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Article

Impact of Selected Starter-Based Sourdough Types on Fermentation Performance and Bio-Preservation of Bread

by
Khadija Atfaoui
1,
Sara Lebrazi
2,
Anas Raffak
3,
Youssef Chafai
4,
Karima El Kabous
1,
Mouhcine Fadil
5,* and
Mohammed Ouhssine
1
1
Laboratory of Natural Resources and Sustainable Development, Faculty of Sciences, Kenitra B.P 242, Morocco
2
Laboratory of Microbial Biotechnology and Bioactive Molecules, Faculty of Sciences and Technologies, Sidi Mohamed Ben Abdellah University, Imouzzer Road, Fez 30000, Morocco
3
Department of Food and Nutritional Sciences, Hassan II Institute of Agronomy and Veterinary Medicine, Rabat 10101, Morocco
4
Department of Process Engineering and Food Technology, Hassan II Institute of Agronomy and Veterinary Medicine, Rabat 10101, Morocco
5
Laboratory of Applied Organic Chemistry, Faculty of Sciences and Techniques, Sidi Mohamed Ben Abdellah University, Road of Imouzzer, Fez. B.P 2202, Morocco
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(8), 449; https://doi.org/10.3390/fermentation11080449
Submission received: 3 July 2025 / Revised: 26 July 2025 / Accepted: 30 July 2025 / Published: 1 August 2025
(This article belongs to the Section Fermentation for Food and Beverages)
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Abstract

The aim of this study is to evaluate the effects of different types of sourdough (I to IV), developed with a specific starter culture (including Lactiplantibacillus plantarum, Levilactobacillus brevis, and Candida famata), on bread fermentation performance and shelf-life. Real-time tracking of multiple parameters (pH, dough rising, ethanol release, and total titratable acidity) was monitored by a smart fermentation oven. The impact of the different treatments on the lactic acid, acetic acid, and ethanol content of the breads were quantified by high performance liquid chromatography analysis. In addition, the bio-preservation capacity of the breads contaminated with fungi was analyzed. The results show that liquid sourdough (D3: Type 2) and backslopped sourdough (D4: Type 3) increased significantly (p < 0.05) in dough rise, dough acidification (lower pH, higher titratable acidity), production of organic acids (lactic and acetic), and presented the optimal fermentation quotient. These findings were substantiated by chemometric analysis, which successfully clustered the starters based on performance and revealed a strong positive correlation between acetic acid production and dough-rise, highlighting the superior heterofermentative profile of D3 and D4. These types of sourdough also stood out for their antifungal capacity, preventing the visible growth of Aspergillus niger and Penicillium commune for up to 10 days after inoculation.

