p < 0.05) of the onset gelatinization temperature (OGT) with 66.20 °C and 66.15 °C, respectively, compared to white wheat flour (63.85 °C). These values were statistically similar; however, the minor rise in WMF and BF in comparison to WF implies that the composition of wholemeal wheat and barley flours (bran, dietary fibers, and associated components) may inhibit the penetration of water into the starch granules, extending by that the initiation of the gelatinization process [38]. Moreover, the non-starch polysaccharides and lipids in wholemeal wheat flour can limit the hydration of starch and enhance its thermal demand by forming complexes with it [39].

In the context of the peak viscosity temperature (PVT), WMF recorded the maximum value (92.05 °C), followed by WF (89.60 °C), and then BF (81.70 °C). The presence of fiber and outer grain layers can be the origin for the higher PVT in wholemeal flour, where it can alter the structure of the granule and slow down the point at which starch reaches maximum swelling [40]. Barley flour’s higher β-glucan content is potentially the origin of its lower PVT, which can decrease temperatures and starch crystallinity and stimulate swelling [41].

The onset gelatinization viscosity (OGV) maintained relatively consistent levels among all samples (p > 0.05), with WMF and BF both recording 19.00 mPas and WF slightly lower at 18.50 mPas. These minor fluctuations indicate that the type of flour doesn’t strongly impact the gelatinization at its early stage of thickening and may possibly demonstrate similarities at low viscosity levels in water uptake behavior [38].

The peak viscosity (PV), which indicates the rupture of starch granules and the maximum swelling capacity, has significantly changed (p < 0.05). WF achieved the highest PV (353.50 mPas), followed by WMF (234.00 mPas) and finally BF (120.50 mPas). The decrease observed in PV of wholemeal flour is potentially related to the presence of fiber and bran particles, which can limit the water availability and reduce the starch granules swelling [38]. Barley flour showed a way higher reduction, connected to the significantly higher β-glucan and fiber content, compromising paste (flour–water suspension) thickening, as also reported by Symons and Brennan [41].

Regarding the final viscosity (FV), which signifies the starch’s ability to retrograde and form a gel during the cooling process, WF exhibited the highest FV (526.50 mPas), followed by WMF (415.00 mPas), and then BF with a significantly lower FV (91.00 mPas). The lower FV in wholemeal flour appears to be connected with the interference of amylose retrogradation with lipids and dietary fibers; this phenomenon may reduce the gel strength and restrict the final flour–water suspension viscosity [38]. In addition, the poor gel-forming potential of barley flours explains the minimal FV value previously reported by Symons and Brennan [39] in their study on barley β-glucan fiber fractions.

3.2. Structural Properties of Flours and Protein Secondary Structure Analysis

To learn more about the molecular structure of white flour, wholemeal wheat, and barley flours used in the preparation of sourdoughs and breads, the chemical groups and the secondary structure of proteins were analyzed. Figure 1 shows FTIR spectra of studied flours.

All recorded spectra demonstrate two bands between 3100 cm⁻¹ and 3500 cm⁻¹ and between 2600 and 3000 cm⁻¹ assigned to amide A and amide B, respectively [29,30]. These spectral features result from NH stretching vibration [42]. Peaks centered at ~1650 cm⁻¹ and at ~1537 cm⁻¹ are characteristic, respectively, of amide I and amide II bands due to CO carbonyl stretch with minor contribution from out-of-plane CN stretching vibration and NH bonding and CH stretching of proteins, respectively Kong and YU, 2007 [42]; Fetouhi et al., 2019 [43]. Amide III (1229–1300 cm⁻¹) band centered at 1249 cm⁻¹ resulting from CN stretching and NH bending also appeared. The spectral region between 800 and 1200 cm⁻¹ of polysaccharides is characteristic for backbone vibrations of CO, CN, and CC bonds [29].

The curve-fitted amide I bands (1590–1710 cm⁻¹) are shown in Figure 2. Peak areas and positions used in the curve fitting of each type of flour were determined according to results indicated by the second derivative (Figure S1). Distribution of secondary structures of flour protein, calculated by deconvolution of FT-IR spectra of the examined flours, was summarized in

Table 2. Comparison of protein secondary structures in white wheat, wholemeal wheat, and barley flours.

	β-Sheet (%)	α-Helix (%)	β-Turn (%)
WF	47	29	24
WMF	51	32	17
BF	47	33	20

. The content of secondary structures of proteins for all flours was evaluated by analysis of amide I differential spectra between flours (Figure S2).

