Next Article in Journal
An Engineered Multi-Enzyme Cascade with Low-Cost ATP Regeneration for Efficient D-Allulose Production from D-Fructose
Previous Article in Journal
Utilization of Non-Saccharomyces to Address Contemporary Winemaking Challenges: Species Characteristics and Strain Diversity
Previous Article in Special Issue
Smart and Functional Probiotic Microorganisms: Emerging Roles in Health-Oriented Fermentation
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Fermentation
Volume 11
Issue 12
10.3390/fermentation11120666
fermentation-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Nutritional and Physicochemical Attributes of Sourdough Breads Fermented with a Novel Pediococcus acidilactici ORE 5 Strain

by
Ioanna Mantzourani
1,
Athanasios Alexopoulos
2,
Gregoria Mitropoulou
3,
Yiannis Kourkoutas
3 and
Stavros Plessas
2,*
1
Department of Chemistry, University of Patras, 26504 Patras, Greece
2
Laboratory of Microbiology, Biotechnology and Hygiene, Department of Agricultural Development, Democritus University of Thrace, 68200 Orestiada, Greece
3
Laboratory of Applied Microbiology and Biotechnology, Department of Molecular Biology & Genetics, Democritus University of Thrace, 68100 Alexandroupolis, Greece
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(12), 666; https://doi.org/10.3390/fermentation11120666
Submission received: 10 October 2025 / Revised: 19 November 2025 / Accepted: 26 November 2025 / Published: 28 November 2025
(This article belongs to the Special Issue Emerging Microbial Technologies in Fermentation: Innovations in Food, Environmental, and Health Bioprocesses)
Browse Figures
Versions Notes

Abstract

This study evaluated the application of Pediococcus acidilactici ORE 5 (applied in free or immobilized form in a prebiotic matrix called “trahanas”) in sourdough bread production. Specific volumes of all the produced bread samples varied at approximately the same levels (2.50 to 2.54 mL/g), indicating a satisfactory dough expansion. In contrast, acidity differed significantly (p < 0.05). Sourdough bread produced with immobilized cells (IB) exhibited the lowest pH (4.30) and the highest titratable acidity (9.13 mL NaOH N/10), followed by the sourdough bread produced with free cells (FB) and the control bread (CB), reflecting the enhanced metabolic activity of immobilized cells supported by the prebiotic matrix. The organic acid analysis revealed higher levels of lactic (2.96 g/kg) and acetic acid (0.99 g/kg) in the IB sample, along with increased minor acids. The high organic acid content contributed to a delayed rope (14.7 days) and mold spoilage (7.3 days) compared to the CB sample, enhancing microbial stability. In addition, the nutritional properties of the produced sourdough breads containing P. acidilactici ORE 5 were improved significantly. The IB sample demonstrated the highest total phenolic content (85 mg GAE/100 g), followed by the FB sample (61 mg/100 g) and the CB sample (48 mg/100 g), while phytic acid levels were markedly reduced (93% in IB and 80% in FB). A sensory evaluation confirmed the maintained overall acceptability, with enhanced flavor scores for sourdough breads containing P. acidilactici ORE 5. These findings indicate that P. acidilactici ORE 5, particularly in immobilized form, could be an effective functional starter culture for sourdough bread, highlighting its potential for applications in functional bakery products.

1. Introduction

The production of foods with functional properties targeting the delivery of health benefits besides nutritional requirements has gained an increased interest in recent decades [1]. Among the food sectors producing functional foods nowadays, the bread industry has also introduced innovative methods in order to meet the growing demands of consumers [2]. The main aims of these innovations are (i) the extension of bread shelf life without the addition of chemical preservatives, (ii) the enhancement of nutritional value, (iii) the improvement of sensorial characteristics and (iv) the delivery of health benefits. In this regard, the improvement of gluten-free bread properties, the application of novel sourdoughs and the application of various functional supplements with nutritional composition are the most characteristic examples [3]. Likewise, many research efforts have been used and valuable knowledge has been produced and exploited by the bread manufacturing industry. Sourdough application in bread production is a widely studied scientific area with significant outcomes, such as (i) the enhancement of shelf life, (ii) the improvement of various sensorial characteristics, (iii) elevated nutritional value (increased levels and accessibility of bioactive substances and bioavailability of minerals; increase in the content of protein and vitamins; decrease in the glycemic index), (iv) improved technological properties and (v) the delivery of health benefits, mainly by the efficient treatment of various disorders and gut microbiota modulation [4, 5]. All the above can explain why the sourdough bread market is estimated to expand to USD 3.30 billion by 2029 [4].
On the other hand, the addition of various supplements with functional properties and novel microorganisms in bread and sourdough production is also an important trend lately, with remarkable outcomes. Thus, several supplements have been used for the fortification of bread with bioactive compounds [6], while various microorganisms have been used in sourdough bread production, most of them belonging to lactic acid bacteria (LAB). In particular, the application of novel LAB with functional properties is gaining attention, since the review of the functional and technological properties of sourdough and its breads mainly relies on the activities of LAB species added and presented in the sourdough environment [7]. However, the commercial application of LAB in sourdough bread making requires preserved viability and functionality for long periods. In this vein, the freeze-drying of LAB is an effective drying method for microbial cultures, while cell immobilization can also contribute to the protection of cells during processing and storage [8, 9].
Even though Lactobacillus strains are commonly applied in sourdough preparations, other members of LAB are also worthy of being examined. Pediococcus strains are seldom examined in sourdough bread making, although they can be used as bio-preservatives with various advantageous features, such as reductions in obesity and fatty liver, high antioxidant activity and high antimicrobial activity through bacteriocin production [10].
The role of different Pediococcus strains in the food industry and also in humans and other animals is well documented. Reports have suggested that P. acidilactici inhabits the entire digestive tract of humans due to its ability to survive in an extreme range of pH values, temperatures and osmotic pressures [11]. The importance of P. acidilactici has increased, with some of the strains of this species having been cited as probiotic bacteria [12]. The application of Pediococcus acidilactici strains are scarcely applied in sourdough bread making, since other LAB are preferred. Likewise, there are reports dealing with sourdough bread making with various strains of P. acidilactici, mainly targeting a reduction of acrylamide content, improved shelf life, reduced phytate content, controlled staling rate and improved sensorial features [13, 14, 15, 16, 17, 18]. However, a comprehensive approach dealing with the assessment of the physicochemical parameters, microbiological data, nutritional features and volatile compound composition of sourdough bread has not been published until now. Apparently, in addition to the aforementioned, the application of the specific strain P. acidilactici ORE 5 is innovative.
Recently, a potentially probiotic P. acidilacti ORE 5 isolated from kefir grains was studied for the functional regulation of the novel katiki domokou-type cheese microbiome, with interesting outcomes in terms of fermentative activity and metabolic functional compound production [19].
In this study, the strain P. acidilactici ORE 5 was assessed for the first time as a starter culture in sourdough bread making in free and immobilized forms. The produced sourdough breads were evaluated in terms of physicochemical and sensory properties, shelf life and nutritional features.