1. Introduction

Lately, consumers are looking to buy natural products without artificial additives and are becoming more selective and aware of the ingredients in their food. Ref. [1] states that consumers now clearly prefer natural products that are unprocessed and contain no chemical additives. This trend is all the stronger as people become more wary of synthetic food additives, which are often considered potentially hazardous to health [2]. Food products formulated from natural ingredients, including preservatives of organic origin, are increasingly in demand on the market. The global market for natural food preservatives is projected to grow at a compound annual growth rate (CAGR) of 6.8% from 2024 to 2030 [3].
In most countries, bread is the most widely consumed foodstuff. Nonetheless, its production is significantly dependent on artificial additives, including leavening agents and chemical preservatives. This fact has motivated manufacturers and researchers to enhance the manufacturing process, organoleptic and nutritional quality, and, primarily, the shelf life by utilizing exclusively natural products [4,5]. Bread can be made without additives by utilizing a traditional sourdough starter. However, its use remains restricted at the industrial level due to its limited and slow bread-making performance [6,7].
Sourdough is a flour–water mixture fermented by a complex microbial community, including both facultative (e.g., Lactiplantibacillus plantarum) and obligate heterofermentative (e.g., Levilactobacillus brevis) lactic acid bacteria, along with yeasts and, occasionally, acetic acid bacteria [8]. The yeasts are the first to initiate the fermentation and produce carbon dioxide, which is released to ensure the dough rises while the bacteria reduce its acidity [9,10]. Sourdough fermentation gives bread unique sensory characteristics—such as a more complex aroma, a softer crumb, and better preservation—as well as proven nutritional and functional benefits [6,11]. The synergistic effects of extended fermentation duration, microbial metabolic processes, and acidification facilitate the degradation of undesirable substances like phytic acid via enzymatic mechanisms, enhance mineral bioavailability by diminishing chelating agents, and potentially improve digestive tolerance through gluten modification [8,12]. The effects arise from intricate interactions among fermentation duration, microbial composition, and dough matrix conditions.
According to De Vuyst et al. and De Vuyst and Comasio [13,14], sourdoughs are divided into three groups based on the nature of the inoculum used in their preparation: type 1 (spontaneously fermented sourdough with successive refreshments), type 2 (initiated with liquid starter cultures), and type 3 (starter culture followed by backslopping). Also, there are four classifications of sourdough based on their preparation methods: type I (traditional sourdough), type II (sourdough utilizing a starter culture), type III (dried sourdough), and type IV (mixed dried) [14,15].
The impact of sourdough type on the organoleptic and antifungal characteristics of the bread is primarily associated with its abundance of heterofermentative lactic acid bacteria, particularly L. plantarum and L. hammesii, which generate substantial amounts of organic acid, specifically acetic and lactic acids. This indicates the establishment of a detrimental environment for the germination and growth of molds for several days [16,17]. Nonetheless, the enhancement of the sensory attributes of the bread, its preservation, and the stability of the final product are contingent upon the proportion of sourdough incorporation and its microbial diversity [18,19].
Aspergillus and Penicillium molds are a major threat to breads’ shelf life. The filament production by these molds accelerates the spoilage of bread within a brief period, presenting significant challenges for bakers and, particularly, the baking industry. Enhancing the microbial flora composition in sourdoughs is a primary focus for researchers in food formulation microbiology. Researchers are especially focused on lactic acid bacteria strains such as Lactiplantibacillus plantarum, Lacticaseibacillus casei, and Lactobacillus acidophilus due to their ability to generate compounds that resist fungi and enhance the acidity of the fermentation process. In this context, various studies [20,21,22] have demonstrated that sourdoughs derived from lactic bacteria can mitigate this issue and function as antifungal agents to prolong bread shelf life.
Although yeast contributes to food preservation by generating extracellular and intracellular antibacterial compounds, toxins, ethanol, and organic acids [23], the antifungal properties of sourdough derived from a starter culture of lactic acid bacteria and selected yeasts remain inadequately researched. However, ref. [24] found that adding a starter culture of lactic acid bacteria along with commercial yeast (Saccharomyces cerevisiae) extended the shelf life of bread.
Despite being less prevalent than other yeasts typically found in traditional sourdoughs, Candida famata can coexist with lactic acid bacteria and endure acidic conditions. Though being less fermentatively active than Saccharomyces cerevisiae, Candida famata exhibits moderate antifungal properties and is significant for the long-term stability of sourdough [22].
The combination of two strains of Lactiplantibacillus plantarum NRRL B-14768T and Levilactobacillus brevis ATCC 14869 and the yeast, Candida famata, has already been tested for the formulation of a bio-organic fertilizer. The resulting formulation was able to preserve the biofertilizer for up to two years. Also, its use in an agricultural trial showed an improvement in the growth parameters and nutritional quality of red beet compared with chemical fertilizer [25]. From this perspective, using this combination in bread fermentation may produce intriguing results. This study is, as far as we know, the first to combine Lactiplantibacillus plantarum NRRL B-14768T, Levilactobacillus brevis ATCC 14869, and Candida famata in Type II (liquid starter), Type III (backslopped starter), and Type IV (lyophilized mixed starter) sourdoughs, to help to improve fermentation efficiency and preservation for industrial use.
The main goal of this study is to examine the impact of various starter types (I to IV) derived from a novel starter culture combining the heterofermentative LAB strains Lactiplantibacillus plantarum NRRL B-14768T and Levilactobacillus brevis ATCC 14869 with the acid-tolerant yeast Candida famata on the efficacy of fermentation. This consortium is specifically selected to enhance acetic acid production (associated with antifungal properties) while preserving dough-rise capability—a balance not previously attained in earlier research. Additionally, we utilize real-time fermentation monitoring (Panigraph) to continuously observe fermentation performance, thereby addressing a significant deficiency in comprehending the kinetic relationships between microbial activity and bread quality.

2. Materials and Methods

2.1. Preparation of the Starter Culture

The starter culture includes two types of bacteria, Lactiplantibacillus plantarum NRRL B-14768T and Levilactobacillus brevis ATCC 14869, as well as the yeast Candida famata. The LAB strains were previously isolated from traditional fermented foods and identified through phenotypic tests, including morphology, carbohydrate fermentation via API 50 CHL, and acidification capacity. Genotypic confirmation through 16S rRNA sequencing (GenBank accessions: MW494522 for L. plantarum, MW494520/MW494521 for L. brevis) demonstrated 99.79–99.93% similarity to type strains. Candida famata was identified via ITS sequencing and physiological assessments (acid tolerance, growth kinetics) [26].
Strains were subsequently preserved in sterile 30% glycerol at −70 °C. Prior to the bread-making trials, lactic acid bacteria (LAB) were reactivated in MRS broth at 37 °C for 24 h under anaerobic conditions at concentration of 4.5 × 108 CFU per ml, while the yeast strain was revitalized in YPD broth for 48 h at 30 °C under aerobic conditions at a concentration of 1.2 × 107 CFU per ml. Each strain was isolated from the growth broth utilizing an ALC PK 120 centrifuge. The cells were washed and re-suspended in isotonic saline (0.85% NaCl) until their optical density attained 2.00 at 650 nm for yeast and 540 nm for LAB; cells at this stage are designated as “C1”.

2.2. Preparation of Traditional Sourdough (Type 1)

Traditional type 1 sourdough (PL4) was produced using regional artisanal techniques, comprising 56% soft wheat flour, 30% water, 12.5% apple pulp, and 1.5% honey. The apple pulp contributes extra sugars and microbiota, yet all comparative doughs (D1–D5) preserved uniform flour–water ratios in the final mixture (Table 1), thereby ensuring that leavening performance was predominantly assessed based on microbial activity rather than substrate variations. The mixture was refreshed daily (10% inoculum transferred to fresh substrate) at 30 °C for 7 days until total titratable acidity (TTA) stabilized (±0.1 mL NaOH 0.1 N/10 g dough) [27,28].

2.3. Preparation of the Liquid Sourdough (Type 2)

To prepare sourdough type 2 “PS,” a formulation consists of 38.5% T55 flour, 1.5% honey, and 60% starter culture (comprises 15% of each lactic acid bacteria preparation “C1” at a concentration of 4.5 × 108 CFU per ml and 30% (v/w) C. famata suspension at a concentration of 1.2 × 107 CFU per mL). Anaerobic fermentation was conducted at 30 °C for 24 h following the preparation of the liquid sourdough.