The deconvoluted amide I band (1590–1710 cm⁻¹) shows the presence of four characteristic peaks for all examined flours. Peaks centered at 1610 cm⁻¹ for white wheat flour, at 1616 cm⁻¹ for wholemeal wheat flour, and at 1614 cm⁻¹ for barley flour were assigned to intermolecular β-sheet due to aggregation [29,30,40]. Bands centered at 1637 cm⁻¹ for white wheat flour and barley flour and at 1638 cm⁻¹ were assigned to extended β-sheets [30,40]. The α-helix secondary structure implicated in the structuration of the protein network of the studied flours was found at 1656 cm⁻¹ for white wheat flour and barley flour and at 1658 cm⁻¹ for wholemeal wheat flour [29,30,44]. Peaks centered at 1680 cm⁻¹ for WF, at 1684 cm⁻¹ for WMF, and at 1683 cm⁻¹ for BF were assigned to β-turns. No spectral features indicating the presence of random structures were found for all types of flours. On the other hand, the presence of broad peaks assigned to the extended β-sheet structure shown by the second derivative results for all flours (centered at 1637 cm⁻¹ for WF and at 1638 cm⁻¹ for WMF and BF) (Figure S1) indicates the high involvement of this type of structure in the formation of the studied protein networks [25,43]. Comparable results were observed in gluten-free doughs by Fetouhi et al. [27], wherein the absence of gluten, the dominance of β-sheet structures over the other conformations was critical for network formation.

Quantitative composition of the secondary structure of protein networks in the studied flours obtained by the deconvolution of amide I bands shown in Table 1 demonstrates that the networks of all flours show a dominance of β-sheet (47% for WF and BF and 51% for WMF) compared to α-helix (29% for WF, 32% for WMF, and 33% for BF) and β-turns (24% for WF, 17% for WMF, and 20% for BF). These results are similar to those found by Bock and Damodaran [28], Van Velzen et al. [30], and Nawrocka et al. [45], who showed the dominance of β-sheet structure in pure gluten proteins. According to Nawrocka et al. [45] and Pézolet et al. [46], the domination of β-sheet structures in the present studies could be connected with the presence of the intermolecular extended β-sheets, which were observed in the highly hydrated solid state of proteins, which was previously demonstrated by the results of the second derivative.

The protein network of wholemeal wheat flour exhibited a higher proportion of β-sheet secondary structure, which was 1.06 times greater than those observed in white wheat flour and barley flour, as shown in Table 2. This dominance was accompanied by a low amount of β-turns, which was 1.41 and 1.17 times less than amounts found for WF and BF, respectively. These findings suggest that the protein network of WMF is more distinctly characterized by the formation of β-sheet structure at the expense of β-turns compared to other flour types [43].

3.3. Effect of Sourdough Incorporation Level on Physical Properties of Breads

Sourdough can improve the loaf volume of bread and reduce crumb stiffness, hence increasing the quality and shelf life of wheat and gluten-free bread [45].

Table 3. Physical and textural characteristics of breads prepared with three sourdough types at different incorporation levels.

	SV (cm3/g)	Moisture (%)	Hardness (N)	Chewiness (N)	Springiness (–)
CB	5.54 ± 0.09 a	31.15 ± 0.37 bcd	3.53 ± 0.26 f	3.4 ± 0.03 g	1.01 ± 0.01 d
GBBS1 20%	3.15 ± 0.15 c	29.65 ± 0.33 cde	20.87 ± 0.90 d	13.22 ± 0.71 d	1.00 ± 0.00 de
GBBS1 30%	3.38 ± 0.18 fg	26.26 ± 1.74 e	14.00 ± 2.01 e	7.94 ± 0.70 e	0.92 ± 0.01 b
GBBS1 40%	3.59 ± 0.06 b	26.55 ± 2.39 e	17.22 ± 0.07 de	11.45 ± 0.75 d	0.99 ± 0.01 e
GBBS2 20%	2.13 ± 0.05 e	27.99 ± 3.01 de	49.27 ± 2.4 a	28.29 ± 1.23 a	1.00 ± 0.00 de
GBBS2 30%	2.23 ± 0.04 de	29.24 ± 0.52 cde	40.96 ± 1.35 b	23.81 ± 0.47 b	1.00 ± 0.001 de
GBBS2 40%	2.32 ± 0.04 d	32.02 ± 2.57 bc	33.21 ± 6.45 c	20.11 ± 3.76 c	1.00 ± 0.001 de
GBBS3 20%	3.31 ± 0.06 g	33.67 ± 0.48 ab	8.41 ± 0.56 f	5.07 ± 0.21 efg	0.95 ± 0.006 c
GBBS3 30%	3.39 ± 0.11 fg	35.93 ± 0.55 a	7.06 ± 0.05 f	4.79 ± 0.24 fg	0.94 ± 0.007 c
GBBS3 40%	3.44 ± 0.11 f	34.23 ± 0.09 ab	6.99 ± 0.22 f	7.33 ± 0.15 ef	1.06 ± 0.007 a

CB—control bread, GBBS1—gluten-based bread with S1 (white wheat sourdough), GBBS2—gluten-based bread with S2 (wholemeal wheat sourdough), GBBS3—gluten-based bread with S3 (barley sourdough); SV—specific volume. Data is presented as mean values ± standard deviation. The ^a–g letters indicate statistically significant differences among groups in columns at p < 0.05.

summarizes the evolution of bread quality attributes at different levels of added sourdough.