2. Materials and Methods

2.1. Microorganism, Raw Materials and Media

The new, potentially probiotic strain P. acidilactici ORE 5 recently isolated from kefir grains [20] was employed as a starter culture for sourdough bread making. It was cultivated in MRS broth at 37 °C for 24–48 h, and the biomass was recovered through centrifugation (Sigma 3K12, Bioblock Scientific, Illkirch, France) at 5000 rpm for 10 min at 25 °C. Media were sterilized through autoclaving at 120 °C for 15 min (1–1.5 atm).
Commercial white flour (Hellenic Biscuit CO S.A., Athens, Greece) containing 11.0% protein, 72.0% carbohydrates, 1.5% fat, 2.2% fiber and 12.0% moisture was used for bread making. Baker’s yeast was supplied as pressed blocks (70% moisture, w/w) from S.I. Lesaffre (Maisons-Alfort, France). The Greek traditional, fermented food trahanas was used as an immobilization support for P. acidilactici ORE 5 cells. Trahanas was prepared by mixing 70% hard wheat flour with sour sheep’s milk, boiling, soaking in fresh sour milk, drying/maturing for 4 days at 30 °C and cutting into ~1 cm3 cubes [21].

2.2. Culture Immobilization and Freeze-Drying

The immobilization procedure was achieved through the mixing of 0.5 g of harvested P. acidilactici ORE 5 cell mass with 5 g of trahanas in 500 mL MRS broth and incubation at 37 °C for 48 h. The immobilized biocatalyst was washed twice with ¼-strength Ringer’s solution to remove unbound cells. Free and immobilized biocatalysts were frozen to −44 °C at 5 °C/min and freeze-dried for 48 h at −45 °C under 5–15 mbar using a FreeZone 4.5 system (Labconco, Kansas City, MO, USA). Freeze-dried biocatalysts contained 8.2 ± 0.2 and 8.1 ± 0.3 log cfu/g of P. acidilactici ORE 5, respectively, and were applied in sourdough bread production.

2.3. Sourdough Making

2.3.1. Sourdough Preparation

Two mother sourdoughs were prepared by mixing 300 g wheat flour, 160 mL tap water and 1% w/w (on flour basis) of either freeze-dried P. acidilactici ORE 5 or freeze-dried immobilized P. acidilactici ORE 5 for 15 min. A third sourdough, containing only flour and water, was prepared in parallel to serve as a reference for evaluating the specific impact of P. acidilactici on the microbial composition. This reference sourdough was not used for bread making. All sourdoughs were fermented for 24 h at 30 °C.

2.3.2. Sourdough Bread Making

Three breads were totally produced by incorporating 30% (w/w) of (i) sourdough prepared with freeze-dried P. acidilactici ORE 5, (ii) sourdough prepared with freeze-dried immobilized P. acidilactici ORE 5 and (iii) traditional sourdough containing wild microflora from a local bakery. The three sourdoughs were added with doughs containing 500 g wheat flour, 270 mL water, 4 g salt and 1% w/w baker’s yeast. Doughs were fermented at 30 °C for 2 h, proofed at 40 °C for 60 min and baked at 230 °C for approximately 40 min.
Finally, the sourdough breads were encoded as (i) bread made with sourdough containing freeze-dried P. acidilactici ORE 5 (FB), (ii) bread made with sourdough containing freeze-dried immobilized P. acidilactici ORE 5 (IB) and (iii) control bread made with 30% traditional sourdough containing wild microflora (CB). All trials were conducted in triplicate.

2.4. Analysis

2.4.1. Analysis of LAB and Yeasts in Sourdough

In total, 10 g of sourdough was added to 90 mL of phosphate buffer (0.25 M solution of KH2PO4 diluted as 1.25 mL/L of distilled water) in order to analyze the viable cell counts in the sourdough prepared with (i) freeze-dried P. acidilactici ORE 5, (ii) freeze-dried immobilized P. acidilactici ORE 5, (iii) wild microflora (traditional) and (iv) only flour and water (without the addition of P. acidilactici ORE 5). After decimal dilutions, the suspensions were plated on MRS agar (Fluka, Buchs, Switzerland) and incubated at 37 °C for 48–72 h for the determination of LAB. Yeasts were determined on malt agar (Fluka, Switzerland) after incubation at 30 °C for 3 days.

2.4.2. pH and Acidity

The pH levels of the sourdough bread samples were evaluated using a Sentron Argus pH-meter (Sentron Europe B.V., Roden, The Netherlands). The total titratable acidity (TTA; expressed as the volume of 0.1 M NaOH used per 10 g of sample) for the sourdough bread samples was determined according to the previously outlined procedure [22].

2.4.3. Specific Loaf Volume

The specific volume of the bread was measured by weighing the loaves and calculating their volume using the rapeseed displacement method [22]. The volume of the rapeseed was deducted from the total container volume, and the specific volume was reported in mL/g.

2.4.4. Organic Acids

The specific organic acids present in the bread samples, such as lactic, acetic, formic, propionic, n-valeric and caproic acids, were quantified through ion-exchange liquid chromatography as previously described [22]. In summary, samples were combined with sterile water, homogenized with a Seward Stomacher 400 blender (London, UK), centrifuged and analyzed using a Shimadzu HPLC system equipped with a Shim-pack ICA1 column, an LC-10AD pump, a CTO-10A oven set at 40 °C and a CDD-6A conductivity detector (Shimadzu Corporation, Kyoto, Japan). The mobile phase was composed of 2.5 mM phthalic acid and 2.4 mM tris(hydroxymethyl)aminomethane (pH 4.0) at a flow rate of 1.2 mL/min. The concentrations of organic acids were calculated using standard calibration curves.