2.4. Preparation of the Liquid Backslopped Sourdough (Type 3)

The backslopped sourdough (PLS, type 3) was produced by first fermenting a type 2 starter culture (38.5% T55 flour, 1.5% honey, 15% of each lactic acid bacteria preparation “C1” at a concentration of 4.5 × 108 CFU per ml and 30% C. famata suspension at a concentration of 1.2 × 107 CFU per ml), anaerobically at 30 °C for 24 h, followed by daily backslopping (20% inoculum transfer to fresh medium) for 7 days until metabolic stabilization was achieved (pH variation < 0.1, TTA variation < 5% between cycles). The final microbial loads were confirmed at 108–109 CFU/g for lactic acid bacteria and 106–107 CFU/g for yeast prior to utilization.

2.5. Preparation of the Lyophilized Inoculum for Sourdough (Type IV)

According to the classification of sourdoughs based on the technological process, a combination of traditional sourdough with freeze-dried starter culture is categorized as type IV sourdough [15]. The lyophilized starter (PL4LYO) was prepared as follows: * Starter Culture Reactivation: Lactiplantibacillus plantarum and Levilactobacillus brevis were cultured in MRS broth (37 °C, 24 h, anaerobic), while Candida famata was grown in YPD broth (30 °C, 48 h, aerobic). Cells were harvested by centrifugation (10,000 rpm, 10 min), washed with 0.85% NaCl, and resuspended to OD~540 nm (LAB) or OD~650 nm (yeast) = 2.0 (“C1” suspension). The “C1” suspension was frozen at −18 °C for 12 h, then freeze-dried (SCIENTZ-12N, 50 °C, 150 μPa, 24 h). Lyophilized powder was stored at −70 °C until use. Sourdough Inoculation: For PL4LYO dough, lyophilized culture included C. famata at 0.5% (w/w) of total starter and 1% (w/w) of Lactiplantibacillus plantarum and Levilactobacillus brevis, was rehydrated in 20% traditional sourdough (PL4, Section 2.2), and was incubated at 30 °C for 2 h prior to bread dough preparation [29].

2.6. Doughs Preparation

The bread was produced as outlined by [30] with some modifications. The composition of the doughs used for fermentation tests (per 100 g) is presented in (Table 1) and, for each trial, 20% of the dough weight is substituted with each type of sourdough, excluding the control. Doughs D3, D4, and D5 incorporate a starter culture comprising both lactic acid bacteria, Lactiplantibacillus plantarum NRRL B-14768T, and Levilactobacillus brevis ATCC 14869, as well as the yeast Candida famata.
All doughs were manually kneaded for 5 min using a standardized technique (approximately 60 strokes/min with consistent fold-press-rotate motions) in stainless steel bowls maintained at 22 ± 2 °C, until gluten development was confirmed by windowpane testing, according to AACC Method 54-50.01. This duration was selected based on preliminary validation trials showing comparable water absorption (70–75% flour basis) and extensibility properties from machine-kneaded control [31], while maintaining traditional artisanal processing conditions [32].

2.7. Fermentation Parameters

Fermentation parameters such as dough rise (cm) and pH were monitored in real time using a device for monitoring dough fermentation, “panigraph”, developed by [32] (patent application no. MA57946).
The fermentation procedure consisted of three stages:
  • Sourdough fermentation: Each sourdough type (I–IV) was fermented for 24 h at 30 °C prior to dough preparation, with pH and TTA monitored hourly until stabilization (ΔpH < 0.1 over 2 h).
  • Dough fermentation: All doughs (containing 20% sourdough) were fermented at 30 °C until peak dough rise (height plateau) and stable pH (<0.05 change over 30 min), as monitored by Panigraph. Fermentation time varied based on sourdough type (Table 2), and was terminated when the above endpoint criteria were met.
  • Post-fermentation: Dough was then formed into round loaves and allowed to ferment for an additional 30 min at 40 °C before being baked in an electric oven (Itimat, Istanbul, Turkey) at 220 °C for about 15 min (Figure S1).

2.8. Total Titratable Acidity “TTA”

To evaluate total titratable acidity (TTA) prior to, and at, the end of dough fermentation, a 0.1 M NaOH solution is prepared to titrate a mixture consisting of a 2 g sample diluted in 20 mL of distilled water, with the addition of 3 to 4 drops of the color indicator phenolphthalein [31].

2.9. Analysis of Organic Acids and Ethanol

The concentrations of organic acids (lactic acid and acetic acid) and ethanol were quantified using an Agilent 1110 series HPLC (Agilent Technologies, Santa Clara, CA, USA). Initially, samples were collected and centrifuged at 10,000 rpm for 10 min, after which the supernatant was filtered through a CHROMAFIL Xtra polyamide (Nylon) membrane (0.2 μm, Merck Millipore, Darmstadt, Germany). Subsequently, HPLC analyses were conducted utilizing an Agilent 1110 series HPLC, fitted with a Supelcogel C610H column (300 × 7.8 mm, 5 µm; Merck KGaA, Darmstadt, Germany) with an Agilent 1260 Infinity RID detector Agilent Technologies, Santa Clara, CA, USA). A mobile phase comprising 0.1% H3PO4 (in Milli-Q water) was employed at a flow rate of 0.5 mL/min. The analysis was conducted in 30 min and performed at a maximum pressure of 60 bar [33]. The assay results were presented as a percentage (g/kg). The standard curve was constructed with standard organic acid solution (0–7.5 g/kg) (Figure 1). The fermentation quotient (FQ) for different sourdough types was calculated as the molar ratio of lactic acid to acetic acid.