One-way ANOVA analysis revealed significant differences among all samples: CB (control bread made with commercial yeast), GBBS1 (bread made with sourdough from white wheat flour), GBBS2 (bread made with sourdough from wholemeal wheat flour), and GBBS3 (bread made with sourdough from barley flour). A statistically significant variation (p < 0.05) was observed across all evaluated parameters, including specific volume, moisture content, hardness, springiness, and chewiness. According to Table 3, the control bread made with yeast recorded the highest (p < 0.05) specific volume (5.54 cm³/g) among all bread samples. Within the sourdough group, GBBS1 at 40% incorporation displays the highest specific volume (3.59 cm³/g), followed by GBBS3 at 40% (3.44 cm³/g) and GBBS3 at 30% (3.39 cm³/g). Conversely, GBBS2 at 20% and 30% incorporation exhibit the lowest values (p < 0.05) of specific volume (2.13 and 2.23 cm³/g, respectively). Research comparing yeast and sourdough breads concludes that, in general, yeast-leavened bread achieves higher specific volumes than sourdough. Verdonck et al. [47] reported that yeast bread achieved a maximum specific volume increase from 2.13 mL/g to 3.26 mL/g over the sourdough bread. This decrease in specific volume of sourdough breads can be explained through proteolysis and the resultant acidic conditions of fermentation since both of them can reduce the gas-retaining capacity of dough and loosen the network of gluten.

These effects are typically observed when using whole wheat or barley flour, which possess lower-quality gluten compared to white wheat flour, Picozzi et al. [48] also reported that reduced gas retention and compact structure in sourdough breads are related to enzymatic and acidic activity during fermentation, especially in flours with limited gluten-forming capacity. On the other hand, the control sample fermented only with commercial yeast benefits from a well-developed gluten network, resulting in increased gas retention and loaf volume. Another supportive study confirms that sourdough-leavened wholemeal wheat breads have lower specific volumes than yeast-leavened breads [47].

In line with previous findings, we discovered that both our yeast and sourdough breads had improved volume characteristics, most likely due to differences in formulation, flour quality, or fermentation conditions, especially considering we used a long fermentation process that alternated between a hot 40 °C phase and a cold overnight fermentation. However, there is a complex link between fermentation duration and specific volume. While some research has found that ideal specific volumes are reached after 6 h of fermentation [49], others have found that volumes drop as fermentation duration increases [47]. However, GBBS1 at 40% incorporation stood out, most likely due to the white flour matrix, which may improve gas retention due to the superior quality of the sourdough, as reported by Arendt et al. [50]. Similarly, Corsetti and Settanni [51] demonstrated how white flour sourdough can improve gas retention when fermented properly.

When it comes to moisture content, we noticed that breads made with barley sourdoughs had higher moisture. GBBS3, incorporated with 30% barley sourdough, had the highest value of moisture (35.93%), followed by GCS3 at 40% (34.23%) and GBBS2 at 40% (32.02%). The control bread had a moderate moisture level (31.15%), while GBBS2 had the lowest moisture content at 20% (27.93%). This increase in moisture is most likely owing to the qualities of barley flour, which is rich in β-glucans and fiber ingredients known for their ability to store water. While baking decreases the overall β-glucan content in barley flour, it enhances its solubility [52]. These soluble fibers have high water-binding capacities, creating gel-like structures that lock water inside the dough matrix [53]. In addition, the lactic acid bacteria in sourdough fermentation are also able to create exopolysaccharides that help with moisture retention due to binding water into the dough. Novotni et al. [54] stated that using barley sourdough could improve moisture retention; moreover. Zhang et al. [55] explained that the exopolysaccharides produced during fermentation can contribute to hydration and softer crumb texture.

In terms of hardness, the control bread CB made with yeast had the lowest value (3.52 N), which indicates a soft and light texture. However, the inclusion of sourdough increased crumb hardness in most cases, depending on the type and amount of sourdough incorporation. Breads made with white wheat sourdough (GBBS1) had the highest (p < 0.05) hardness at 20% (20.87 N), then softened at 30% (14.00 N) and slightly increased again at 40% (17.22 N). Sourdough breads made with wholemeal wheat (GBBS2) possessed the firmest crumbs among all the samples, especially at 20%, with a hardness of 49.27 N, then reduced to 40.96 N at 30% and to 33.21 N at 40%. GBBS3 breads were noticeably softer, and the hardness decreased progressively as we ascended the level of incorporation. At 20%, hardness was 8.41 N, dropping at 30% to 7.06 N and then decreasing further at 40% to 6.99 N. These differences in hardness between breads made with wholemeal wheat sourdough and the others made with barley can majorly be attributed to the type and the behavior of the flour during the fermentation and cooking process. The bran and the germ in the wholemeal wheat flour interfere with the development of the gluten, absorb a lot of water, and make the dough less elastic and the bread’s crumb harder and denser. Moreover, using wheat bran or wholegrain wheat flour instead of refined wheat flour increases bread hardness [56].