2.4.5. Total Phenolic Content (TPC)

After baking, breads were cooled to room temperature for 3 h and sliced, and the crumb samples were freeze-dried for 48 h (FreeZone 4.5, Labconco, Kansas City, MO, USA). One gram of the freeze-dried sample was extracted with 20 mL phosphate-buffered saline (PBS, pH 7.4) under shaking at 37 °C for 1 h. The extract was separated by decanting, and the residue was re-extracted with another 20 mL PBS. The combined extracts were stored at −20 °C until analysis. TPC was determined using the Folin–Ciocalteu method with slight modifications [23]. In summary, 200 μL of the sample extract was combined with 800 μL of Folin–Ciocalteu reagent and allowed to incubate in the dark for 2 min. Subsequently, 2 mL of 7.5% (v/v) sodium carbonate was introduced, and the total volume was adjusted to 10 mL using distilled water. The resulting mixture was then incubated in the dark at room temperature for 60 min, after which the absorbance was recorded at 765 nm. Standard solutions of gallic acid (GA) and a blank were also prepared, with the total phenolic content (TPC) expressed as mg GA equivalents per 100 g of dried sample (mg GAE/100 g).

2.4.6. Phytic Acid

Phytic acid (as myo-inositol-1,2,3,4,5,6-hexakisphosphate) was determined based on the amount of phosphorus liberated through enzymatic hydrolysis. The determination was carried out through the Megazyme K-PHYT assay kit (Bray, Ireland), a well-established and reliable tool for evaluating phytic acid levels. Phytic acid is enzymatically degraded, releasing inorganic phosphorus, which is subsequently measured as an indicator of its concentration in the sample. The protocol was performed in accordance with the manufacturer’s guidelines (Megazyme, Bray, Ireland) to guarantee precision and reproducibility.

2.4.7. Sensory Evaluation

A consumer hedonic test was conducted to evaluate the sensory quality of the sourdough breads. A panel of 26 untrained consumers evaluated the breads for flavor, taste, appearance and overall quality using a 9-point hedonic scale, where 1 represented “dislike extremely” and 9 represented “like extremely”. All panelists provided voluntary informed consent prior to participation. Twenty untrained consumers at a local bakery conducted a blind sensory evaluation of the sourdough breads immediately after production The evaluation followed standard sensory analysis procedures, adapted from Mantzourani et al., 2019 [24]. Rope and mold spoilage were also evaluated through macroscopic observation. Rope spoilage was assessed based on the presence of a ripe cantaloupe-like aroma, discoloration and sticky threads, whereas mold spoilage was identified by the appearance of visible fungal colonies.

2.4.8. Statistical Analysis

Analysis of variance (ANOVA) followed by Duncan’s post hoc multiple range test was used to identify significant differences among treatments, specifically evaluating the effects of different sourdoughs on the physicochemical and sensory properties of the breads. The analysis was performed using the SPSS Statistics 20.0 (IBM Corp., Armonk, NY, USA) software at an alpha level of 5%.

3. Results and Discussion

3.1. Viable Cell Counts in the Sourdoughs

Viable LAB and yeast cells (cfu) were enumerated in all of the prepared sourdoughs, and the results are shown in Table 1. The addition of P. acidilactici ORE 5 in free or immobilized forms led to higher LAB viabilities in the final sourdough compared to the commercial sourdough and to the sourdough prepared with the same conditions, only without the addition of P. acidilactici ORE. In particular, the highest LAB average viabilities were observed in the sourdough containing immobilized P. acidilactici ORE 5 (10.7 log cfu/g) and free P. acidilactici ORE 5 (9.4 log cfu/g), followed by the traditional sourdough (8.3 log cfu/g) and the sample that did not contain P. acidilactici ORE 5 (5.1 log cfu/g). On the other hand, no statistically significant differences were observed between all sourdoughs regarding the levels of yeasts, varying between 7.0 and 7.2 log cfu/g. These results showed that the two types of P. acidilactici ORE prevailed microbiologically in the produced sourdoughs.

3.2. Bread Volume and Acidity Levels

The results of the proximate analysis of the produced sourdough breads are presented in Table 2. The use of P. acidilactici ORE 5 in free or immobilized form in sourdough bread making did not negatively influence the dough expansion. Specifically, no statistically significant differences were recorded regarding the specific loaf volume of all the samples (2.51 to 2.54 mL/g), which, in general, reflects good rising. On the contrary, statistically significant differences were observed in the case of pH and TTA values of all the samples. In particular, IB possessed the highest acidity (9.13 mL NaOH N/10) and the lowest pH value (4.30), followed by FB and finally the control sample (CB). These differences can be attributed to the matrix of immobilization, which consisted of prebiotic compounds such as starch that enhance the viability and metabolic activity of P. acidilactici ORE 5. Notably, enhancements in the fermentation processes have been documented earlier concerning the immobilization carrier utilized in this research (trahanas), which were linked to their prebiotic attributes, boosting the variability and fermentation potential of LAB [22]. This outcome has been verified by many other researchers, who have revealed the respective effects of prebiotic matrices (as immobilization support) on the LAB viability and fermentative activity [25, 26].
Therefore, P. acidilactici ORE 5 probably more effectively fermented the sourdough, leading to a higher organic acid production, which resulted in the higher observed TTA values. Regardless of the effect of cell immobilization, the use of free P. acidilactici ORE 5 led to higher TTA values compared to CB, demonstrating its effectiveness in lactic acid fermentation and therefore its suitability to be used as a starter culture in sourdough fermentation. In particular, the TTA value of FB reached 7.61 mL NaOH N/10, while the respective value for CB was 6.80 mL NaOH N/10.