2.10. Bio-Preservation of Breads

Following the methodology established by [30], the bio-preservation effectiveness of breads produced with various types of sourdough was examined. Prior to baking, the bread was cooled and sliced within a sterile environment. Each slice was inoculated with 10 μL of pure cultures of the fungal species Aspergillus niger and Penicillium commune. Samples were preserved in petri dishes and incubated at 25 °C for a duration of 10 days. The duration of bio-preservation was evaluated following the initial manifestation of fungal growth on the bread slices.

2.11. Multivariate Analysis

Multivariate analysis, including Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA), was conducted to assess the relationships between fermentation parameters. PCA served to reduce dimensionality and highlight key variations within the dataset [34], while HCA grouped samples with similar extraction profiles based on their measured characteristics, as illustrated by a dendrogram. Together, PCA and HCA provided insights into the complex interactions governing the extraction process, facilitating the identification of dominant factors for further optimization. Multivariate analysis was carried out using JMP software (version 17 PRO). The multivariate analysis dataset comprised five sourdough starters (B1–B5) assessed across seven parameters: fermentation time, dough-rise, pH change (%), total titratable acidity (ATT) change (%), lactic acid change (%), acetic acid change (%), and ethanol change (%). Most variables were expressed as percentage changes from initial values, calculated as [(post-fermentation value − pre-fermentation value)/pre-fermentation value] × 100. However, pH was treated as an exception, with absolute final values used instead to avoid negative results from its consistent decline during fermentation.

2.12. Statistical Analysis

To assess notable differences among trials, one-way ANOVA and Tukey’s post hoc test were conducted at a significance level of 5 percent (p < 0.05). Statistical analyses were performed using IBM SPSS STATISTICS (25th edition, Armonk, NY, USA). Results were presented as the mean of three repetitions ± standard deviation. Graphs were generated using the software GRAPHPAD Prism 9 (Version 9.3.1; GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Fermentation Parameters and TTA

The findings regarding the impact of sourdough type on dough produced at the end of the fermentation process are illustrated in (Table 2). Fermentation duration ranged from 664 min (D1, yeast-only control) to 1214 min (D5, freeze-dried starter), reflecting metabolic rates of the respective cultures. Despite time differences, all doughs (excluding control) reached comparable endpoint criteria (pH ≤ 3.9, TTA ≥ 10 mL NaOH), ensuring valid comparisons.
According to the results in (Table 2), the type of sourdough had a significant effect on dough leavening, with averages ranging from 3.68 to 5.52 cm. The rise of the PSC control dough (3.74 cm) was not significantly different (p > 0.05) from that of the freeze-dried sourdough dough PL4LYO (3.68 cm). Nonetheless, both doughs showed a reduced leavening in comparison to the other types.
The rise of dough D4, fermented with type 3 sourdough PLS (5.52 cm) was significantly (p < 0.05) higher than that of dough made with type 1 sourdough PL4 (4.54 cm) and dough made with type 2 sourdough PS (4.13 cm) (Figure 2).
As shown in Table 2, doughs containing a specific type of sourdough reduce pH (values below 4) and increase acidic titration values. However, the initial pH values of the different doughs were similar in D2 and D4. D3 and the dough control demonstrated the highest initial pH. On the other hand, final pH was similar in D2, D4, and D5, and the lowest compared to D1 and D3. The control dough exhibited minimal pH variation. The pH variation of doughs D2, D4, and D5, which were obtained from sourdough types I, III, and IV, respectively, was not statistically significant and showed less variation than that of sourdough type 2 (1.11).
At the end of fermentation, the dough, D4, with the highest acidity variation (16 mL NaOH per 10 g dough after 740 min) was fermented with type 3 sourdough PLS, followed by dough D3 (13.5 mL NaOH per 10 g dough after 940 min). The control dough exhibited the lowest initial and final acidity among the samples (0.8 mL NaOH per 10 g dough after 664 min). The results indicated no significant difference in pH and TTA variation between doughs D2 (fermented exclusively by traditional sourdough) and D5 (utilizing the same sourdough and freeze-dried starter culture) (Table 2). The findings suggest that incorporating a freeze-dried starter culture into traditional sourdough does not enhance its metabolic activity.
Fermentation durations differed among sourdough varieties (Table 2), indicating inherent metabolic distinctions among starters. Type 3 sourdough (D4) attained the most significant dough rise and acidity in the briefest duration (740 min), demonstrating that daily backslopping enhanced microbial synergy, thereby expediting acidification and rise. In contrast, the prolonged duration for D5 (1214 min) corresponds with the diminished viability of freeze-dried cells, highlighting the compromises inherent in starter stabilization techniques. All endpoints were meticulously regulated (pH, TTA, dough rise), guaranteeing that the measured properties were comparable and biologically significant.