The water competition from the high level of fiber also tends to lead to harder bread in general [57]. As mentioned above, the very high levels of non-starch polysaccharides and β-glucans in the barley flour not only enhance water-holding capacity but also provide exopolysaccharides (EPS) produced during sourdough fermentation by the lactic acid bacteria. Through the fixation of water and inhibition of firming activity of organic acids, the EPS makes the crumb softer [58]. As for springiness, yeast bread presented a low springiness (1.01), indicating a flexible and cohesive crumb structure. Particularly, breads made with barley sourdough (GBBS 3) have shown an enhanced springiness, with the 40% incorporation level reaching the highest value (1.06). This implies that a higher amount of barley sourdough can enhance crumb flexibility due to its unique fiber composition. Also, Terrazas et al. [59] mentioned in their research that fermentation time in sourdough breads can significantly affect bread texture, including springiness. Yet, white wheat sourdough breads (GBBS1) maintained a stable springiness throughout all inclusion levels: (1.00) at 20%, (0.92) at 30%, and (0.99) at 40%.

Breads made with wholemeal wheat sourdough (GBBS2) presented consistent springiness values across all incorporation levels with no significant difference (p > 0.05): 1.00 at 20%, 1.00 at 30%, and 1.00 at 40%. Although these values are slightly less than those observed in bread made with barley sourdough, they still maintained adequate springiness. This reduction may be attributed to the presence of bran and higher fiber content in wholemeal wheat flour, which can interfere with gluten development and dough elasticity. Our results match what Casado et al. [60] concluded in their research: that sourdough-incorporated bread had a higher springiness than non-sourdough breads, indicating an elastic crumb due to fermentation.

Chewiness is also one of the primary elements of texture in bread and other common foods. The type of flour, the bread making techniques, and the additives all affect how chewy sourdough bread is. As shown in Table 3, the control bread displayed the lowest chewiness value (3.40 N), and the incorporation of wholemeal wheat sourdough (GBBS2) significantly decreased the chewiness with values recorded at 28.29 N (20% inclusion), 23.81 N (30%), and 20.11 N (40%). Recent research conducted by Islam and Islam [61] found that the chewiness of sourdough bread can be affected by many factors, including the dietary fiber composition. Breads made with white wheat sourdough (GBBS1) maintained intermediate chewiness levels: 13.22 N at 20% inclusion, 7.94 N at 30%, and 11.45 N at 40%. These results suggest that white wheat sourdough can offer a balanced textural profile, delivering a moderate level of chewiness between very soft bread and overly hard bread, potentially appealing to a broad range of consumer preferences. The barley sourdough breads (GBBS3) also revealed a significant decrease in the chewiness of the crumb: 5.07 N at 20% inclusion, 4.79 N at 30%, and 7.33 N at 40%. The lower chewiness of barley sourdough breads could be due to the soluble fibers (e.g., β-glucans) that could modify the crumb porosity and lower the perceived chewiness. Based on these findings, we opted to keep the 40% incorporation level for each sourdough type throughout the rest of this study. This choice was guided by the fact that 40% incorporation consistently showed the most balanced bread quality across all sourdoughs, including high moisture content, desirable specific volume, and a generally acceptable crumb texture. As a result, all subsequent analysis in this research was conducted on loaves with a 40% sourdough inclusion.

3.4. Shelf Life and Technological Quality Evaluation of Selected Sourdough Breads

The impact of three sourdough types on shelf life and quality of sourdough wheat breads (at 40% of each sourdough incorporation) stored in plastic zip bags at room temperature was investigated over a period of 72 h. The determined parameters included specific volume, moisture content, hardness, springiness, and chewiness at 1, 24, 48, and 72 h after baking. Results are presented in Figure 3, Figure 4 and Figure 5.

According to Figure 3, at 1 h post baking, the control bread had the highest specific volume (SV) (4.91 cm³/g), followed by GBBS1 (3.92 cm³/g), GBBS2 (3.80 cm³/g), and GBBS3 (3.507 cm³/g). The higher SV of the control bread results from the quick gas production of commercial yeast, as lactic acid bacteria and wild yeast in sourdough-fermented bread are both relatively slower. Within 72 h, control bread fell to 3.29 cm³/g, but GBBS1 remained higher (3.71 cm³/g). This suggests that sourdough fermentation, especially in GBBS1, can better preserve bread structure due to exopolysaccharides (EPS) and organic acids that improve dough stability and volume retention, as supported by studies such as Xu et al. [62], who highlighted the contributions of EPS to improved crumb structure.

Regarding the moisture content of breads (Figure 4), GBBS3 retained the most, beginning at 41.88% at 1 h and staying at 38.83% after 72 h. CB went down from 35.42% to 32.86%. This superior moisture retention in sourdough breads, particularly GBBS3, is always attributed to the water-binding capacity of EPS produced by lactic acid bacteria through fermentation, which has been shown to minimize the loss of moisture and delay staling.