3.3. Organic Acids Content

The qualitative profile of organic acids determined in the bread samples seems to correspond to the previously observed acidity levels (Table 3). The two major organic acids, lactic and acetic acid, were produced in significantly higher (p < 0.05) levels in IB compared to FB and CB. Specifically, IB contained approximately 2.96 g/kg lactic acid and 0.99 g/kg acetic acid, followed by FB with 2.59 g/kg and 0.85 g/kg, respectively, while the concentrations of lactic and acetic acid in CB were 2.19 g/kg and 0.74 g/kg, respectively. In addition, IB contained higher levels of minor organic acids such as formic (0.09 g/kg), propionic (0.06 g/kg), n-valeric (0.08 g/kg) and caproic acid (0.06 g/kg) compared to FB, while these acids were detected only in trace amounts in CB.
The calculated quotient of fermentation (QF, lactic-to-acetic acid molar ratio) was 1.99 for IB, 2.03 for FB and 1.97 for CB. These values fall within the recommended range for sourdough breads (QF < 5.0) reported in the literature [27], indicating a well-balanced fermentation profile. Notably, the relatively higher concentrations of both lactic and acetic acids in IB contribute to a more pronounced sourdough-like sensory profile.
Beyond their sensory impact, lactic and acetic acids—together with formic, propionic, valeric and caproic acids—are known to exert strong antimicrobial and antifungal effects, contributing to the natural preservation of sourdough bread [28]. The higher levels of these acids in IB indicate that cell immobilization enhanced the metabolic activity of P. acidilactici ORE 5, resulting in high expectations for improved flavor development and potentially prolonged shelf life compared to the control breads [3].

3.4. Spoilage of the Baked Breads During Storage

The daily macroscopic monitoring of the sourdough breads (Figure 1) revealed clear and reproducible differences in spoilage onset between treatments. Rope spoilage was first detected after 14.7 days in the immobilized cell bread (IB), 14.3 days in the free cell bread (FB) and 11.7 days in the control bread (CB). Mold development occurred approximately after 7.3 days in IB, 7.0 days in FB and 6.0 days in CB. Both FB and IB bread samples containing P. acidilactici ORE 5 showed a significantly (p < 0.05) greater resistance to both types of spoilage than the control, with IB demonstrating the highest resistance.
The higher preservation times observed for IB can be mainly linked to its higher organic acid content (lactic and acetic acids).
Rope spoilage in bread is commonly caused by spore-forming Bacillus spp., while mold spoilage is typically due to airborne filamentous fungi (e.g., Penicillium, Aspergillus). The observed delay of rope onset in IB (≈3.0 days later than CB) is notable, since Bacillus spores are intrinsically heat- and acid-resistant; therefore, an acid-mediated delay likely arises from the inhibition of vegetative growth after spore germination, reduced initial contamination (e.g., antagonism during dough maturation) or from other antimicrobial metabolites that limit the outgrowth of spoilage Bacillus strains. The delay in mold growth (≈1.3 days longer in IB vs. CB) is consistent with acid and minor acid activity; however, other factors (packaging, water activity, microstructure) can modulate mold growth and should be measured in follow-up experiments. Beyond organic acids, lactic acid bacteria (LAB) commonly produce other antimicrobial compounds, including bacteriocins, antifungal peptides, H2O2 and cyclic dipeptides. These metabolites can enhance protection, particularly against Gram-positive bacteria and some fungi. In the case of Pediococcus acidilactici, many strains are known to produce pediocins, which are bacteriocins that contribute to antimicrobial activity and potentially to the extended shelf life of fermented products. Even though data regarding the P. acidilactici ORE 5 strain are limited, it is likely that antimicrobial metabolites may be produced, contributing to the shelf life extension. For instance, studies have demonstrated that certain strains of P. acidilactici exhibit antimicrobial activity through the production of bacteriocins and other metabolites, which play a role in preserving food products.

3.5. Total Phenolic Content (TPC)

The evaluation of total phenolic content (TPC) in the different bread samples revealed clear and statistically significant differences (p < 0.05) (Figure 2). The sourdough bread produced with immobilized P. acidilactici ORE 5 on trahanas (IB) displayed the highest levels of TPC (85 mg GAE/100 g), followed by the FB (61 mg GAE/100 g) and the CB (48 mg GAE/100 g). These findings underline the combined role of the immobilization support and probably the metabolic activity of P. acidilactici in enhancing the phenolic profile of the breads. Previous research has emphasized that substrates containing prebiotic compounds can stimulate LAB growth and drive enzymatic conversions of initial phenolic compounds into smaller derivatives with a higher activity and functionality. Such processes often result in elevated TPC values [29]. Consistent with these observations, it has been widely documented that the addition of phenolic- and antioxidant-rich supplements in bread formulations increases the overall TPC [30,31].
In the present work, the immobilization support is a starch gluten matrix (trahanas), which is a natural source of prebiotics and possibly other functional compounds that can enhance the TPC of the bread [22,32,33]. The highest impact was recorded in IB, confirming that the immobilization of P. acidilactici ORE 5 on trahanas provides favorable conditions for enzymatic activity and phenolic release. FB also showed a significant increase compared to the control, suggesting that free cells of P. acidilactici ORE 5 can positively influence TPC, although to a lesser extent compared to immobilized cells. This outcome implies the possible effect of P. acidilactici ORE 5 in the development of enhanced TPC levels of sourdough bread, as has been revealed earlier for other Pediococcus strains [34,35]. Nonetheless, more detailed studies are necessary to fully elucidate the transformations and specific phenolic metabolites formed during sourdough fermentation and baking.

3.6. Content of Phytic Acid

Breads produced with the proposed sourdoughs exhibited underlined reduced contents of phytic acid, probably due to the better action of intrinsic cereal phytases (Figure 3). Initially, all dough samples contained 4.6 mg/g phytic acid. After baking, the levels of phytic acid were 0.3 mg/g for IB, 0.6 mg/g for FB and 0.9 mg/g for CB. Likewise, both sourdough bread samples containing P. acidilactici ORE 5 displayed a higher reduction in phytic acid compared to CB; IB especially exhibited a higher reduction (93%). Research has shown that Pediococcus strains play a crucial role in reducing phytic acid during sourdough fermentation, due to their high phytase activity. In particular, P. acidilactici (L19) has demonstrated a strong ability to degrade phytate reaching up to ~89%, whereas other strains of Pediococcus achieved reduction rates higher than 70%. Consequently, P. acidilactici ORE 5 also exhibited a high phytase activity that can lead to improvements regarding mineral bioavailability and bread quality. This effect can be attributed not only to the direct action of microbial phytases, but also to sourdough acidification, which enhances substrate accessibility and activates endogenous phytases [36].