3.2. Analysis of Organic Acids and Ethanol

Prior to fermentation, all sourdough-based doughs showed varying lactic acid values, except for the control, where the value was almost zero (Figure 3A). The varying proportions and types of sourdoughs added may contribute to the presence of these quantities in the doughs before fermentation begins. Only doughs D3 and D4 recorded the presence of acetic acid, with a higher value for dough based on type 2 sourdough (D3) (Figure 3B).
The ethanol produced from the D1 control dough (which was fermented only by S. cerevisiae yeast) was twice as much as that from D2 and D5 doughs, and over 6 times more than D3 and D4 doughs, reaching a level of 2.22 ± 0.14% after 20 h of fermentation (Figure 3C). It can also be noted that there is no significant difference in the variation of ethanol production between doughs based on the fresh starter cultures D3 and D4, with low values not exceeding 0.23 ± 0.19 and 0.28 ± 0.02, respectively.
(Table 3) showed a significant difference between the variation in acid production of dough D3 (which demonstrated the highest variation throughout the fermentation period) and the other dough types. There was no significant difference between control D1 and D4, which showed the lowest variation.

3.3. Multivariate Analysis and Correlation Study

Principal Component Analysis (PCA), CHA, and a correlation study were conducted utilizing the percentage change values of the bread-making parameters presented in Table 4.
To elucidate the complex relationships between the five starter cultures and their fermentation performance, a multivariate analysis was conducted as shown in Figure 4.
Principal Component Analysis (PCA) successfully captured 91.4% of the total variance within the first two components (PC1: 50.2%, PC2: 41.2%), providing a robust model for interpretation. The loading plot (Figure 4A) revealed that PC1 separated the starters based on their dominant metabolic pathway, contrasting a heterofermentative profile (positively loaded with dough-rise, acetic acid, and ATT) against a homofermentative/ethanolic profile (negatively loaded with lactic acid, ethanol, and time of fermentation). PC2 represented the overall fermentation vigor, with high production of key metabolites (lactic acid, acetic acid, ethanol, and dough-rise) loading positively and indicators of inefficiency (long fermentation times) loading negatively. Figure 4B shows that the PCA score plot clearly discriminated the performance of the starters, positioning B4 as the most potent and efficient starter in the top-right quadrant, distinguished by its superior dough-rise (5.52 cm) and the highest production of acetic acid (1723.47%), which is crucial for bio-preservation. In contrast, B1 (top-left quadrant) typified a classic homofermentative/yeast profile, yielding the highest level of lactic acid (978.05%) and high ethanol but no acetic acid. Starter B3 showed a similar, though less potent, heterofermentative profile for B4. Conversely, starters B2 and B5 were identified as inefficient, characterized by their position in the lower half of the plot, reflecting their prolonged fermentation times and comparatively poor metabolite production and leavening capacity. These differentiations were strongly corroborated by the correlation heat map matrix (Figure 4D), which showed a significant positive correlation between dough-rise and acetic acid and a negative correlation between time of fermentation and dough-rise, quantitatively confirming that the most effective leavening was achieved by efficient heterofermenters. Furthermore, Hierarchical Cluster Analysis (Figure 4C) reinforced these findings by revealing two primary and distinct super-clusters. The first cluster groups B2 and B5 together, characterized by their shared profile of low overall effectiveness. The heatmap shows that both are defined by long fermentation times (red/orange cells) but exhibit low values (blue cells) for key performance indicators such as dough-rise and the production of lactic acid, acetic acid, and ethanol. This confirms their identity as slow and inefficient starters. The second, more effective super-cluster consists of B1, B3, and B4, but it further subdivides to highlight their distinct metabolic profiles. Starters B3 and B4 are grouped most closely, reflecting their shared identity as potent heterofermenters, both displaying high values (red cells) for dough-rise and acetic acid. Baker yeast B1 joins this pair at a greater distance, indicating it is also an effective starter but with a fundamentally different profile, distinguished by its high production of lactic acid and ethanol, and a lack of acetic acid. Collectively, this analysis demonstrates that the choice of starter culture has a profound impact on fermentation outcomes, with starter B4 being optimal for producing bread with both strong leavening and enhanced bio-preservative potential.

3.4. Shelf Life Improvement

The pieces of bread from the control inoculated with A. niger and P. commune showed the appearance of the first fungal growth after only 3 and 2 days, respectively (Table 4).
The breads produced with type II and type III sourdoughs exhibited no visible signs of moisture throughout the 10-day observation period, signifying the longest preservation duration among all tested treatments (Figure 5). In addition, the shelf life of the bread is ensured by trials based on traditional sourdough alone (B2) or combined with starter culture (B5), which were able to improve the shelf life of the bread pieces compared to the control (Table 5).