Concerning the hardness, control bread had the softest crumb (4.19 N) at 1 h. After 72 h, GBBS3 sourdough bread had the highest hardness (31.93 N), followed by GBBS2 (24.50 N) and GBBS1 (24.03 N), whereas control bread with yeast only achieved 6.25 N. Despite the expected increase in crumb hardness over time, the sourdough breads, particularly those made with wholemeal sourdough (GBBS2) and barley sourdough (GBBS3), retained a softer texture in the earlier stages and were more resistant to staling. This aligns with the findings of Wang et al. [23], who reported that sourdough fermentation helps limit starch retrogradation, helping maintain crumb softness [63]. According to Novotni et al. [54], adding barley sourdough to bread can slow down mold growth, improve flavor, and keep it fresh for up to 30 days when combined with vacuum refrigeration and modified environment packaging.

As for springiness, it was the highest in barley sourdough bread GBBS3 (0.95) 1 h after baking, while GBBS2 had the lowest springiness (0.720). The control bread and GBBS1 showed intermediate outcomes (~0.85 to 0.87). The control bread’s springiness increased over time, peaking at 72 h (1.22). This suggests that moisture retention or starch retrogradation may have positively influenced texture. Sourdough bread underwent a decrease in springiness over 72 h, with GBBS2 revealing the most significant loss at 0.50. The decreasing trend of springiness in sourdough breads is more likely due to the acidification that proceeds during fermentation and modifies the gluten network properties, producing a firmer crumb that may appear less springy [64]. The lower springiness shown in wholemeal wheat sourdough bread (GBBS2) may also be caused by a high fiber content obstructing the gluten network from stretching [65]. Over time, the springiness seemingly tended to decrease, while chewiness, in contrast, showed an opposing trend. After an hour upon baking, the control had the lowest chewiness values (2.36), whereas the sourdough breads GBBS1 (6.64) and GBBS2 (5.49) prominently soared in terms of chewiness, making the crumb denser and stiffer just after baking. This suggests a denser and therefore more resilient crumb in sourdough breads shortly after baking.

Within 72 h, chewiness in the control bread developed significantly (6.69 N), indicating crumb firming related to staling. In contrast, chewiness in white wheat sourdough (GBBS1) declined from 6.64 N to 2.46 N, indicating softening with time. Even after 72 h, barley sourdough bread (GBBS3) remained chewy (5.04 N), indicating improved crumb texture preservation. At 72 h, the chewiness of wholemeal sourdough bread (GBBS2) decreased moderately, reaching 3.00 N. The initial increase in chewiness in sourdough breads is consistent with previous observations that organic acids and fermentation increase crumb firmness but could delay starch retrogradation and crumb hardness over time, thereby increasing shelf life [66].

3.5. Enzymes’ Effect on the Textural Properties of Sourdough Breads

The textural properties of sourdough breads incorporating enzymes (laccase, hemicellulose, and lipase) and their combinations are presented in

Table 4. Texture profile parameters of GBBS1 sourdough bread treated with different enzymes and combinations.

	Hardness (N)	Gumminess (–)	Cohesiveness (–)	Springiness (–)	Chewiness (N)
GBBS1	17.22 ± 0.07 c	11.53 ± 0.62 ab	0.67 ± 0.03 a	0.99 ± 0.01 a	11.45 ± 0.74 a
Lacc 10 ppm	19.27 ± 1.36 ab	11.04 ± 1.16 bcd	0.61 ± 0.02 b	0.94 ± 0.04 b	11.07 ± 0.75 a
Lacc 20 ppm	18.59 ± 1.39 bc	11.52 ± 0.80 abc	0.59 ± 0.02 bc	0.92 ± 0.01 bc	10.64 ± 0.83 a
Lipa 30 ppm	19.27 ± 1.20 ab	8.27 ± 1.39 ef	0.55 ± 0.06 cd	0.85 ± 0.05 c	6.94 ± 0.81 c
Lipa 50 ppm	20.08 ± 0.94 ab	9.43 ± 0.49 de	0.40 ± 0.03 d	0.74 ± 0.02 d	6.98 ± 0.56 c
Hemi 5 ppm	20.40 ± 0.88 a	10.09 ± 1.24 cde	0.55 ± 0.04 c	0.90 ± 0.03 c	9.02 ± 0.95 b
Hemi 10 ppm	19.25 ± 1.28 ab	13.04 ± 2.24 a	0.58 ± 0.04 bc	0.91 ± 0.03 bc	10.84 ± 1.06 a
EnzComb ppm	12.68 ± 1.28 d	7.01 ± 0.29 f	0.56 ± 0.04 c	0.89 ± 0.01 c	6.21 ± 0.18 c

GBBS1—Gluten-based bread with S1, with ^a–f letters indicating statistically significant differences among groups in columns at p < 0.05.

,

Table 5. Texture profile parameters of GBBS2 sourdough bread treated with different enzymes and combinations.