3.7. Sensory Evaluation

The results obtained through the preliminary sensory evaluation of the sourdough breads are presented in Table 4, while the images of all the bread samples are illustrated in Figure 4.
There were no significant differences among the samples for taste, appearance or overall quality, indicating that the incorporation of P. acidilactici ORE 5, whether free (FB) or immobilized (IB), did not negatively affect these attributes compared to the commercial sourdough bread (CB). In contrast, significant differences were observed in the flavor scores, with both FB and IB receiving higher ratings than CB (p < 0.05). These findings suggest that the use of P. acidilactici ORE 5, either free or immobilized, not only maintains the overall acceptability of the sourdough breads, but may also enhance specific sensory attributes such as flavor.
The unique fermentation characteristics that P. acidilactici ORE 5 achieves can be directly linked to the improved flavor perception in breads fermented with this strain. IB especially exhibited considerably increased amounts of lactic and acetic acids, as well as minor volatile acids such formic, propionic, valeric and caproic acids, as shown in Section 3.1 and Section 3.2. These substances are widely known for playing crucial roles in creating the distinctive flavor complexity and scent of sourdough. The excellent sensory scores were attributed to the balanced lactic-to-acetic acid molar ratio (QF = 2) obtained throughout treatments, which guaranteed a nice sourness without excessive acidity. Aside from acidification, the increased total phenolic content (TPC) found in sourdough breads containing P. acidilactici ORE 5 most likely improved the flavor depth and antioxidant-associated freshness perception. Phenolic chemicals, whether natural or biotransformed during LAB fermentation, can combine with Maillard reaction products during baking to produce aromatic notes associated with roasted, nutty or slightly caramelized tones. This conclusion is supported by the fact that IB has a greater TPC (85 mg GAE/100 g) than FB (61 mg GAE/100 g) and CB (48 mg GAE/100 g). In addition, the decrease in phytic acid compounds detected in sourdough breads containing P. acidilactici ORE 5 (p ≤ 0.05, up to 93% in IB) might indirectly affect the sensory aqueous view by retarding the mineral bioavailable inlet and buffering capacity within the dough, which modulate yeast and bacterial enzymatic activity during the fermentation process. This could help to balance fermentation and give a more complex volatile pattern. The good organoleptic properties are also in accordance with the better keeping quality and slow rancidity development found between both the FB and IB samples (Section 3.3). A longer microbial stability period retains the freshness and minimizes off-flavor development in bread at an early stage, which ultimately leads to better flavor acceptance and overall perceived quality.
From a product development perspective, this highlights the potential of P. acidilactici ORE 5 as a functional starter culture to improve the sensory quality of sourdough bread without compromising consumer acceptance, offering a competitive advantage over traditional commercial sourdough formulations.

3.8. Industrial Application and Scale-Up Potential

The experimental results indicate that P. acidilactici ORE 5 has a high potential for industrial applications. The strain displayed a respectable metabolic activity, leading to sourdough breads with higher levels of organic acids, increased TPC and a considerable phytic acid reduction. These outcomes can support its application in larger-scale sourdough operations. Immobilization on trahanas offers technological benefits for large-scale manufacturing, such as an increased cell stability, reusability and fermentation efficiency. Fermentation kinetics (mild pH reduction, balanced QF) are consistent with traditional industrial sourdough processes, implying that P. acidilactici ORE 5 might replace or supplement typical Lactobacillus starters without requiring significant process adjustments. Likewise, the results obtained support that P. acidilactici ORE 5 has practical significance for health-promoting, functional bread production. Pilot-scale studies are recommended to ensure culture stability, fermentation repeatability and shelf life in commercial settings.

4. Conclusions

The employment of P. acidilactici ORE 5, either free or immobilized on trahanas, in sourdough bread making effectively improved the quality of the final products. Both FB and IB maintained a good loaf volume and exhibited a higher titratable acidity and organic acid content than the control. It seems that immobilization further enhanced the levels of lactic, acetic and minor organic acids in the bread, thereby contributing to a prolonged resistance against rope and mold spoilage. The sensory evaluation confirmed that P. acidilactici ORE 5 not only did not negatively affect the sensorial features of the bread, but even enhanced the flavor of the final product compared to the control bread. Nutritional improvements were observed in both breads made with P. acidilactici ORE 5, due to the increased total phenolic content and the reduction in phytic acid of the final products. These results highlight the potential of P. acidilactici ORE 5, particularly in immobilized form, as a functional starter culture for sourdough fermentation capable of improving sensory qualities, bioactive content and shelf life without compromising consumer preference.

Author Contributions

Conceptualization, S.P.; methodology, S.P. and I.M.; validation, I.M., G.M. and Y.K.; formal analysis, I.M. and A.A.; investigation, S.P., A.A., G.M., Y.K. and I.M.; resources, S.P.; data curation, A.A. and I.M.; writing—original draft preparation, S.P.; writing—review and editing, S.P.; visualization, S.P.; supervision, S.P.; project administration, S.P.; funding acquisition, S.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The sensory analyses involved adult volunteers who participated voluntarily and without the collection or processing of sensitive data. Therefore, the study does not fall within the scope of mandatory ethical review according to the Law on Clinical Trials of Medicinal Products and Medical Devices (Zakon o lekovima i medicinskim sredstvima, Official Gazette of the RS, no. 105/2017).

Informed Consent Statement

Informed consent for participation was obtained from all subjects involved in the study.