4. Discussion

The reduced leavening rate of type IV sourdoughs, in comparison to other sourdough types, is mainly due to the inactivation of bacterial strains resulting from the freeze-drying process, which compromises cell integrity and diminishes strain vitality [35].
All of the breads were made with the same proportion and type of flour, so the difference in leavening at the end of fermentation was primarily due to the type of microorganisms present in the dough. This finding is supported by [36], who found that doughs incorporating a pure culture of lactic acid bacteria and yeasts leaven better than doughs made with traditional sourdoughs due to their increased activity. Furthermore, ref. [14] asserted that the inclusion of heterofermentative LAB species (Levilactobacillus brevis) within the starter culture, along with the practice of daily backslopping, significantly improves the leavening of dough throughout the fermentation process.
Ref. [37] demonstrated that bread fermentation using a single selected yeast strain was less efficient than the baker’s yeast used in the study. The combined use of the selected yeast strain with lactic acid bacteria strains in the present research improved dough leavening capacity. The findings are supported by [38] who showed that combining yeast with lactic acid bacteria and a starter culture improves the fermentation process. Also, the combination of LAB (L. plantarum, L. brevis) with yeast in the presence of a carbon source increases lactic acid production at the end of fermentation [39].
The key difference among the different sourdough types examined in this study lies in the capacity of microbial strains to increase titratable acidity or reduce pH during a specified incubation duration. Type III sourdough is characterized by its capacity for an extended fermentation of the starter mixture, initiated through the addition of specific strains of lactic acid bacteria, primarily aimed at achieving the rapid acidification of the dough and the development of aromas. These factors may explain the high final TTA of the B4 dough at a reduced breadmaking time (740 min) compared with the other types of sourdough tested [40].
As stated by [41], the fast growth of yeasts does not account for the acidification of the dough, which is primarily due to the synthesis of lactic and acetic acids by LAB strains. This occurrence elucidates the minor decrease in pH and TTA in the control dough, predominantly resulting from the production of succinic acid [13].
The composition of the starter culture in two heterofermentative bacteria (L. plantarum and L. brevis), known for their ability to produce components other than lactic acid, notably acetic acid and ethanol, may explain the detection of acetic acid in type 2 and 3 sourdough doughs (D3 and D4). Also, ref. [42] found a correlation between acetic acid production and the presence of Candida humilis yeast. This study may explain the acetic acid levels recorded only in D3 and D4 doughs (which contain the starter culture) and which contain a selected Candida famata yeast strain.
To our knowledge, this is the first study to correlate real-time fermentation parameters (via Panigraph) with chemometric clustering of starter performance. This approach revealed a strong positive correlation between acetic acid and dough-rise (Figure 4D), underscoring the heterofermentative advantage of our consortium. Traditional methods relying on endpoint measurements [31,43] could not capture these dynamic relationships, which are critical for industrial process optimization.
The D3 and D4 doughs produced fermentation quotients (FQ) ranging from 1.5 to 4, ensuring optimal sourdough production and improving the aroma and shelf life of the final product [44,45]. Several studies have reported fermentation quotient values that exceed those obtained in this study. This increase can be explained by the prevalence of homofermentative and facultative heterofermentative LAB [43,46]. Additionally, the strain Lactiplantibacillus plantarum produced higher levels of acetate compared to the mixed LAB cultures [47].
The degradation of the starch present in the flours by the yeasts mainly engenders the production of ethanol and carbon dioxide (CO2). Commercial yeasts like S. cerevisiae accelerate this mechanism. In contrast, yeasts that are indigenous to traditional sourdough exhibit slower activity as they adapt to the acidic environment created by bacteria [24]. However, the low levels of ethanol in doughs D3 and D4 (Table 3) can be explained by the oxidation of the ethanol produced into acetic acid by yeasts [9,48] and acetic bacteria [49].
The estimated shelf life of the bread, following the application of type II and III sourdoughs from this study, surpasses the findings of numerous studies that have attempted to prolong bread shelf life exclusively with sourdoughs. However, the research conducted by [30] indicated that optimal conservation performance was attainable solely with the inclusion of the synthetic additive, calcium propionate. Notably, the antifungal capacity of our starter culture surpassed that of [50], who reported mold growth after 6 days despite using three LAB strains, highlighting the importance of strain selection and consortium design. Conversely [20] employed a poolish-type sourdough supplemented with a singular lactic bacterial strain, Lactiplantibacillus plantarum, to prolong the shelf life to 10 days for only 50% of the bread samples.
Lactic acid bacteria are known for their ability to inhibit fungal growths, specifically those that develop on bread. Ref. [51] found that among 12 strains of LAB, the species L. brevis has the most potent inhibitory effect against A. niger. This ability is explained by its belonging to the group of obligate heterofermentative LAB, which are known for their highly advanced antifungal activity due to the synergistic effect of several acids produced during fermentation [52]. Shortly thereafter, ref. [24] showed that acetic acid and phenyllactic acid have an inhibitory effect (MIC50) on the germination of conidia of three species of molds: Penicillium sp., Fusarium graminearum, and Aspergillus niger at pH 3.5 and 6.0, which is more significant than lactic acid. It can be concluded that lactic acid is responsible for the acidification of the medium, due to high levels (Figure 3A), while acetic acid plays a key role in preserving against fungal growth. Also, ref. [23] has shown that the synergy between yeast and LAB inhibits mold proliferation for up to 14 days due to yeast production of ethanol and acid mycotoxins.
The production of antifungal substances by the starter culture of LAB and yeast may be linked to their predominance following the direct inoculation of strains into the dough (B3) or after several backslopings of the sourdough (B4). This process contributed to the stability of the breads made from type 2 and 3 starters. Moreover, this combination of LAB and yeast generates better quantities of acetic acid than yeast extract alone [53].
This study is the first to systematically assess the combined efficacy of these strains in Type II–IV sourdoughs for industrial bread production, despite prior research examining their individual or paired applications in other contexts, such as biofertilizers [25] or traditional sourdoughs [8,13]. The exceptional efficacy of Type II and III sourdoughs in leavening and bio-preservation highlights the capability of this consortium to substitute synthetic additives in large-scale applications.