	Hardness (N)	Gumminess (–)	Cohesiveness (–)	Springiness (–)	Chewiness (N)
GBBBS2	33.21 ± 6.40 a	18.08 ± 0.92 a	0.60 ± 0.004 ab	1.00 ± 0.001 a	20.11 ± 3.70 a
Lacc 10 ppm	23.24 ± 1.44 c	14.66 ± 0.63 ab	0.63 ± 0.01 ab	1.00 ± 0.01 a	14.63 ± 0.60 b
Lacc 20 ppm	12.97 ± 1.75 f	8.30 ± 1.10 c	0.64 ± 0.02 ab	0.94 ± 0.01 ab	7.76 ± 1.02 c
Lipa 30 ppm	26.11 ± 1.42 b	11.59 ± 1.57 bc	0.50 ± 0.02 b	0.81 ± 0.03 c	9.34 ± 0.94 c
Lipa 50 ppm	23.59 ± 0.37 c	16.65 ± 7.23 a	0.69 ± 0.33 a	0.81 ± 0.01 c	14.12 ± 1.75 b
Hemi 5 ppm	20.39 ± 1.58 d	11.32 ± 1.97 bc	0.62 ± 0.02 ab	0.94 ± 0.04 ab	10.69 ± 2.36 bc
Hemi 10 ppm	9.69 ± 0.36 g	8.38 ± 2.05 c	0.65 ± 0.07 ab	0.91 ± 0.02 b	7.61 ± 1.70 c
EnzComb ppm	17.56 ± 1.12 e	7.92 ± 1.53 c	0.51 ± 0.02 b	0.87 ± 0.02 bc	6.87 ± 1.29 c

GBBS2—Gluten-based bread with S2, with ^a–g letters indicating statistically significant differences among groups in columns at p < 0.05.

and

Table 6. Texture profile parameters of GBBS3 sourdough bread treated with different enzymes and combinations.

	Hardness (N)	Gumminess (–)	Cohesiveness (–)	Springiness (–)	Chewiness (N)
GBBS3	6.99 ± 0.21 e	8.53 ± 0.29 d	1.01 ± 0.03 a	1.06 ± 0.007 a	7.33 ± 0.15 c
Lacc 10 ppm	19.99 ± 0.79 c	12.78 ± 0.48 a	0.64 ± 0.02 b	0.98 ± 0.03 b	12.55 ± 0.77 a
Lacc 20 ppm	16.95 ± 1.09 d	10.61 ± 1.56 bc	0.61 ± 0.01 b	0.93 ± 0.01 bc	9.89 ± 1.62 b
Lipa 30 ppm	35.72 ± 0.96 a	12.26 ± 1.66 ab	0.39 ± 0.03 d	0.73 ± 0.10 d	7.62 ± 0.80 c
Lipa 50 ppm	23.48 ± 1.18 b	10.18 ± 0.39 cd	0.43 ± 0.03 d	0.76 ± 0.06 d	7.49 ± 0.67 c
Hemi 5 ppm	16.9 ± 1.20 d	10.26 ± 1.44 cd	0.61 ± 0.05 b	0.92 ± 0.05 b	10.3 ± 1.51 b
Hemi 10 ppm	15.71 ± 0.77 d	11.17 ± 1.65 abc	0.61 ± 0.03 b	0.91 ± 0.02 bc	9.57 ± 0.24 b
EnzComb ppm	16.69 ± 1.60 d	8.54 ± 0.63 d	0.48 ± 0.03 c	0.85 ± 0.05 c	7.24 ± 0.45 c

GBBS3—Gluten-based bread with S3, with ^a–e letters indicating statistically significant differences among groups in columns at p < 0.05.

for GBBS1, GBBS2, and GBBS3, respectively. The addition of enzymes to wheat bread prepared with various sourdoughs had a significant (p < 0.05) impact on texture parameters. Laccase performs best in the acidic circumstances created by sourdough fermentation since its enzymatic activity is at its peak at pH 4 and 40 °C [67].

Laccase treatment dramatically reduced hardness in GBBS2, which had a hardness of 33.21 N in the control sample (no enzyme) and dropped to 23.24 N at 10 ppm and 12.97 N at 20 ppm. The phenolic chemicals and fibers found in wholemeal flours likely aid laccase’s oxidative cross-linking ability, which results in this softening effect. The compatibility of laccase and bran-rich sourdough matrices was confirmed by these improvements, which were also demonstrated by a sharp decrease in chewiness from 20.11 N for GBBS1 to 7.76 N at 20 ppm. However, because refined flour has less fiber and phenol, the hardness of GBBS1 (white wheat bread) increased slightly from 17.22 N (control) to 18.59 N at 20 ppm, indicating a reduced effect. A comparable behavior in GBBS3 (barley sourdough), where laccase reduced hardness from 19.99 N at 10 ppm to 16.95 N at 20 ppm, this moderate decrease is likely limited by the β-glucan-rich matrix of barley sourdough, which restricts enzyme access compared to arabinoxylans [68].