Data Availability Statement

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

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Sgroi, F.; Sciortino, C.; Baviera-Puig, A.; Modica, F. Analyzing Consumer Trends in Functional Foods: A Cluster Analysis Approach. J. Agric. Food Res. 2024, 15, 101041. [Google Scholar] [CrossRef]
  2. Zarzycki, P.; Wirkijowska, A.; Teterycz, D.; Łysakowska, P. Innovations in Wheat Bread: Using Food Industry By-Products for Better Quality and Nutrition. Appl. Sci. 2024, 14, 3976. [Google Scholar] [CrossRef]
  3. Plessas, S. Innovations in Sourdough Bread Making. Fermentation 2021, 7, 29. [Google Scholar] [CrossRef]
  4. Islam, M.A.; Islam, S. Sourdough Bread Quality: Facts and Factors. Foods 2024, 13, 2132. [Google Scholar] [CrossRef]
  5. Alkay, Z.; Falah, F.; Cankurt, H.; Dertli, E. Exploring the Nutritional Impact of Sourdough Fermentation: Its Mechanisms and Functional Potential. Foods 2024, 13, 1732. [Google Scholar] [CrossRef]
  6. Hasan, M.M.; Islam, M.R.; Haque, A.R.; Kabir, M.R.; Hasan, S.K. Fortification of Bread with Mango Peel and Pulp as a Source of Bioactive Compounds: A Comparison with Plain Bread. Food Chem. Adv. 2024, 5, 100783. [Google Scholar] [CrossRef]
  7. Luti, S.; Mazzoli, L.; Ramazzotti, M.; Galli, V.; Venturi, M.; Marino, G.; Lehmann, M.; Guerrini, S.; Granchi, L.; Paoli, P. Antioxidant and Anti-Inflammatory Properties of Sourdoughs Containing Selected Lactobacilli Strains Are Retained in Breads. Food Chem. 2020, 322, 126710. [Google Scholar] [CrossRef]
  8. Carvalho, A.S.; Silva, J.; Ho, P.; Teixeira, P.; Malcata, F.X.; Gibbs, P. Relevant Factors for the Preparation of Freeze-Dried Lactic Acid Bacteria. Int. Dairy J. 2004, 14, 835–847. [Google Scholar] [CrossRef]
  9. Gul, L.B.; Con, A.H.; Gul, O. Storage Stability and Sourdough Acidification Kinetic of Freeze-Dried Lactobacillus Curvatus N19 under Optimized Cryoprotectant Formulation. Cryobiology 2020, 96, 122–129. [Google Scholar] [CrossRef]
  10. Todorov, S.D.; Dioso, C.M.; Liong, M.-T.; Nero, L.A.; Khosravi-Darani, K.; Ivanova, I.V. Beneficial Features of Pediococcus: From Starter Cultures and Inhibitory Activities to Probiotic Benefits. World J. Microbiol. Biotechnol. 2023, 39, 4. [Google Scholar] [CrossRef]
  11. Fugaban, J.I.I.; Bucheli, J.E.V.; Park, Y.J.; Suh, D.H.; Jung, E.S.; Franco, B.D.G.d.M.; Ivanova, I.V.; Holzapfel, W.H.; Todorov, S.D. Antimicrobial Properties of Pediococcus Acidilactici and Pediococcus Pentosaceus Isolated from Silage. J. Appl. Microbiol. 2022, 132, 311–330. [Google Scholar] [CrossRef] [PubMed]
  12. Bhagat, D.; Raina, N.; Kumar, A.; Katoch, M.; Khajuria, Y.; Slathia, P.S.; Sharma, P. Probiotic Properties of a Phytase Producing Pediococcus Acidilactici Strain SMVDUDB2 Isolated from Traditional Fermented Cheese Product, Kalarei. Sci. Rep. 2020, 10, 1926. [Google Scholar] [CrossRef] [PubMed]
  13. Bartkiene, E.; Vizbickiene, D.; Bartkevics, V.; Pugajeva, I.; Krungleviciute, V.; Zadeike, D.; Zavistanaviciute, P.; Juodeikiene, G. Application of Pediococcus Acidilactici LUHS29 Immobilized in Apple Pomace Matrix for High Value Wheat-Barley Sourdough Bread. LWT-Food Sci. Technol. 2017, 83, 157–164. [Google Scholar] [CrossRef]
  14. Huang, J.; Omedi, J.O.; Huang, C.; Chen, C.; Liang, L.; Zheng, J.; Zeng, Y.; Xu, Y.; Huang, W. Effect of Black Bean Supplemented with Wheat Bran Sourdough Fermentation by Pediococcus Acidilactici or Pediococcus Pentosaceus on Baking Quality and Staling Characteristics of Wheat Composite Bread. Appl. Food Res. 2024, 4, 100425. [Google Scholar] [CrossRef]
  15. Wang, H.-E.; Hwang, C.-F.; Tzeng, Y.-M.; Hwang, W.-Z.; Mau, J.-L. Quality of white bread made from lactic acid bacteria-enriched dough: Quality of lactic acid bacteria-enhanced bread. J. Food Process. Preserv. 2012, 36, 553–559. [Google Scholar] [CrossRef]
  16. Karaman, K.; Sagdic, O.; Durak, M.Z. Use of Phytase Active Yeasts and Lactic Acid Bacteria Isolated from Sourdough in the Production of Whole Wheat Bread. LWT 2018, 91, 557–567. [Google Scholar] [CrossRef]
  17. Nachi, I.; Fhoula, I.; Smida, I.; Taher, I.B.; Chouaibi, M.; Jaunbergs, J.; Bartkevics, V.; Hassouna, M. Assessment of Lactic Acid Bacteria Application for the Reduction of Acrylamide Formation in Bread. LWT 2018, 92, 435–441. [Google Scholar] [CrossRef]
  18. Cizeikiene, D.; Juodeikiene, G.; Paskevicius, A.; Bartkiene, E. Antimicrobial Activity of Lactic Acid Bacteria against Pathogenic and Spoilage Microorganism Isolated from Food and Their Control in Wheat Bread. Food Control 2013, 31, 539–545. [Google Scholar] [CrossRef]
  19. Pavlatou, C.; Nikolaou, A.; Prapa, I.; Tegopoulos, K.; Plesssas, S.; Grigoriou, M.E.; Bezirtzoglou, E.; Kourkoutas, Y. Effect of Immobilized Pediococcus Acidilactici ORE5 Cells on Pistachio Nuts on the Functional Regulation of the Novel Katiki Domokou-Type Cheese Microbiome. Appl. Sci. 2023, 13, 8047. [Google Scholar] [CrossRef]