5. Limitations

This study demonstrates significant advancements in fermentation efficiency and bio-preservation; however, certain issues warrant attention. In the absence of sensory evaluation, the extent of individuals’ preference for high-acidity breads (pH ≤ 3.9) remains unknown. This is particularly accurate as [54] demonstrated that excessive sourness can diminish the palatability of food. [55] indicates that scaling effects can alter microbial and dough behavior, rendering laboratory-scale results potentially inapplicable to industrial production. [56] emphasizes that the efficacy of starter cultures may vary depending on the quality of the ingredients and their processing methods. This indicates that our specialized microbial consortium may behave differently in conventional bakery environments. Subsequent research should address these deficiencies through sensory evaluations, pilot-scale validation, and exploring alternative mechanisms of food spoilage beyond mold inhibition.

6. Conclusions

Our finding indicates that the combination of heterofermentative lactic acid bacteria (L. plantarum, L. brevis) and Candida famata yeast in type 2 (liquid starter) and type 3 (backslopped) sourdoughs significantly improved fermentation efficacy and bio-preservation relative to traditional (Type I) and freeze-dried (Type IV) variants. Type III sourdough attained the greatest dough rise (5.52 cm) and the most rapid acidification (740 min), concurrently generating optimal concentrations of acetic acid (0.28%) for antifungal efficacy. Both Type II and III sourdoughs effectively inhibited the growth of Aspergillus niger and Penicillium commune for 10 days, confirming their potential as natural preservatives. The diminished efficacy of Type IV underscores the necessity for enhanced lyophilization protocols. These results endorse the industrial implementation of specific starter cultures as substitutes for chemical additives; however, subsequent research must examine scalability and sensory compromises linked to heightened acidity.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11080449/s1, Figure S1: Dough and bread production process.

Author Contributions

Conceptualization, A.R. and K.A.; methodology, M.F. and K.A.; software, Y.C.; validation, M.F. and A.R.; formal analysis, K.A.; writing—original draft preparation, S.L. and K.A.; writing—review and editing, K.A. and K.E.K.; supervision, M.O. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Acknowledgments

We express our gratitude to Anas Raffak for granting us access to his laboratory to conduct the practical part of this study. We would like to thank Ghita Almourid for her assistance in conducting this experiment. The authors have reviewed and edited the submitted manuscript and are fully responsible for the published content.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AAAcetic acid
AACCAmerican Association of Cereal Chemists
FQFermentation quotient
LALactic acid
LABLactic acid bacteria
TTATotal titratable acidity

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Fermentation 11 00449 g001
Figure 1. Chromatogram of an aqueous standard solution with a concentration of 7.5 g/kg (%) of the various compounds (lactic acid, acetic acid, and ethanol).
Figure 1. Chromatogram of an aqueous standard solution with a concentration of 7.5 g/kg (%) of the various compounds (lactic acid, acetic acid, and ethanol).
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Figure 2. Evolution of dough rise during fermentation of the various tested doughs.
Figure 2. Evolution of dough rise during fermentation of the various tested doughs.
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Figure 3. Comparison of the content of lactic acid (A), acetic acid (B), and ethanol (C) before and after the fermentation for each dough.
Figure 3. Comparison of the content of lactic acid (A), acetic acid (B), and ethanol (C) before and after the fermentation for each dough.
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Figure 4. Multivariate statistical analysis of fermentation parameters for five different sourdough starters (B1–B5) with (A) Principal Component Analysis (PCA) loading plot; (B) PCA score plot; (C) Two-way Hierarchical Cluster Analysis (CHA) heatmap; (D) Correlation heatmap matrix.
Figure 4. Multivariate statistical analysis of fermentation parameters for five different sourdough starters (B1–B5) with (A) Principal Component Analysis (PCA) loading plot; (B) PCA score plot; (C) Two-way Hierarchical Cluster Analysis (CHA) heatmap; (D) Correlation heatmap matrix.
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Figure 5. Fungal growth on the control loaves of bread (B1) and bread of different type of sourdough. (B1): Bread control with only Saccharomyces cerevisiae; (B2): traditional sourdough bread control (type 1); (B3): liquid sourdough bread (type 2); (B4): bread with liquid sourdough backslopped for seven successive days (type 3); (B5): bread with apple sourdough and 1.5% of lyophilized starter (Type IV).
Figure 5. Fungal growth on the control loaves of bread (B1) and bread of different type of sourdough. (B1): Bread control with only Saccharomyces cerevisiae; (B2): traditional sourdough bread control (type 1); (B3): liquid sourdough bread (type 2); (B4): bread with liquid sourdough backslopped for seven successive days (type 3); (B5): bread with apple sourdough and 1.5% of lyophilized starter (Type IV).
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Table 1. Composition of doughs in grams of ingredients per 100 g of product.
Table 1. Composition of doughs in grams of ingredients per 100 g of product.
Dough CodeSourdough CodeSourdough TypeDurum Wheat Flour (g)Soft Wheat Flour (g)Water (g)Salt (g)Sugar (g)Baking Yeast (g)Sourdough (g)
D1PSCType 030.0030.0033.701.302.003.000.00
D2PL4Type I30.0030.0026.701.300.000.0020
D3PSType II30.0030.0016.701.302.000.0020
D4PLSType III30.0030.0018.701.300.000.0020
D5PL4LYOType IV30.0030.0018.701.300.00.0020
D1: Bread control with only Saccharomyces cerevisiae; D2: apple sourdough bread control (type 1); D3: liquid sourdough bread (type 2); D4: bread with liquid sourdough backslopped for seven successive days (type 3); D5: bread with apple sourdough and 1.5% of lyophilized starter (Type IV).
Table 2. Properties of the produced dough at the end of bread-making process.
Table 2. Properties of the produced dough at the end of bread-making process.
Dough CodeSourdough CodeFermentation Time (min)Leavening Capacity (cm)pHTTA (ml NaOH 0.1 N (10 g)−1)
Initial pHFinal pHVariationInitial TTAFinal TTAVariation
D1PSC664 ± 21.28 a3.74 ± 2.15 a4.924.740.18 ± 0.071.220.8
D2PL41087 ± 20.42 d4.54 ± 0.08 c4.673.690.53 ± 0.093.5128.5
D3PS940 ± 38.30 c4.13 ± 0.13 b5.033.921.11 ± 0.093.513.510
D4PLS740 ± 13.87 b5.52 ± 0.08 d4.723.690.50 ± 0.144.51611.5
D5PL4LYO1214 ± 16.52 e3.68 ± 0.11 a4.333.720.50 ± 0.053.5117.5
Data are presented as mean ± standard deviation, with different lowercase letters in the same column indicating significant differences at p < 0.05. Fermentation time = duration to reach peak dough rise and pH stabilization.
Table 3. Variation of the lactic acid, acetic acid, and ethanol production for the tested doughs during 20 h of fermentation and the fermentation quotient FQ.
Table 3. Variation of the lactic acid, acetic acid, and ethanol production for the tested doughs during 20 h of fermentation and the fermentation quotient FQ.
Dough TypeΔ Lactic Acid (%)Δ Acetic Acid (%)Δ Ethanol (%)Fermentation Quotient FQ
D10.76 ± 0.08 an.d.2.12 ± 0.03 dn.d.
D20.74 ± 0.01 an.d.0.43 ± 0.04 bn.d.
D31.03 ± 0.09 c0.43 ± 0.03 a0.23 ± 0.19 a2.40
D40.92 ± 0.02 b0.28 ± 0.04 b0.28 ± 0.02 a3.29
D50.91 ± 0.05 bn.d.0.60 ± 0.05 cn.d.
Mean ± standard deviation (n = 3). Different superscript letters within columns indicate significant differences (p < 0.05). n.d. = not detected. FQ = molar ratio of lactic acid to acetic acid. Optimal range: 1.5–4.0.
Table 4. Fermentation performance indicators of sourdoughs prepared with four starter cultures (D2–D5) and the control (D1). Values correspond to the fermentation time (min), final dough rise (cm), final absolute pH, and the percentage change (%) for Total Titratable Acidity (ATT), lactic acid, acetic acid, and ethanol.
Table 4. Fermentation performance indicators of sourdoughs prepared with four starter cultures (D2–D5) and the control (D1). Values correspond to the fermentation time (min), final dough rise (cm), final absolute pH, and the percentage change (%) for Total Titratable Acidity (ATT), lactic acid, acetic acid, and ethanol.
Time of Fermentation (min)Dough-Rise (cm)pH Change (%)ATT Change (%)Lactic Acid Change (%)Acetic Acid Change (%)Ethanol Change (%)
D16643.743.6662.16978.050.001967.13
D210874.5420.99241.5195.910.0066.17
D39404.1222.07282.08123.23448.852184.87
D4740.675.5221.88255.56408.241723.472148.12
D512143.6814.22213.21104.670.00170.25
Table 5. Appearance of fungal growth on different types of bread contaminated with spores of A. niger and P. commune.
Table 5. Appearance of fungal growth on different types of bread contaminated with spores of A. niger and P. commune.
BreadsFungiAppearance of Fungal Growth (Days)
12345678910
B1A. niger---+++++++
B2-----+++++
B3----------
B4----------
B5------++++
B1P. commune--++++++++
B2----++++++
B3----------
B4----------
B5-----+++++
B1: Bread control with only Saccharomyces cerevisiae; B2: traditional sourdough bread control (type 1); B3: liquid sourdough bread (type 2); B4: bread with liquid sourdough backslopped for seven successive days (type 3); B5: bread with apple sourdough and 1.5% of lyophilized starter (Type IV).
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MDPI and ACS Style

Atfaoui, K.; Lebrazi, S.; Raffak, A.; Chafai, Y.; Kabous, K.E.; Fadil, M.; Ouhssine, M. Impact of Selected Starter-Based Sourdough Types on Fermentation Performance and Bio-Preservation of Bread. Fermentation 2025, 11, 449. https://doi.org/10.3390/fermentation11080449

AMA Style

Atfaoui K, Lebrazi S, Raffak A, Chafai Y, Kabous KE, Fadil M, Ouhssine M. Impact of Selected Starter-Based Sourdough Types on Fermentation Performance and Bio-Preservation of Bread. Fermentation. 2025; 11(8):449. https://doi.org/10.3390/fermentation11080449

Chicago/Turabian Style

Atfaoui, Khadija, Sara Lebrazi, Anas Raffak, Youssef Chafai, Karima El Kabous, Mouhcine Fadil, and Mohammed Ouhssine. 2025. "Impact of Selected Starter-Based Sourdough Types on Fermentation Performance and Bio-Preservation of Bread" Fermentation 11, no. 8: 449. https://doi.org/10.3390/fermentation11080449

APA Style

Atfaoui, K., Lebrazi, S., Raffak, A., Chafai, Y., Kabous, K. E., Fadil, M., & Ouhssine, M. (2025). Impact of Selected Starter-Based Sourdough Types on Fermentation Performance and Bio-Preservation of Bread. Fermentation, 11(8), 449. https://doi.org/10.3390/fermentation11080449

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