In contrast, the acidic conditions of sourdough bread hinder lipase activity, which functions best at an alkaline pH around 8 [69]. Its impact across the three bread types was mixed and often suboptimal. In GBBS1, although gumminess and chewiness improved at the 30 ppm level of incorporation (dropping from 11.53 (control bread) to 8.27 N and from 11.45 to 6.94 N, respectively), hardness increased slightly to 19.27 N and 20.08 N compared to the control bread (17.22 N). In GBBS2, lipase addition led to a notable decrease in hardness, from 33.21 N (control) to 26.11 N at 30 ppm and 23.59 N at 50 ppm. Springiness was reduced from 1.00 (control) to 0.81 in both levels of incorporation, indicating poor structural performance. However, lipase at 50 ppm improved cohesiveness, reaching a value of 0.69, compared to 0.60 scored in the bread without enzyme addition (GBBS2). GBBS3 displayed the most adverse response, with lipase increasing hardness from 19.99 N to 35.72 N (30 ppm). This poor outcome may be attributed to barley β-glucans interfering with lipase diffusion and sourdough acidity reducing its catalytic efficiency [70,71].

Hemicellulase demonstrated much greater compatibility with sourdough systems. In GBBS2 (wholemeal sourdough), the incorporation of various enzymes and their combinations significantly (p < 0.05) reduced the hardness, chewiness, and gumminess of the breads. Hemicellulase at 5 and 10 ppm yielded the most desirable hardness values, at 20.39 N and 9.69 N, respectively. Chewiness decreased from 20.11 N to 10.69 (5 ppm) and 7.61 N (10 ppm), and gumminess decreased from 18.08 N to 8.38 N (10 ppm), indicating efficient softening due to fiber hydrolysis. In GBBS1, hemicellulase led to a moderate reduction in hardness (from 20.40 N at 5 ppm to 19.25 N at 10 ppm) and stable springiness (0.91). Regarding GBBS3, which contains mainly β-glucans rather than arabinoxylans, the addition of enzymes to barley sourdough bread did not enhance its textural parameters. In fact, hardness, chewiness, and gumminess generally increased following enzyme treatment. Table 6 results showed how hardness went from 6.99 to 16.90 N to 15.71 N in both incorporation levels; as for chewiness, it slightly increased from 7.33 (control) to 10.30 N (5 ppm) and 9.57 N (10 ppm), confirming some degree of non-starch polysaccharide solubilization.

Depending on the type of flour used in sourdough, the enzyme combination (EnzComb) displayed different results. It reduced chewiness from 11.45 N to 6.21 N and hardness from 17.22 N to 12.68 N in GBBS1, offering the best overall texture improvement. In GBBS2, it resulted in a significant decrease in chewiness from 20.11 N to 6.87 N and a noticeable softening (hardness went from 33.21 N to 17.56 N). Additionally, improvements were noted in gumminess, which dropped from 18.08 to 7.92. Combining enzymes in GBBS3 did not enhance texture; chewiness decreased slightly (from 7.33 N to 7.24 N), but hardness stayed high at 16.69 N. Compensatory enzyme interactions, such as the cross-linking activity of laccase counteracting the softening action of lipase to produce more balanced textural results, are typically the cause of these intermediate effects.

Lastly, each enzyme’s effectiveness differed depending on the sourdough’s flour type and mechanism of action of separated and combined enzymes. Lipase was usually less efficient because of pH incompatibility, whereas laccase and hemicellulase yielded the most promising outcomes, especially in wholemeal breads. Although it was limited in barley-based formulations, the enzyme combination produced balanced outcomes in white and wholemeal wheat bread. These results highlight the necessity of tailoring the kind and dosage of enzymes to the specific flour matrix in order to attain the ideal bread texture.

3.6. Principal Component, Cluster, and Hierarchical Classification Analysis

Important connections between the textural parameters are shown in the correlation matrix (Figure 6). Chewiness and gumminess have a very significant correlation (R² = 0.91), implying that chewiness rises as gumminess does. Gumminess (R² = 0.77) and chewiness (R² = 0.56) both show a positive correlation with hardness, while cohesiveness (R² = −0.66) and springiness (R² = −0.48) indicate that harder breads are less cohesive and less springy, providing a negative correlation. Additionally, cohesiveness and springiness have a moderate association (R² = 0.71). The strong positive correlations observed demonstrate that when the gluten structure is strengthened or modified, all textural attributes respond in concert, regardless of whether that modification is caused by enzymatic crosslinking or lipid-based plasticization.

PCA visualized 92.9% of the overall variation in the dataset utilizing the first two components, indicating how enzyme treatments and various sourdough types affect bread texture (Figure 7). Each texture parameter’s contribution is illustrated by a vector (arrow), and the bread samples’ positions reflect how similar or dissimilar their textural properties are. The right side of the first principal component (PC1) is dominated by chewiness, gumminess, and hardness. On the left side, cohesiveness and springiness are more pronounced. Breads located to the left of the axis are distinguished by a softer, more cohesive crumb, while those to the right have a firmer texture that is more resistant to chewing. The upper left quadrant is dominated by springiness, with nearby samples, including GBBS1 and BS2 hemi 5, suggesting these breads have a high ability to recover their shape after compression. Cohesiveness lies toward the left side, with GBBS3 positioned closer to it, indicating a more internally integrated crumb structure. The upper right quadrant is associated with chewiness and gumminess. GBBS2 lies near the chewiness vector, showing a denser crumb requiring more mastication. Several laccase-treated breads (BS2 lacc 10, BS3 lacc 10, BS1 lacc 10) are also in this region, implying an increase in both chewiness and gumminess. The lower right quadrant corresponds to hardness, where high-dose lipase treatments (BS1 lipa 50, BS2 lipa 50, BS3 lipa 50, BS2 lipa 30, and BS3 lipa 30) are located. This indicates that lipase, particularly at higher levels, produces firmer, denser bread.