  20. Mantzourani, I.; Daoutidou, M.; Terpou, A.; Plessas, S. Evaluation of a Novel Potentially Probiotic/Pediococcus Acidilactici ORE5 in Lactic Acid Fermentation of Cornelian Cherry Juice: Assessment of Nutritional Properties, Physicochemical Characteristics, and Sensory Attributes. Fermentation 2024, 10, 650. [Google Scholar] [CrossRef]
  21. Plessas, S.; Bekatorou, A.; Kanellaki, M.; Psarianos, C.; Koutinas, A. Cells Immobilized in a Starch–Gluten–Milk Matrix Usable for Food Production. Food Chem. 2005, 89, 175–179. [Google Scholar] [CrossRef]
  22. Mantzourani, I.; Daoutidou, M.; Plessas, S. Impact of Functional Supplement Based on Cornelian Cherry (Cornus mas L.) Juice in Sourdough Bread Making: Evaluation of Nutritional and Quality Aspects. Appl. Sci. 2025, 15. [Google Scholar] [CrossRef]
  23. Plessas, S.; Mantzourani, I.; Alexopoulos, A.; Alexandri, M.; Kopsahelis, N.; Adamopoulou, V.; Bekatorou, A. Nutritional Improvements of Sourdough Breads Made with Freeze-Dried Functional Adjuncts Based on Probiotic Lactiplantibacillus plantarum Subsp. plantarum and Pomegranate Juice. Antioxidants 2023, 12, 1113. [Google Scholar] [CrossRef] [PubMed]
  24. Mantzourani, I.; Terpou, A.; Alexopoulos, A.; Bezirtzoglou, E.; Plessas, S. Assessment of Ready-to-Use Freeze-Dried Immobilized Biocatalysts as Innovative Starter Cultures in Sourdough Bread Making. Foods 2019, 8, 40. [Google Scholar] [CrossRef] [PubMed]
  25. Bonik, S.K.; Tamanna, S.T.; Happy, T.A.; Haque, M.N.; Islam, S.; Faruque, M.O. Formulation and Evaluation of Cereal-Based Breads Fortified with Natural Prebiotics from Green Banana, Moringa Leaves Powder and Soya Powder. Appl. Food Res. 2024, 4, 100377. [Google Scholar] [CrossRef]
  26. Terpou, A.; Bekatorou, A.; Bosnea, L.; Kanellaki, M.; Ganatsios, V.; Koutinas, A.A. Wheat Bran as Prebiotic Cell Immobilisation Carrier for Industrial Functional Feta-Type Cheese Making: Chemical, Microbial and Sensory Evaluation. Biocatal. Agric. Biotechnol. 2018, 13, 75–83. [Google Scholar] [CrossRef]
  27. Arora, K.; Ameur, H.; Polo, A.; Di Cagno, R.; Rizzello, C.G.; Gobbetti, M. Thirty Years of Knowledge on Sourdough Fermentation: A Systematic Review. Trends Food Sci. Technol. 2021, 108, 71–83. [Google Scholar] [CrossRef]
  28. Katina, K.; Sauri, M.; Alakomi, H.-L.; Mattila-Sandholm, T. Potential of Lactic Acid Bacteria to Inhibit Rope Spoilage in Wheat Sourdough Bread. LWT-Food Sci. Technol. 2002, 35, 38–45. [Google Scholar] [CrossRef]
  29. Graça, C.; Lima, A.; Raymundo, A.; Sousa, I. Sourdough Fermentation as a Tool to Improve the Nutritional and Health-Promoting Properties of Its Derived-Products. Fermentation 2021, 7, 246. [Google Scholar] [CrossRef]
  30. Balli, D.; Cecchi, L.; Pieraccini, G.; Venturi, M.; Galli, V.; Reggio, M.; Di Gioia, D.; Furlanetto, S.; Orlandini, S.; Innocenti, M. Millet Fermented by Different Combinations of Yeasts and Lactobacilli: Effects on Phenolic Composition, Starch, Mineral Content and Prebiotic Activity. Foods 2023, 12, 748. [Google Scholar] [CrossRef] [PubMed]
  31. Balli, D.; Bellumori, M.; Paoli, P.; Pieraccini, G.; Di Paola, M.; De Filippo, C.; Di Gioia, D.; Mulinacci, N.; Innocenti, M. Study on a Fermented Whole Wheat: Phenolic Content, Activity on PTP1B Enzyme and in Vitro Prebiotic Properties. Molecules 2019, 24, 1120. [Google Scholar] [CrossRef] [PubMed]
  32. Koc, F.; Sabuncu, M.; Yavuz, G.G.; Düven, G.; Abdo, R.M.K.; Bağci, U.; Şahan, Y.; Toğay, S.Ö.; Ross, R.P.; Stanton, C. Exploring Tarhana’s Prebiotic Potential Using Different Flours in an in Vitro Fermentation Model. Food Funct. 2025, 16, 4754–4771. [Google Scholar] [CrossRef]
  33. Atlama, K.; Sengun, I.Y.; Nalbantsoy, A. Assessment of Probiotic, Biosafety and Technological Properties of Lactic Acid Bacteria Isolated from Tarhana. Biologia 2024, 80, 415–432. [Google Scholar] [CrossRef]
  34. Hassan, A.A.A.; Jin, Y.H.; Mah, J.-H. Influence of Pediococcus Pentosaceus Starter Cultures on Biogenic Amine Content and Antioxidant Activity in African Sourdough Flatbread Fermentation. Antioxidants 2024, 13, 1204. [Google Scholar] [CrossRef]
  35. Mantzourani, I.; Chondrou, P.; Bontsidis, C.; Karolidou, K.; Terpou, A.; Alexopoulos, A.; Bezirtzoglou, E.; Galanis, A.; Plessas, S. Assessment of the Probiotic Potential of Lactic Acid Bacteria Isolated from Kefir Grains: Evaluation of Adhesion and Antiproliferative Properties in in Vitro Experimental Systems. Ann. Microbiol. 2019, 69, 751–763. [Google Scholar] [CrossRef]
  36. Reffai, Y.M.; Fechtali, T. A Critical Review on the Role of Lactic Acid Bacteria in Sourdough Nutritional Quality: Mechanisms, Potential, and Challenges. Appl. Microbiol. 2025, 5, 74. [Google Scholar] [CrossRef]
Fermentation 11 00666 g001
Figure 1. Appearance of rope and mold spoilage in the sourdough breads. Different superscripts in columns indicate statistically significant differences (ANOVA, Duncan’s multiple range test, p < 0.05).