The central area contains several low-dose treatments (e.g., BS1 hemi 10, BS2 hemi 10, BS3 hemi 10, and BS1 hemi 5), which are not strongly associated with any extreme texture parameter, suggesting a more balanced crumb profile. Enzyme combination samples (BS1 EnzComb, BS3 EnzComb) are located in the lower left quadrant, far from hardness and chewiness vectors, indicating that combining enzymes results in neither excessively firm nor overly gummy bread.

Figure 7 partitions the breads into three distinct clusters that are consistent with the PCA-based patterns of hardness, gumminess, chewiness, cohesiveness, and springiness. Cluster 1 (blue) is a singleton containing only GBBS2 (wholemeal wheat sourdough control), indicating that this sample is texturally distinct from all others. Its isolation is in line with the PCA loading structure, where higher hardness/gumminess/chewiness and lower cohesiveness/springiness differentiate GBBS2 from the rest.

The second cluster groups the softest breads, including CB and GBBS3, characterized by comparatively lower hardness and chewiness and higher cohesiveness/springiness, consistent with their positions near the “soft/cohesive” side of the PCA map.

The third cluster contains the enzyme-treated breads, which display intermediate, tailored textures. Within this cluster, samples treated with higher lipase doses tend to align toward the firmer sub-branch (greater hardness/gumminess/chewiness), whereas lower-dose laccase and hemicellulase treatments group nearer the balanced-texture region (reduced hardness while maintaining springiness). Overall, HCA confirms that enzyme strategy and sourdough type produce reproducible groupings: GBBS2 forms a distinct class, CB/GBBS3 represents the soft/cohesive profile, and enzyme variants span a continuum of intermediate structures rather than collapsing into a single extreme.

Overall, the panels in Figure 7 provide a more comprehensive overview of textural changes in gluten-based breads resulting from enzymatic treatments. Unlike in gluten-free systems, where the goal is to recreate missing structure, the enzymes in this system act directly on a viscoelastic gluten network present. This distinction is critical: in gluten-based systems, laccase can be used to improve structure via protein crosslinking, lipase to increase crumb density and durability through lipid reorganization, and hemicellulase to adjust hydration and extensibility through polysaccharide degradation. Multivariate data analysis not only confirms these mechanical effects but also demonstrates that enzymatic strategies can be targeted to achieve specific textural properties in wheat- and barley-based sourdough breads.

4. Conclusions

The results of this study underscore the significant influence of flour type, sourdough fermentation, and enzymes on the technological quality of wheat bread. The high content of dietary fibers and β-glucans in wholemeal and barley flours contributed to distinct behaviors in viscosity profiles and protein secondary structures, leading to notable changes in starch gelatinization and retrogradation. FT-IR analyses revealed significant variations in protein secondary structures, especially an increase in β-sheet content to the detriment of β-turns, reflecting a structural influence of fiber components on protein conformation. These molecular changes were manifested in texture properties, notably hardness, springiness, and chewiness. In terms of storage and shelf life, trials showed that sourdough breads, especially those made with barley and wholemeal flours, performed better in moisture retention and slowing staling due to the production of exopolysaccharides and the water-binding ability of fibers.

Finally, synergistic and antagonistic effects were demonstrated through the combined activity of laccase, lipase, and hemicellulose, depending on enzyme dosage and the flour type used for sourdough. Breads made with white wheat sourdough and wholemeal sourdough showed improved texture compared to those made with barley sourdough. Enzyme combinations significantly decreased hardness, chewiness, and gumminess in white and wholemeal sourdough breads, while in barley sourdough bread, they significantly decreased cohesiveness and springiness and increased hardness. High-dose laccase and hemicellulase show the most promising results by reducing hardness and chewiness, especially in wheat and wholemeal formulations. In contrast, lipase is less effective except for some chewiness reduction in white and wholemeal sourdough breads due to the sourdough’s acidic conditions. The result of multivariate analysis further confirmed that these enzymatic strategies can be intentionally selected to optimize specific textural attributes of bread, such as hardness, chewiness, and springiness, according to the flour type and fermentation method used.

This study provides encouraging prospects for the baking sector, particularly in the development of high-fiber, clean-label, and functionally enhanced bread products. The findings highlight the importance of a multifactorial strategy to bread formulation, integrating flour quality, targeted enzyme application and fermentation type to improve the technological quality of the final product and extend its shelf life. Despite the presentation of these essential characteristics in this study, further research is necessary, including pilot-scale trials and an evaluation of the practical feasibility of the proposed approach on an industrial scale.