Figure 1. Appearance of rope and mold spoilage in the sourdough breads. Different superscripts in columns indicate statistically significant differences (ANOVA, Duncan’s multiple range test, p < 0.05).
Fermentation 11 00666 g001
Fermentation 11 00666 g002
Figure 2. Total phenolic content (TPC) of the produced sourdough breads. Different superscripts in columns indicate statistically significant differences (ANOVA, Duncan’s multiple range test, p < 0.05).
Figure 2. Total phenolic content (TPC) of the produced sourdough breads. Different superscripts in columns indicate statistically significant differences (ANOVA, Duncan’s multiple range test, p < 0.05).
Fermentation 11 00666 g002
Fermentation 11 00666 g003
Figure 3. Phytic acid reduction in sourdough bread samples. Different superscript letters in a column indicate statistically significant differences (ANOVA, Duncan’s multiple range test, p < 0.05).
Figure 3. Phytic acid reduction in sourdough bread samples. Different superscript letters in a column indicate statistically significant differences (ANOVA, Duncan’s multiple range test, p < 0.05).
Fermentation 11 00666 g003
Fermentation 11 00666 g004
Figure 4. Sourdough bread samples after baking.
Figure 4. Sourdough bread samples after baking.
Fermentation 11 00666 g004
Table 1. Yeast and LAB counts in the produced sourdoughs.
Table 1. Yeast and LAB counts in the produced sourdoughs.
SourdoughsLABYeasts
Log cfu/g
Free P. acidilactici ORE 59.4 ± 0.1 b7.1 ± 0.1 a
Immobilized P. acidilactici ORE 510.7 ± 0.1 a7.0 ± 0.2 a
Traditional8.3 ± 0.2 c7.2 ± 0.1 a
Absence of P. acidilactici ORE 55.1± 0.2 c7.0 ± 0.1 a
LAB: lactic acid bacteria; cfu: colony-forming units. Different superscript letters in a column indicate statistically significant differences (p < 0.05).
Table 2. Physicochemical characteristics of breads made with sourdoughs prepared with freeze-dried, free and immobilized P. acidilactici ORE 5 and with the control sourdough.
Table 2. Physicochemical characteristics of breads made with sourdoughs prepared with freeze-dried, free and immobilized P. acidilactici ORE 5 and with the control sourdough.
Bread SamplepHTTA
(mL NaOH N/10)		SLV
(mL/g)
FB4.51 ± 0.05 b7.61 ± 0.07 b2.51 ± 0.05 a
IB4.30 ± 0.05 a9.13 ± 0.05 a2.50 ± 0.03 a
CB4.76 ± 0.04 c6.80 ± 0.03 c2.54 ± 0.05 a
Different superscripts in columns indicate statistically significant differences (ANOVA).
Table 3. Content of organic acids in breads made with sourdoughs prepared with freeze-dried, free and immobilized P. acidilactici ORE 5 and with the control sourdough.
Table 3. Content of organic acids in breads made with sourdoughs prepared with freeze-dried, free and immobilized P. acidilactici ORE 5 and with the control sourdough.
Bread SampleOrganic Acids (g/kg Bread)
 LacticAceticFormicPropionicn-ValericCaproic
FB2.59 ± 0.04 b0.85 ± 0.01 c0.05 ± 0.01 b0.04 ± 0.01 a0.04 ± 0.01 b0.03 ± 0.01 b
IB2.96 ± 0.08 a0.99 ± 0.03 a0.09 ± 0.01 a0.06 ± 0.01 a0.08 ± 0.01 a0.06 ± 0.01 a
CB2.19 ± 0.05 c0.74 ± 0.03 d0.04 ± 0.01 btrtrtr
Different superscripts in columns indicate statistically significant differences (ANOVA, Duncan’s multiple range test, p < 0.05). tr: traces (<0.01 g/kg).
Table 4. Sensory evaluation test (consumer preference) of sourdoughs breads.
Table 4. Sensory evaluation test (consumer preference) of sourdoughs breads.
Bread Sample FlavorTasteAppearanceOverall Quality
FB8.9 ± 0.1 a8.7 ± 0.2 a8.9 ± 0.1 a8.8 ± 0.2 a
IB8.9 ± 0.2 a8.8 ± 0.1 a8.9 ± 0.2 a8.8 ± 0.1 a
CB8.3 ± 0.2 b8.2 ± 0.1 a8.2 ± 0.1 b8.3 ± 0.1 b
Different superscript letters in a column indicate statistically significant differences (ANOVA, Duncan’s multiple range test, p < 0.05).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Mantzourani, I.; Alexopoulos, A.; Mitropoulou, G.; Kourkoutas, Y.; Plessas, S. Nutritional and Physicochemical Attributes of Sourdough Breads Fermented with a Novel Pediococcus acidilactici ORE 5 Strain. Fermentation 2025, 11, 666. https://doi.org/10.3390/fermentation11120666

AMA Style

Mantzourani I, Alexopoulos A, Mitropoulou G, Kourkoutas Y, Plessas S. Nutritional and Physicochemical Attributes of Sourdough Breads Fermented with a Novel Pediococcus acidilactici ORE 5 Strain. Fermentation. 2025; 11(12):666. https://doi.org/10.3390/fermentation11120666

Chicago/Turabian Style

Mantzourani, Ioanna, Athanasios Alexopoulos, Gregoria Mitropoulou, Yiannis Kourkoutas, and Stavros Plessas. 2025. "Nutritional and Physicochemical Attributes of Sourdough Breads Fermented with a Novel Pediococcus acidilactici ORE 5 Strain" Fermentation 11, no. 12: 666. https://doi.org/10.3390/fermentation11120666

APA Style

Mantzourani, I., Alexopoulos, A., Mitropoulou, G., Kourkoutas, Y., & Plessas, S. (2025). Nutritional and Physicochemical Attributes of Sourdough Breads Fermented with a Novel Pediococcus acidilactici ORE 5 Strain. Fermentation, 11(12), 666. https://doi.org/10.3390/fermentation11120666

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop