A Critical Review on the Role of Lactic Acid Bacteria in Sourdough Nutritional Quality: Mechanisms, Potential, and Challenges

Abstract

Sourdough fermentation, driven by the biochemical activity of lactic acid bacteria (LAB), presents a scientifically promising approach to addressing nutritional limitations in cereal-based staples. This review critically examines both the underlying mechanisms by which LAB enhance the nutritional profile of sourdough and the translational challenges in realizing these benefits. Key improvements explored include enhanced mineral bioavailability (e.g., up to 90% phytate reduction), improved protein digestibility, an attenuated glycemic response (GI ≈ 54 vs. ≈75 for conventional bread), and the generation of bioactive compounds. While in vitro and animal studies extensively demonstrate LAB’s potential to reshape nutrient profiles (e.g., phytate hydrolysis improving iron absorption, proteolysis releasing bioactive peptides), translating these effects into consistent human health outcomes proves complex. Significant challenges hinder this transition from laboratory to diet, including the limited bioavailability of LAB-derived metabolites, high strain variability, and sensitivity to fermentation conditions. Furthermore, interactions with the food matrix and host-specific factors, such as gut microbiota composition, contribute to inconsistent findings. This review highlights methodological gaps, particularly reliance on in vitro or animal models, and the lack of long-term, effective human trials. Although LAB hold significant promise for nutritional improvements in sourdough, translating these findings to validated human benefits necessitates continued efforts in mechanism-driven strain optimization, the standardization of fermentation processes, and rigorous human studies.

1. Introduction

Since ancient times, staple cereal grains have been a fundamental part of global diets [1]. As major sources of dietary energy, fiber, and key nutrients such as proteins, minerals, and vitamins, they play crucial roles in human health [2]. Despite these vital contributions, inherent compositional factors limit their full nutritional potential, particularly concerning micronutrients [2]. While these grains are rich in minerals like iron and zinc, bioavailability is typically low (e.g., ~10% for Fe, ~25% for Zn from wheat) [3], largely due to chelation by phytic acid (Myo-inositol hexakisphosphate), especially in whole grains [4,5]. Additionally, the complex structure of gluten proteins can present digestive challenges for some individuals [6,7]. Moreover, fermentable carbohydrates, such as fructans (FODMAPs), in widely consumed grains like wheat and rye can trigger gastrointestinal symptoms in susceptible individuals [8,9]. These inherent limitations are often not mitigated by rapid processing methods that use solely baker’s yeast in modern breadmaking [10]. This is particularly relevant given the recognized prevalence of gluten sensitivities [11].

Sourdough fermentation, one of the oldest biotechnological approaches to bread leavening [12], offers a scientifically grounded strategy for addressing these nutritional challenges. This process relies on a complex microbial ecosystem, typically dominated by lactic acid bacteria (LAB) and yeasts [12,13], whose metabolic activities are crucial for enhancing bread quality. LAB act as critical biochemical agents, reshaping the cereal matrix via acidification, extensive enzymatic activity, and metabolite synthesis [6,14]. Specifically, strain-dependent production of phytase can degrade phytic acid, thereby improving mineral solubility [15,16,17]. Meanwhile, proteolytic activity modifies the gluten structure, potentially enhancing protein digestibility [7,11,18] and generating bioactive compounds [19]. These diverse activities position LAB as central mediators in unlocking the nutritional potential of cereals through sourdough fermentation. However, translating these potential benefits into consistent and predictable in vivo human health outcomes proves challenging [20]. This difficulty arises because the efficacy of microbial interactions and their consequent nutritional impacts are highly context-dependent. Intrinsic cereal properties, the specific microbial strains used, process parameters (such as temperature, time, and dough yield), and host-specific factors all significantly influence these outcomes [7,12,21,22]. This inherent complexity presents critical translational challenges. It underscores the frequently observed gap between promising in vitro findings and demonstrable benefits in human consumers [20,23], thus necessitating a careful evaluation of the available evidence.

Given the nutritional limitations of cereals, this review critically examines the potential of LAB-driven sourdough fermentation to enhance nutritional quality using the updated post-2020 LAB taxonomy. It evaluates the strength and limitations of current evidence across three core objectives: (1) elucidating key LAB metabolic pathways relevant to nutritional outcomes, including phytase activity, proteolysis, EPS biosynthesis, FODMAP hydrolysis, and GABA synthesis; (2) critically evaluating the translational evidence for LAB-mediated modulation of macronutrient digestibility (protein, starch, fiber), micronutrient bioavailability (minerals, vitamins), and levels of anti-nutrients and bioactive compounds (phytate, FODMAPs, phenolics, peptides); and (3) identifying key barriers hindering clinical translation, such as interindividual variability and food matrix effects.

2. Lactic Acid Bacteria in Sourdough: Diversity, Taxonomy, and Relevant Metabolism

2.1. Taxonomy and Diversity of Lactic Acid Bacteria in Sourdough

The taxonomy of lactic acid bacteria (LAB), particularly the broad Lactobacillus group, has been recently reclassified based on whole-genome analyses [24]. Earlier classifications relying on 16S rRNA sequencing showed limited resolution and failed to capture major genomic and phenotypic differences [24,25]. This old system incorrectly grouped distinct species with different metabolic and ecological profiles. The broad term ‘Lactobacillus’ often obscures precise metabolic functions, hindering the reliable linkage of specific taxonomic units to their roles in sourdough fermentation. Reorganizing the Lactobacillaceae family into multiple, more coherent genera effectively addressed this issue [24,26].

This new taxonomic framework is powerful because it is predictive, allowing researchers to infer a genus’s functional potential from its phylogenetic identity. The contrast between key sourdough genera illustrates this principle. On one hand, a genus like Lactiplantibacillus employs a highly adaptable, generalist strategy. Its members, such as the resilient Lactiplantibacillus plantarum, are facultative heterofermenters with a broad metabolic repertoire, capable of utilizing diverse carbohydrates for rapid acidification, making them highly adaptable to different flour types [12,27]. On the other hand, a genus like Fructilactobacillus is a highly specialized microorganism. Its species, particularly the key Fructilactobacillus sanfranciscensis, are obligate heterofermenters defined by a unique metabolism that often utilizes fructose to enhance acetic acid production—a trait essential for traditional sourdough flavors [12,28].

Beyond this primary distinction, the framework reveals other specialized functional potentials now attributable to distinct genera. For instance, genomic analyses of the Limosilactobacillus genus highlight a high prevalence of genes for bacteriocin production, a genetic trait that may confer innate competitive advantages [29], while species like Companilactobacillus paralimentarius are noted for their ability to ferment pentoses from dietary fibers [28]. While these examples underscore key specializations,

Table 1. Key lactic acid bacteria genera in sourdough ecosystems: taxonomy, example species/strains, and relevant functional traits. Note: functional traits are often highly strain-specific and cannot be generalized to the entire species or genus.

Genus	Example Species	Key Relevant Function(s)/Trait(s)	Key Reference(s)
Pediococcus	P. pentosaceus	EPS production (alpha-glucan/Reuteran)	[30]
Lactiplantibacillus	Lp. plantarum (e.g., A16, A25, B11, B15)	Phytase activity/antifungal activity	[15]
Lacticaseibacillus	Ls. rhamnosus (e.g., RW-9595M, GG)	High EPS production (RW-9595M); potential probiotic activity	[30]
Fructilactobacillus	Fr. sanfranciscensis SB52	Proteolytic activity targets soluble proteins (e.g., β-amylase)	[31]
Levilactobacillus	Lv. brevis (e.g., A6)	proteolytic activity A6; phytase activity MW831027	[31,32]
Limosilactobacillus	Lm. Fermentum YL-11/Lm. reuteri	EPS production (galactose-rich; antioxidant, texturizing properties, bioactive and flavor peptide generation (e.g., kokumi/umami)	[22,28,29,30,33]
Leuconostoc	Lc. mesenteroides (e.g., subsp. mesenteroides SJRP55)	High EPS yield (dextran); dough properties, prebiotic potential.	[30,34]
Weissella	W. cibaria/W. confusa (e.g., MG1, Ck15, OF126)	High EPS yield (dextran); bread properties	[30,34]
Companilactobacillus	Co. alimentarius G4/Co. paralimentarius	High peptide count, targets β-amylase/ATIs; Pentose fermentation → Faster acidification and increased acetic acid	[28,31]

provides a broader overview of the key LAB genera found in sourdough and summarizes their diverse—and often highly strain-specific—functional traits.

Concurrently, the redefined ‘Lactobacillus’ genus (sensu stricto) is now much smaller, primarily comprising homofermentative species adapted to vertebrate or invertebrate hosts, such as the Lactobacillus delbrueckii group [24]. This distinct ecological niche helps to explain its lower prevalence in the plant-associated sourdough environment compared to the key genera detailed in Table 1 [24].

Ultimately, this predictive taxonomy provides a direct roadmap for operationalizing strain selection and design. It enables the rational formulation of multi-strain starter cultures by combining strains with complementary metabolic functions, a synergistic approach exemplified by the co-fermentation of functionally distinct LAB strains in food processing [27]. This genomic understanding also informs targeted strain engineering to enhance desired traits, such as the synthesis of specific aromatic compounds, vitamins, or antimicrobial bacteriocins [27,35]. Consequently, this represents a methodological shift away from traditional, often random, screening methods towards a more strategic and directed development of next-generation starters for improved product quality, nutritional value, and extended shelf-life [35,36,37]. The key to this strategic development lies in a deeper understanding of the specific metabolic pathways that underpin these functional traits

2.2. Relevant Metabolism and Acidification

Building on the taxonomic framework outlined above, lactic acid bacteria (LAB) drive sourdough fermentation primarily through their metabolic activities, with carbohydrate fermentation being particularly crucial for both leavening and acidification [38,39]. LAB are classified based on their glucose metabolism into three groups: obligately homofermentative, facultatively heterofermentative, and obligately heterofermentative 38].

Obligately homofermentative LAB, exclusively use the Embden–Meyerhof–Parnas pathway [40]. They produce lactic acid from hexoses, which acidifies the dough [41,42]. Obligately heterofermentative species (e.g., Fr. sanfranciscensis, Levilactobacillus brevis), on the other hand, use the 6-phosphogluconate/phosphoketolase pathway. This pathway generates lactic acid, CO₂, ethanol, or acetate [12,41]. This metabolic diversity creates a functional synergy: while homofermenters act as the primary acidifiers, heterofermenters provide the CO₂ essential for leavening [39], along with a complex mix of flavor compounds [12,41] (Figure 1).

Beyond these primary pathways, LAB exhibit remarkable metabolic flexibility, a key trait for their adaptation and success in the competitive sourdough environment [12,43,44]. A primary example is the substrate versatility of facultative heterofermenters like Lp. plantarum. These microbes can engage the phosphoketolase (PK) pathway to metabolize pentoses released from the hydrolysis of flour’s dietary fibers, a capability that contributes to its widespread presence and adaptation in diverse sourdough environments [12,28,43,44]. A different, highly specialized form of metabolic adaptation is seen in some obligate heterofermenters, which optimize energy yield by using external electron acceptors. Fr. sanfranciscensis exemplifies this strategy; when fructose is available, it is used as an electron acceptor to redirect metabolic flux away from ethanol and towards acetate production via acetate kinase, a process that yields an additional ATP molecule and enhances the formation of acetic acid—a key driver of sourdough’s characteristic flavor [12,28,43,44].

While LAB possess this diverse metabolic toolkit, the balance of these pathways during fermentation is not random; it is strongly governed by environmental conditions. This interplay is often quantified by the Fermentation Quotient (FQ)—the molar ratio of lactic to acetic acid [12,41]. Factors such as initial dough pH and hydration (dough yield) significantly impact microbial succession and acidification rates [12,45]. Temperature is a particularly critical lever; warmer temperatures (e.g., 35 °C) favor lactic acid accumulation (high FQ, often >4), while cooler temperatures (e.g., 28 °C) promote heterofermentative activity and, thus, acetic acid production (low FQ, ~2–3) [46]. The overall impact on the final aroma profile is complex, as different temperatures selectively promote the formation of distinct volatile compounds, including both desirable esters and potentially undesirable short-chain fatty acids [12,46].

The key outcome of these different metabolic pathways is the production of organic acids. This resulting acidification is not merely a byproduct; it is the central event that triggers sourdough’s nutritional enhancements. An acidic environment (pH 3.5–4.5):

-

Optimizes the activity of endogenous cereal enzymes (phytases, proteases) and LAB-derived enzymes, facilitating the breakdown of anti-nutrients like phytic acid and protein modification [11,47].

-

Enhances mineral solubility (iron, zinc), improving bioavailability [11,47]

-

Alters starch properties (e.g., structure, susceptibility to enzymes, possibly reducing gelatinization), contributing to a lower glycemic response [48,49].

Collectively, these acid-mediated modifications significantly reshape the cereal matrix, influencing nutrient release and digestibility [50].

3. Nutritional Benefits of Sourdough

3.1. Impact of LAB-Mediated Sourdough Fermentation on Mineral Bioavailability: Mechanisms and Challenges

Whole grains, especially unrefined grains, contain considerable amounts of essential minerals such as iron, zinc, calcium, and magnesium [51,52]. However, the human body’s absorption of these minerals is remarkably low; for example, it is estimated that only about 10% of the iron and 25% of the zinc in wheat are bioavailable [3]. Phytic acid (myo-inositol hexakisphosphate), concentrated in the bran layers, acts as the primary inhibitor by strongly chelating essential minerals [4,5]. Sourdough fermentation by lactic acid bacteria (LAB) is a well-recognized strategy to address this limited mineral uptake, holding significant potential to improve the nutritional value of whole grains. These benefits are primarily rooted in several key biochemical transformations. For instance, the pH reduction during fermentation directly enhances iron solubility (e.g., by promoting the conversion from Fe³⁺ to Fe²⁺), improving its release from the food matrix [53,54]. This acidic environment is also crucial for activating and optimizing endogenous phytases naturally present in the flour, thereby accelerating the hydrolysis of phytic acid [54,55,56]. Indeed, studies have shown that even mild acidification to pH 5.5 during sourdough fermentation can degrade approximately 70% of phytate in whole-wheat flour [56].

Beyond these pH-mediated effects, many LAB strains directly contribute to phytic acid degradation through their intrinsic microbial phytase activity [54,57]. Several studies have identified various LAB with significant phytase capabilities, with strains belonging to the Lactiplantibacillus genus, particularly Lp. plantarum, which is frequently highlighted for its efficacy [16,58]. Other species, including those from the Weissella, Pediococcus, and Leuconostoc genera, also play a valuable role in this process [57,59,60]. The synergistic action of these microbial phytases can lead to substantial reductions in phytate levels in sourdough, often ranging between 62% and 90%. This overall performance is markedly higher than what is typically achieved with yeast fermentation alone (25–75%) [56,59,61,62,63]. Importantly, phytase activity varies significantly even between strains of the same species, highlighting the importance of selecting the optimal strain for achieving optimal results [16,63]. To further complement these actions, organic acids like lactic and acetic acid, produced abundantly during fermentation, can also function as enhancers, improving overall mineral solubility and extractability [54]. This role for organic acids is substantiated by direct evidence, showing that lactic acid fermentation can significantly enhance iron absorption from bread [64], thus challenging a sole focus on phytate degradation.

Even though sourdough fermentation often succeeds in breaking down phytate, achieving real, measurable improvements in mineral bioavailability is far from guaranteed. However, this biochemical success does not consistently translate into predictable physiological outcomes. This gap between biochemical potential and physiological outcome represents a central theme in the modern understanding of sourdough’s nutritional impact. For instance, a study using a Caco-2 cell model found that even after near-complete phytate degradation, iron uptake remained low unless iron was supplemented [62]. This highlights a significant limitation of in vitro models: they often lack critical physiological context, including digestive enzymes, microbiota interactions, and systemic absorption dynamics [65]. This disconnect between phytate degradation and mineral uptake is also observed in vivo. A remarkable study observed a significant decrease in iron status indicators (serum ferritin and total body iron) in healthy women consuming low-phytate sourdough rye bread over 12 weeks [66]. The authors attributed this paradoxical outcome primarily to confounding dietary interactions (e.g., displacement of iron-enhancing foods) within the free-living study design, which complicated direct causal inferences about phytate removal alone. In addition, no significant improvements in mineral status (Mg, Fe, Ca) were observed in mice fed sourdough bread, according to Kwon et al. [67], who suggested that altered gut microbiota might compete with the host for mineral uptake. This discrepancy reveals that quantitative degradation does not necessarily equate to bioavailability gains, as demonstrated in in vitro and in vivo studies [62,66,67]. Therefore, these studies collectively suggest that a singular focus on phytate is insufficient, compelling a shift towards a more holistic view that accounts for dietary context, host physiology, and microbiome interactions.

Further complicating the picture, findings from in vitro models reveal that phytate reduction alone may not be the dominant factor in determining mineral bioaccessibility. In fact, the scientific evidence suggests that the influence of phytate can be overshadowed by other critical variables, including the composition of the raw materials and, most notably, the specific metabolic activity of the fermenting microbes themselves. Martinez Tuppia et al. [68], for example, reported similar mineral bioaccessibility between sourdough and yeast-leavened bread under standardized laboratory conditions. This suggests that other variables (like flour composition) can sometimes outweigh the effects of fermentation. Supporting this idea, research outside the sourdough context has shown that micronizing whole wheat significantly improves iron bioavailability, as measured by Caco-2 cell uptake, even though in vitro bioaccessibility metrics remain unchanged [69]. These findings highlight a fundamental limitation of relying solely on bioaccessibility data to predict actual absorption outcomes. Moreover, the specific microbial strains used in fermentation may unexpectedly influence mineral bioavailability. In an animal study, Gil-Cardoso et al. (2021) [70] compared multigrain bread fermented with different sourdough starters. Remarkably, one starter dominated by Fr. sanfranciscensis (SF) showed no detrimental effects on mineral absorption relative to white bread. In contrast, bread fermented with two other distinct sourdough starters (Carla (Ca) and Rebola (Re), with different microbial compositions) significantly decreased the absorption of Fe, Cu, and/or Zn [70]. This strain-dependent outcome reinforces the conclusion that factors beyond phytate—such as specific microbial metabolites or altered matrix interactions—can critically modulate mineral absorption from sourdough [70]. This evidence signals a potential paradigm shift, where the focus must broaden from what is removed during fermentation (i.e., phytate) to what is produced or altered by the specific microbial consortia involved. These complex and sometimes conflicting findings are summarized in

Table 2. Summary of key studies on the effect of sourdough fermentation (or related factors) on mineral bioavailability/Bioaccessibility.

Study Type/Model	Key Intervention/Comparison	Mineral(s) Studied	Main Outcome re: Mineral Bioavailability/Bioaccessibility/Status	Key Message/Implication/Reported Limitation	References
In vitro Caco-2 cells	Wheat sourdough (low phytate) vs. yeast/CBP bread	Iron (Fe)	No increase in Fe uptake despite phytate degradation (unless supplemental Fe added).	Phytate removal alone may be insufficient to drive absorption in this specific model.	[62]
Human intervention (healthy women)	Low-phytate sourdough rye bread vs. high-phytate rye bread (total diet context)	Iron (Fe) (status biomarkers)	Significant decrease in Fe status indicators (serum ferritin, total body Fe).	The outcome was likely caused by the displacement of other dietary iron sources by the intervention bread in a free-living context.	[66]
In vitro digestion model	Sourdough vs. yeast breads (white and whole grain)	Fe, Zn, Mg, Ca (bioaccessibility)	Comparable mineral bioaccessibility between sourdough and yeast breads.	The effect of flour composition may outweigh the fermentation-driven phytate reduction on bioaccessibility.	[68]
In vitro digestion and Caco-2 cells	Micronized vs. standard whole-wheat flour dough	Iron (Fe) (bioaccessibility and bioavailability)	Increased Fe bioavailability (Caco-2 uptake) despite unchanged in vitro bioaccessibility.	Highlights limitations of bioaccessibility measures; bioavailability can improve via other mechanisms.	[69]
Animal model (mice)	Sourdough bread vs. yeast bread vs. control diet	Mg, Fe, Ca (status)	Non-significant trends in mineral status.	Proposed gut microbiota competition for iron (Fe) might offset absorption benefits	[67]
Animal model (rats)	Multigrain bread fermented with different sourdough starters (SF, Ca, Re) with distinct microbial compositions vs. white bread.	Fe, Cu, Zn (apparent absorption)	Significantly decreased Fe, Cu, and/or Zn absorption with starters Ca and Re, but not with starter SF.	Strain-dependent negative effects exist; factors beyond phytate critically modulate absorption.	[70]

.

The factors governing mineral bioavailability are complex, extending beyond single components to include the dietary matrix, host physiology, and gut microbiota [65]. To advance beyond current research discrepancies, the use of advanced analytical techniques is essential. Notably, stable isotopic labeling (e.g., ⁵⁷Fe, ⁵⁸Fe, ⁶⁷Zn, ⁷⁰Zn) serves as a highly accurate tool for tracking mineral fate in vivo, providing precise quantification of absorption and distinguishing between phytate-dependent and independent mechanisms [53,71,72]. This methodological precision is vital for definitively assessing bioavailability from complex food matrices like sourdough. In parallel, in vitro cell models, particularly Caco-2 cell assays, are crucial for investigating cellular-level dynamics. These models are instrumental in identifying non-phytate inhibitory factors (e.g., polyphenols) and, crucially, provide a robust platform to systematically identify specific LAB-derived compounds that act as either enhancers or inhibitors of mineral transport [53,71,72]. Ultimately, this integrated approach, combining precise in vivo data with mechanistic in vitro insights, is essential for dissecting the numerous variables and validating the true nutritional impact of sourdough.

3.2. Sourdough Proteolysis by LAB: Enhancing Nutritional Quality and Modulating Gluten Immunogenicity

3.2.1. Nutritional Enhancement Through Sourdough Proteolysis

As consumer interest in nutritional quality grows, understanding the biological processes of sourdough fermentation, particularly its role in proteolysis, is crucial [6]. This traditional fermentation activates proteolytic activity, which significantly enhances the nutritional value of bread by improving protein digestibility, increasing the content of free amino acids, and releasing bioactive peptides [7,73]. These effects arise from the synergistic action of LAB-derived enzymes (e.g., proteinases, peptidases) and endogenous flour proteases activated by the acidic fermentation environment (pH 3.5–4.5) [6,74,75].

LAB-driven proteolysis profoundly reshapes the nutritional landscape of sourdough bread, offering several key enhancements. A primary contribution is the enrichment of sourdough with essential amino acids, notably lysine, a limiting amino acid in cereals [76]. Indeed, sourdough fermentation has been shown to increase free amino acid (FAA) levels, with variations attributed to flour type [7]. Traditionally fermented sourdoughs (e.g., t-SB) have been shown to exhibit markedly higher total FAA concentrations compared to yeast-leavened bread [77]. However, it is essential to consider that the bioavailability of this liberated lysine in the final bread can be partially compromised during the baking stage due to its susceptibility to the Maillard reaction [78]. Critically, this enrichment translates into tangible in vivo benefits, as the consumption of such sourdough leads to significantly higher and more sustained plasma FAA levels, indicating enhanced bioavailability of these protein constituents [77]. The extensive ‘pre-digestion’ of cereal proteins by LAB, already shown to enhance free amino acid profiles, is also widely reported to improve overall protein digestibility [77,78]. However, the evidence supporting this latter claim is considerably more complex and method-dependent, creating significant interpretational challenges. For instance, simpler in vitro protein digestibility (IVPD) assays frequently report notable increases under controlled conditions [7]. These findings, however, are not always corroborated by more physiologically relevant models. Standardized protocols, such as INFOGEST, for example, which more closely simulate the multi-phase human digestion process, can reveal minimal differences between sourdough and yeast-fermented bread [68]. This method-dependent variability highlights the need for harmonized and physiologically relevant testing protocols [78]. Another significant outcome of this extensive proteolysis is the generation of potentially bioactive peptides, whose specific functions and translational challenges are discussed in detail in Section 3.5, “Bioactive Compounds.”

3.2.2. Gluten Detoxification Strategies

While the proteolytic activity discussed previously enhances overall nutritional value, it faces a significant obstacle: the inherent resistance of certain cereal proteins to complete enzymatic breakdown, notably gluten proteins [74]. This resistance is attributed mainly to their high content of proline and glutamine residues, which necessitate specific peptidase activities for effective hydrolysis [79]. To counteract this, lactic acid bacteria employ a repertoire of cell envelope-associated proteinases and intracellular peptidases, alongside specialized transporters, to break down gluten into smaller peptides and free amino acids [11]. The selection of specific LAB strains is a critical determinant of proteolytic efficacy, precisely because this capacity is a highly variable trait, even at the intra-specific level [80]. Moreover, even when employing carefully selected strains, the action of a single LAB type typically results in only partial hydrolysis of the complex gluten network [79]. Peptidomic analyses provide further evidence, showing that LAB extensively degrades abundant soluble proteins (e.g., β-amylase, serpins, triticin), while gluten proteins are less affected [31].

The global prevalence of gluten-related disorders (GRDs), including celiac disease, wheat allergy, and non-celiac gluten sensitivity, is increasing, positioning gluten consumption as a significant public health concern [81]. The issue stems from gluten proteins, particularly gliadins, which contain immunogenic peptide sequences rich in proline and glutamine that are resistant to complete digestion by humans [81]. This resistance allows immunogenic peptides to persist, triggering adverse reactions in susceptible individuals. Consequently, strategies to mitigate gluten’s immunotoxicity are of paramount importance, and sourdough fermentation is positioned as a key technological advancement to address these challenges [82].

The primary mechanism by which sourdough fermentation counters gluten immunogenicity is through LAB-mediated hydrolysis that targets key immunodominant epitopes implicated in celiac disease, including the highly resistant 33-mer gliadin fragment [83]. This degradation is further facilitated by structural modifications, such as gluten depolymerization, which can alter epitope accessibility and enhance subsequent enzymatic breakdown [74,75]. While certain individual strains have shown a remarkable capacity for broad gliadin hydrolysis in vitro [84], the general efficacy of single strains is often limited [11,85]. This has led to the investigation of defined mixed cultures and enzyme combinations, which have proven more effective. For instance, in vitro studies have shown that specific probiotic co-cultures can extensively degrade gliadin, and this effect is further underscored by ex vivo findings where gluten hydrolyzed by advanced multi-strain consortia failed to elicit pro-inflammatory responses in duodenal biopsies from celiac patients [86].

A well-controlled clinical study provided a key demonstration of this approach. A specific sourdough fermentation protocol, extensively characterized in vitro and ex vivo for its ability to degrade the 33-mer epitope to very low levels (e.g., <10 ppm) and eliminate its immunotoxicity, was used to produce baked goods. A subsequent clinical trial demonstrated that these extensively hydrolyzed products were well-tolerated by celiac disease patients over 60 days without eliciting clinical symptoms or adverse histological changes [87].

However, translating these promising results into widespread, reliable solutions presents significant challenges. A primary issue is the conflict between safety and quality; extensive gluten degradation required to ensure safety for celiac individuals frequently compromises bread-making quality and gluten’s technological functionality (Figure 2). Strategies of ‘partial hydrolysis’ may preserve texture but render the products unsafe for celiac patients [75]. Furthermore, a rigorous double-blind, crossover clinical trial in individuals with self-reported non-celiac wheat sensitivity (NCWS) found no significant difference in gastrointestinal symptoms between sourdough and yeast-fermented bread, underscoring the translational gap between in vitro observations and consistent clinical benefits [88].

Safety assessment is further complicated by methodological challenges, as standard ELISA kits can be contradicted by more accurate mass spectrometry results due to epitope unmasking during proteolysis [74,89]. Even after optimized fermentation, residual peptides with intact epitopes often persist [31,90]. Research using typical artisanal cultures has shown no significant decrease in prominent immunogenic peptide families [74]. This highlights a critical gap between optimized lab processes and common practice. Finally, significant inter-patient variability in tolerance has been observed even with modified gluten, underscoring that patient-specific factors play a crucial role [91].

In conclusion, while sourdough fermentation offers clear nutritional benefits by enhancing protein digestibility and enriching the free amino acid profile, its application as a gluten detoxification strategy remains complex and limited. Despite promising research on mitigating gluten immunogenicity, sourdough bread remains unsafe for individuals with celiac disease for general consumption outside of tightly controlled clinical studies. Current technology, while capable of targeted epitope degradation, cannot consistently reduce gluten below the 20 ppm ‘gluten-free’ threshold. This gap is driven by significant translational challenges, including the inherent quality–safety trade-offs, methodological variability, and inter-patient specificity.

3.3. Sourdough LAB-Mediated Modification of Cereal Fibers: Pathways to Enhanced Prebiotic Potential

The well-recognized benefits of dietary fiber, pivotal in promoting digestive function and preventing chronic diseases [11,92,93], are not solely dependent on its quantity, but critically on its bioaccessibility and fermentability by the gut microbiota. Many native cereal fibers, such as the arabinoxylans abundant in whole grains [94], present structural complexities that can limit their optimal utilization by gut microbes and thus, their full prebiotic potential [93]. In this context, Sourdough fermentation emerges as a unique biological process capable of profoundly remodeling these intricate fiber structures [95]. But does this microbial transformation consistently translate into a significantly enhanced and demonstrable prebiotic effect in the final bread?

Indeed, a primary pathway by which sourdough fermentation begins to address the question of enhanced prebiotic potential is through the extensive structural remodeling of native cereal fibers, particularly the aforementioned water-extractable arabinoxylans (WE-AX) [94]. This depolymerization is driven by a combination of LAB-driven acidification and the direct action of microbial enzymes, notably xylanases produced by common sourdough strains, such as Lp. plantarum [96,97]. This results in marked reductions in their molecular weight—studies confirm a drop from a range of 400–800 kDa to as low as 100–200 kDa during prolonged fermentation [95], potentially leading to increased solubility. The crucial outcome of this enzymatic breakdown is the generation of a diverse pool of arabinoxylan-oligosaccharides (AXOS). These AXOS are increasingly recognized for their significant prebiotic activity; for instance, in vitro studies using lab-generated AXOS have demonstrated their capacity to selectively stimulate the growth of beneficial gut bacteria, including Bacteroides ovatus and Bifidobacterium adolescentis [98], and to concurrently promote the production of health-associated short-chain fatty acids (SCFAs) [98,99].

Beyond the structural remodeling of existing plant fibers, LAB employs another significant strategy to enrich the prebiotic landscape of sourdough bread: the de novo synthesis of exopolysaccharides (EPS) [96]. These microbially derived polymers are typically synthesized from simpler carbohydrates, such as sucrose, via enzymes like glycosyltransferases (GTFs) [96,100]. Microbial EPS encompass both homopolysaccharides (HoPS), such as various α-glucans (e.g., dextran, reuteran) and fructans (e.g., levan, inulin) often produced by genera like Leuconostoc, Limosilactobacillus, and Fructilactobacillus [30,101] (as detailed in

Table 3. Examples of homopolysaccharides (HoPS) produced by lactic acid bacteria relevant to sourdough.

LAB	Homopolysaccharides (HoPS)	References
Leuconostoc mesenteroides	Dextran	[30,105,106]
Leuconostoc citreum	Dextran	[30,106,107]
Lacticaseibacillus casei	Dextran	[30]
Latilactobacillus sakei	Dextran	[30,108]
Limosilactobacillus reuteri	Dextran, Reuteran, Inulin, Levan	[30,106,109]
Weissella (cibaria, confusa)	Dextran	[30,103,106,110]
Limosilactobacillus frumenti	Levan	[30]
Fructilactobacillus sanfranciscensis	Levan	[30,103,106]

), and structurally diverse heteropolysaccharides (HePS). These EPS make a valuable contribution to the bread’s soluble dietary fiber (SDF) content, with studies reporting substantial increases (up to ~40%) in SDF under optimized EPS-producing fermentation conditions (e.g., using selected high-yield strains like Weissella confusa SLA4 and significant sucrose supplementation) [102]. More critically, many of these EPS fractions exhibit potent prebiotic properties. For instance, in vitro fecal fermentation studies have shown that purified HePS from strains such as Lactobacillus helveticus can selectively stimulate beneficial Bifidobacterium and Lactobacillus populations, while concurrently enhancing the production of health-promoting short-chain fatty acids (SCFAs) like acetate and butyrate [103,104]. Notably, the prebiotic efficacy of some of these EPS is so pronounced that their selectivity index (SI) can, at specific time points, rival or even surpass that of the well-established prebiotic inulin [103].

The significance of LAB activity within the sourdough matrix, therefore, extends beyond the individual modifications of arabinoxylans or the singular synthesis of exopolysaccharides [11]. It is the co-occurrence and combined effect of these two distinct microbial processes that culminate in a profoundly altered dietary fiber landscape [11,95,102]. Consequently, sourdough bread is no longer characterized by its original, relatively simple cereal fiber profile [94]. Instead, it presents a novel, heterogeneous mixture of fermentable substrates [98,103]. This enriched signature is fundamentally different from that of unfermented cereals, comprising both structurally diverse AXOS derived from plant cell wall remodeling [95,98] and newly synthesized microbial EPS [30,96]. It is hypothesized that such a complex and varied array of oligosaccharides and polysaccharides provides a broader nutritional niche for gut microbes [93]. This diversity of substrates, in turn, could support a more diverse and resilient gut microbiome compared to simpler or single-source fiber inputs [92,99].

The in vitro bioactivity of sourdough-derived arabinoxylan-oligosaccharides (AXOS) and newly synthesized exopolysaccharides (EPS) is indeed compelling. Foundational laboratory evidence suggests their potential to favorably modulate gut microbial activity and promote the production of health-associated short-chain fatty acids (SCFAs) [98,99,103,111]. However, the actual profile and concentration of these potentially beneficial modified fibers in the final sourdough bread are critically dependent on factors such as the specific LAB strains utilized and the precise fermentation conditions employed [20,34,106]. Translating such in vitro-observed fiber modifications and their hypothesized prebiotic effects into tangible in vivo human health benefits, however, presents significant challenges. These challenges arise primarily from the complexity of the human gut ecosystem and considerable inter-individual variability in responses to dietary fibers [20,34]. Thus, well-designed clinical trials directly linking the modified fiber profile of sourdough bread to consistent prebiotic effects or validated health outcomes are still limited and often yield inconclusive results [20,34]. Future research must, therefore, prioritize well-designed human intervention trials. These trials should move beyond simple compositional analyses of bread to assess tangible and sustained impacts on human gut microbiome structure and its metabolic output (e.g., SCFA profiles). Particularly when assessing the gut microbiota, such interventions should involve prolonged periods to capture stable and meaningful shifts. Crucially, they must also evaluate associated host physiological responses and validated health markers to fully ascertain the actual prebiotic value of these sourdough-fermented cereal fibers for practical application [92,112].

3.4. Effect of Sourdough Fermentation on the Glycemic Response

Another benefit of sourdough fermentation is its ability to reduce bread’s glycemic index (GI) through multiple biochemical and structural mechanisms [22]. Compared to conventional white bread (GI ≈ 75), sourdough wheat bread often exhibits a lower GI (≈54) due to fermentation-induced changes in starch digestibility [22,113,114]. However, the underlying mechanisms are complex, multifactorial, and their relative contributions are still debated (Figure 3) [20].

3.4.1. Impact on Starch Structure and Resistant Starch Formation

Several mechanisms related to starch structure and properties are implicated. Firstly, researchers have postulated that chemical changes during fermentation in the acidic sourdough environment may diminish the degree of starch gelatinization during baking compared to conventional bread [48], which would inherently slow subsequent enzymatic digestion. However, the structural mechanisms influencing starch digestion are complex. While acidity may reduce gelatinization, other factors, such as increased crumb porosity, noted particularly in sourdough fermentation, could potentially enhance enzymatic access to starch [115]. This interplay underscores the complex nature of these changes and the need for further research on starch structural changes during fermentation [49,115]. Secondly, the modified conditions during sourdough fermentation, including the acidic environment, favor conditions for starch retrogradation upon cooling (forming resistant starch type 3, RS3) [116,117]. This retrogradation increases the overall resistant starch (RS) content, and studies indicate that sourdough bread often contains higher levels of RS compared to conventional bread. The magnitude of this increase varies considerably depending on multiple factors, such as flour type and starter culture, which may, in turn, influence glucose metabolism. Although sourdough fermentation consistently increases resistant starch content, with some studies reporting significant relative gains [116,118], a critical assessment of the absolute values suggests the overall quantitative contribution is often modest. This perspective is supported by findings that attribute sourdough’s glycemic benefits to other mechanisms, such as delayed gastric emptying, rather than the increase in RS itself, thus highlighting that direct evidence for this specific pathway’s role remains limited [116]. Furthermore, storage time and conditions significantly impact final RS levels, with storage durations of up to 5 days, for instance, shown to substantially increase RS content [119].

3.4.2. Role of Organic Acids and Dietary Fibers

Organic acids produced by LAB (e.g., Fr. sanfranciscensis, Leuconostoc spp.) are also major contributors [120]. Their physiological mechanisms appear varied; lactic acid has been suggested to lower the rate of starch digestion within the bread matrix itself, while acetic acid acts by delaying the gastric emptying rate [22]. However, the role of delayed gastric emptying has been challenged, as some studies using specific methodologies found no delay or even faster emptying with certain sourdough products [77,121]. This indicates the mechanism may not be universally applicable, possibly because other significant fermentation-induced changes occur, such as enhanced protein digestibility [77], and potential direct effects of organic acids on starch bioavailability [121]. Additionally, the increased content of soluble dietary fibers, such as microbially produced exopolysaccharides (EPS) and solubilized arabinoxylans (discussed in Section 3.3), contributes another layer of complexity. These fibers may lower the glycemic response by increasing digesta viscosity, which can slow both gastric emptying and glucose absorption in the small intestine [122]. However, it is worth noting that enzymatic or processing-induced degradation of other viscous fibers (like β-glucans and arabinoxylans) can reduce their molecular weight and viscosity, potentially weakening this beneficial effect [123]. Furthermore, beyond these viscosity-mediated effects, the dietary fibers generated or modified during sourdough fermentation lead to the production of short-chain fatty acids (SCFAs) [99,103]. Among these, butyrate has been suggested to potentially improve insulin sensitivity [124]. Notably, combining sourdough fermentation with added dietary fiber (5–10%) has been shown to potentially achieve very low GI values (<55) [118]. This finding suggests a potential synergistic effect between the fermentation process and the added fiber.

Collectively, the modifications to starch, the production of organic acids, and the enhanced functionality of dietary fibers form the basis of sourdough’s potential glycemic benefits [20]. The primary scientific challenge, however, lies in quantifying the precise individual contribution of each of these pathways, an issue consistently highlighted in recent critical and systematic reviews [20,125]. This difficulty stems from the significant interplay between mechanisms and the well-documented discrepancy between in vitro tests and complex in vivo responses [20,116]. Therefore, to move from mechanistic hypotheses to empirical results,

Table 4. Summary of experimental studies on the impact of sourdough fermentation on glycemic index and postprandial glucose/insulin response.

Fermentation Type (Duration, Temp)	LAB Strain(s)	Flour Type (in Final Product)	GI Impact/% GI Reduction	Comments/Key Findings
Sourdough starter refreshed for 10 days (14 °C).  Final dough raised 4–5 h (28 °C).	- Yeasts: Saccharomyces cerevisiae, Saccharomyces exiguous (1:250) - LABs (dominant): Lactobacillus acidophilus, Lacticaseibacillus casei	Various wheat flours (Commercial, Organic, Einkorn)	Lower postprandial glucose and insulin AUCs	Human study. Significant reduction in insulin responses across all tested wheat flours. Significant reduction in glucose responses for organic and einkorn flours only. Einkorn flour bread showed the most favorable metabolic responses [113].
Sourdough starter used.  (No specific duration or temp provided in paper)	(Not specified in paper)	1. Whole-grain flour (mean of various types, likely rye, oats, buckwheat)	Mean GI: 50.50 (significantly lower than refined flour: 60.50)	Human study. Focus on “functional breads.” 65% of tested breads were low GI (<55). Sourdough, whole-grain flour, and oilseeds contribute to GI reduction [114].
		2. Wheat/spelt and other flours (oat, buckwheat, rice, or mixed)	Wheat/spelt mean GI: 60.6  Other mean GI: 56.0	Human study. Part of the functional bread study. Rye breads generally had a lower GI compared to wheat/spelt [114].
Sourdough fermentation with added oat fibre (~8.7% of flour weight)	Lp. plantarum P1; Lv. brevis P2	WF/WMF (50:50) + Oat Fibre	GI: 53.7 (vs. 72 for yeast control bread made with 100% white flour)	Human study. Sourdough fermentation combined with oat fibre significantly reduced the GI of bread [118].
Type-2 Sourdough (24 h at 30 °C)	Lp. plantarum ELB75, Lv. brevis ELB99, Saccharomyces cerevisiae TGM55	Whole wheat flour	Control eGI: 76.93  Sourdough eGI: 54.05 (~29.7 Reduction)	In vitro study (Estimated GI). Type-2 fermentation at 30 °C provided the greatest reduction in eGI [126].
Multi-stage fermentation using an in-house starter (40 h for starter preparation).	Living LABs and Yeasts (Specific strains not specified)	Whole-grain rye flour	Glucose iAUC: No significant difference between the three bread types Insulin iAUC: Unfermented Rye bread gave the lowest response. Sourdough bread showed no improvement vs. yeast wheat control.	Human study. Sourdough fermentation did not improve glucose response. Unfermented rye bread showed the most favorable insulin response. The study suggests that sourdough fermentation in these conditions might lead to ‘less favourable’ metabolic responses compared to the unfermented version [127].
Sourdough fermentation (process details not mentioned)	(Not explicitly mentioned in paper)	White and whole-wheat flour	Both sourdough breads (white and whole-wheat) showed a statistically significant reduction in glucose iAUC compared to their yeast-leavened counterparts (p < 0.001).	Human study. Both white and whole-wheat sourdough breads showed significantly lower glycemic responses and higher resistant starch compared to yeast-leavened counterparts. The study concluded that the fermentation method (sourdough effect) was the primary factor for the reduction [116].

Notes: GI values and reductions are typically presented in comparison to a control bread (e.g., yeast-leavened bread or unfermented bread) or a glucose reference. GI values below 55 are generally considered low, 56–69 medium, and 70+ high. “GI Impact/% GI Reduction” column may present direct GI values, incremental area under the curve (iAUC) for glucose/insulin, or percentage reductions in these parameters. Lower values or higher percentage reductions indicate a more favorable glycemic response. Studies categorized as “Human study” (or “in vivo”) directly measure responses in human subjects. “In vitro study” (e.g., estimated GI) provide valuable insights but are not direct human clinical measurements. Where “N/A” or “Not specified in paper” is noted, the original publication did not provide that specific detail for the fermentation parameters or microbial strains. Abbreviations: GI, glycemic index; iAUC, incremental area under the curve; eGI, estimated glycemic index; LAB, lactic acid bacteria; RS, resistant starch; SD-WB, sourdough white bread; SD-WhB, sourdough whole-meal bread.

provides a comparative summary of the final glycemic impact reported in key clinical studies.

3.4.3. Influencing Factors, Broader Implications

The final glycemic impact of sourdough bread is not a fixed value but is rather modulated by a complex interplay of formulation, processing, and host-related factors [13]. As suggested by the varied outcomes in Table 4, the choice of starter culture is crucial in the glycemic response, as microbial strains vary greatly in their overall metabolic activities [115]. This has been confirmed in animal studies, where different sourdough starters led to distinct glucose and insulin responses, even when the same flour was used [70]. These variations may be linked to differences in acidification patterns or the formation of resistant starch (RS) [70,115]. Flour type and fermentation method are both crucial influencing factors. For instance, one recent study on functional breads demonstrated that using whole grain flour leads to a significantly lower glycemic response compared to non-whole grain flour [114]. The same study also found that using sourdough fermentation was a more effective strategy for lowering the bread’s GI than using commercial yeast alone [114]. The effect of gluten-free bread is inconsistent; however, sourdough can be a strategy to lower the typically high glycemic index (GI) of some glute-free products [128,129]. Furthermore, the overall complexity of the bread formulation can also play a role, with one study on commercial functional breads finding that a higher number of ingredients correlated with a higher GI [114]. This result might appear counterintuitive, considering the potential benefits often associated with functional ingredients. Fermentation conditions, such as temperature (e.g., 25 vs. 30 °C), and the type of fermentation method (e.g., spontaneous vs. added starter), also significantly influence the estimated GI and related properties of sourdough bread [126]. However, the subsequent effect of reheating appears complex; while in vitro studies clearly show that reheating can increase starch digestibility compared to the cooled state, potentially reversing RS formation [130,131], whether this translates to a consistent in vivo glycemic effect remains poorly understood. Finally, individual host responses, influenced by factors such as gut microbiota composition, significantly modulate the perceived glycemic effect [132].

Sourdough fermentation can favorably modulate key factors such as starch behavior, the resulting organic acid profile, and overall fiber complexity [50,133]. However, translating these mechanistic advantages into predictable clinical outcomes remains complex. The ultimate glycemic impact is highly variable, influenced by a wide array of factors including starter culture, flour type, and processing conditions. This complexity is also reflected in the nuanced effectiveness of sourdough compared to other grain processing methods [134]. Therefore, standardized human clinical trials are essential to move beyond mechanistic hypotheses and definitively validate the metabolic benefits of sourdough bread.

3.5. Modulation of Phytochemicals and Bioactive Compounds by Sourdough Fermentation: Mechanisms, Bioaccessibility, and Translational Challenges

Sourdough fermentation acts as a natural bioreactor, profoundly modulating the profiles of various phytochemicals and bioactive compounds [14,135]. Through their enzymatic and metabolic activity, lactic acid bacteria (LAB) are capable of synthesizing novel molecules, releasing compounds from the food matrix, and degrading others [6]. However, the extent of these transformations, and their ultimate impact on the bread’s final nutritional and functional properties, are highly strain- and substrate-dependent [135]. The following table (

Table 5. Summary of key bioactive compounds modulated by sourdough fermentation: mechanisms, in vitro bioactivities, and translational challenges.

Bioactive Compound Class	Primary Modulation Mechanism	Reported Biological Activity (In Vitro/Ex Vivo)	Key Translational Challenge	Key Reference(s)
Phenolic Compounds	Release from bound forms via microbial/endogenous enzymes (e.g., esterases, β-glucosidases). Use of enriched substrates (e.g., germinated grains) to boost initial content.	Chemical antioxidant capacity (e.g., DPPH, ABTS). - Cellular effects (e.g., anti-inflammatory, anti-hemolytic), though translation to in vivo outcomes is not always direct.	Overcoming multiple hurdles: Complex food matrix effects hindering bioaccessibility. Poor correlation between simple chemical assays and cellular outcomes.	[136,137,138,139]
Bioactive Peptides	Proteolytic release driven by the combined action of endogenous flour proteases and specific LAB-derived peptidases.	Cellular antioxidant (ROS reduction) and anti-inflammatory (NF-κB pathway) effects. Demonstrated thermal stability, with bioactivity retained after baking	Overcoming multiple hurdles: Managing biological variability (strain-specificity).Addressing poor systemic bioavailability. Limited generalizability due to high strain and substrate specificity. Incomplete understanding of the precise mechanisms of action.	[137,140]
Vitamins (B-group)	Can result in a net decrease in content for some vitamins (e.g., folic acid), likely due to microbial consumption.	Primarily compositional analysis; specific biological activity is not typically the focus.	Need for in vivo studies to confirm the actual bioavailability and physiological impact of the final vitamin profile.	[137,141]
GABA (γ-Aminobutyric acid)	De novo synthesis from glutamate via the GAD system, enhanced by using enriched substrates (e.g., germinated rice).	Diverse neuroactive and metabolic effects reported in the literature (e.g., anti-hypertensive).	Ensuring efficient and safe production: - Overcoming low yields by screening for hyper-producing strains. - Avoiding co-production of harmful biogenic amines.	[139,142]
Anti-nutrients (Phytate, RFOs)	Enzymatic degradation by microbial and endogenous enzymes (e.g., phytase, α-galactosidase), activated by low pH.	Indirect nutritional benefit: improves the potential bioavailability of minerals and reduces compounds causing digestive discomfort.	Ensuring sufficient degradation, which is highly dependent on strain selection and fermentation conditions.	[135,138]

Note. TPC, total phenolic content; DPPH, 1,1-diphenyl-2-picrylhydrazyl; ACE, angiotensin-converting enzyme; ROS, reactive oxygen species; NF-κB, nuclear factor kappa B; GAD, glutamate decarboxylase. The table summarizes key findings and is not an exhaustive list of all reported activities or challenges. The listed references are illustrative examples supporting the summarized points.

) provides a comparative overview of the primary compound classes affected, detailing their generation mechanisms, potential activities, and key translational hurdles.

3.5.1. Phenolic Compounds: Content, Profile, and Bioaccessibility

Sourdough fermentation enhances the concentration and diversity of phenolic compounds, as demonstrated in pearl millet sourdough which showed increased levels of gallic acid, quercetin, and vanillic acid, thereby contributing to an enhanced antioxidant capacity [19]. LAB possess enzymatic activities, primarily esterases (e.g., feruloyl esterases, tannases) and glycosidases, which facilitate the release of free phenolic acids and aglycones from their bound forms (esters and glycosides). This release significantly enhances their bioaccessibility and subsequent bioavailability, given that these simpler phenolic structures are generally more readily absorbed in the gastrointestinal tract compared to their complex precursors [137,143]. Once absorbed, phenolics can undergo biotransformation, which may affect their biological activity, potentially enhancing, diminishing, or changing it qualitatively [144]. Furthermore, LAB can subsequently metabolize these free phenolic acids through reductases and decarboxylases, impacting other biological activities [6]. The specificity of individual strains, as well as defined microbial consortia, plays a critical role. For instance, Lp. plantarum LG1034 increased total phenolic content (TPC) by approximately 83% and antioxidant activity (via DPPH) by 3.41-fold in medium gluten bread flour dough compared to the unfermented dough control [145]. Similarly, consortia of LAB strains (e.g., Lp. plantarum and Lc. mesenteroides) demonstrated synergistic effects, yielding higher total polyphenol content (TPC) and flavonoid content in quinoa sourdoughs [146].

The altered phenolic profile resulting from fermentation is by no means final; it is further subjected to complex transformations during processing [137]. Fermentation modifies the phenolic profile, but baking introduces complexity by potentially degrading or releasing compounds, such as flavonoids [147]. Interestingly, some thermal degradation products may contribute to antioxidant activity, while sourdough acidity improves phenolic retention during baking [147]. Even if phenolic compounds are favorably influenced during fermentation and baking, their biological relevance is largely determined by their bioaccessibility and behavior during digestion [137,143]. For instance, while sourdough fermentation may increase total phenolics, studies employing common in vitro static digestion models often report a reduced proportional release of these compounds from the bread matrix due to interactions with fiber, proteins, or lipids [148]. Fortified bread, however, may retain higher absolute amounts of bioaccessible ferulic and sinapic acids [148]. Crucially, these static models may not fully account for the extensive metabolism of unabsorbed phenolic compounds by the gut microbiota in the colonic phase, which can lead to the formation of different, potentially bioactive, metabolites [137,143]. This complex interplay involving matrix effects during digestion, subsequent microbial biotransformation in the colon, and eventual host metabolism significantly influences which compounds, and in what forms, are available for absorption and systemic activity [137]. Given these complex transformations, it becomes evident that in vitro antioxidant activity is an imperfect proxy for biological efficacy. While often used in studies, this metric does not always correlate with outcomes in complex biological systems [19,20]. For example, fermented pearl millet showed no significant benefits over unfermented millet in cell-based assays despite having a higher phenolic content and in vitro antioxidant capacity [19]. In contrast, fermented emmer wheat exhibited ex vivo antihemolytic effects and reduced inflammatory markers (IL-8, COX-2, ICAM-1) in intestinal cells, which is linked to fermentation-induced increases in free phenolic acids, such as gallic and ferulic acid [149]. This difference underscores the challenge of extrapolating lab-based antioxidant data to complex biological systems, and suggests that the specific profile of released phenolics—rather than their total concentration—may be the key determinant of biological efficacy. However, positive correlations are possible, as demonstrated by a specific sourdough fermentation that exhibited both high in vitro antioxidant capacity and enhanced cellular protection ex vivo [136].

In summary, while sourdough fermentation effectively increases the bioaccessibility of phenolic compounds in vitro, their ultimate physiological relevance is challenged by processing losses, matrix interactions during digestion, and the poor predictive power of simple antioxidant assays.

3.5.2. Bioactive Peptides, GABA, and Other Key Metabolites Modulated by Sourdough Fermentation

LAB proteolysis during fermentation generates low-molecular-weight (LMW) bioactive peptides, which have been demonstrated to possess antioxidant, anti-inflammatory, and antihypertensive properties [137,150,151]. These peptides have been shown to modulate inflammatory pathways in macrophage cell models by inhibiting NF-κB signaling and reducing the secretion of pro-inflammatory cytokines, such as IL-1β [151]. Additionally, they exhibit potent antioxidant activity ex vivo by reducing reactive oxygen species, in some cases more effectively than reference antioxidants [151]. Specific strains, such as Co. farciminis and Fr. sanfranciscensis, have been reported to generate peptides that not only inhibit NF-κB but also downregulate iNOS and COX-2 expression while retaining their activity post-baking [140]. Furthermore, peptides generated during sourdough fermentation of barley and pigmented wheat, using specific LAB/yeast combinations, exhibited superior COX-2 inhibition [152]. This inhibitory effect was greater than that observed for peptides derived from conventional wheat fermented with a different microbial consortium [140]. Notably, emmer wheat fermentation doubled angiotensin-converting enzyme (ACE) inhibitory activity, suggesting antihypertensive potential [149].

While in vitro studies show promise, the clinical relevance of these peptides remains uncertain. Extensive degradation by digestive enzymes severely limits systemic bioavailability [20,23]. This limitation is particularly pronounced for peptides larger than tripeptides, with only trace amounts typically reaching circulation. However, recent evidence indicates that the final biological activity of peptides depends heavily on their amino acid composition, not necessarily due to resistance to digestion, but because digestion can release smaller, potentially more bioactive, fragments from the original peptide precursor [153]. Some researchers propose that these peptides might still exert beneficial effects locally within the gastrointestinal tract itself, even without significant systemic absorption [23].

Beyond peptides, sourdough fermentation can enrich bread with other bioactive compounds. A key example is the enrichment of gamma-aminobutyric acid (GABA), a neuroactive compound synthesized via LAB glutamate decarboxylase (GAD) activity [142]. For instance, strains such as Latilactobacillus sakei exhibit significant GABA-producing potential [139]. This potential is particularly realized when using specific substrates, such as germinated brown rice in sourdough starters, leading to higher final GABA levels compared to controls [139]. Levels of other beneficial metabolites, such as the short-chain fatty acids lactate and acetate, which are known for their gut health benefits, can also be modulated. Significant increases in SCFAs can be achieved through targeted fermentation using specific microbial consortia [136].

LAB-mediated sourdough fermentation significantly modifies the phytochemical and bioactive profile of bread, with effects being highly strain- and substrate-dependent [70]. These transformations have been observed not only in traditional breads but also in alternative matrices such as sorghum biscuits, where fermentation led to improved antioxidant profiles and enhanced amino acid composition [154]. While many studies highlight the enrichment of compounds such as GABA, short-chain fatty acids, and phenolics, this modulation is not invariably positive across all bioactive components. For example, while total phenolic content (TPC) increased in one study of legume-enriched sourdoughs, concurrent losses of other bioactive compounds (e.g., carotenoids) were observed [138]. Such compound-specific outcomes underscore the complexity of fermentation effects. Preliminary in vivo animal studies further suggest potential systemic relevance, showing the modulation of inflammatory markers and lipid metabolism by certain sourdoughs, although results were sourdough-specific [70]. However, while the in vitro bioactivities of many of these compounds (such as phenolics and peptides) are well-documented, their overall translational efficacy in humans is constrained by bioaccessibility limitations and clinical evidence [20,150]. Specific strategies, such as optimized fermentation using selected strains, show promise for enhancing yields of potentially beneficial peptides like lunasin [155]. Future research should prioritize in vivo validation and optimization of fermentation protocols to maximize the delivery of bioactive compounds and their physiological efficacy.

3.6. Impact of Sourdough Fermentation on Vitamin Content: Synthesis, Stability, and Nutritional Reality

Although sourdough fermentation is frequently associated with enhanced B-vitamin content, its effects are nuanced and context-dependent [156]. While certain vitamins, such as riboflavin (B2), folate (B9), and cobalamin (B12), often increase due to microbial synthesis, others, like thiamin (B1) and pyridoxine (B6), exhibit variability depending on the microbial strains and fermentation conditions [156,157]. Specific lactic acid bacteria (LAB) can synthesize water-soluble vitamins, including riboflavin (B2) and folate (B9) [133]. Additionally, specific LAB strains, such as Lm. reuteri, are known to produce cobalamin (B12), offering a pathway for in situ food fortification [158]. Consistent B12 production in cereal fermentations can be effectively achieved using specialized adjunct cultures, such as Propionibacterium freudenreichii [159].

The substrate choice also plays a role, as using flour from germinated grains can improve the initial content and bioavailability of vitamins [133]. However, net vitamin levels in the final product are not consistently increased. Studies have reported decreases in folic acid content after fermentation with specific LAB inoculants compared to spontaneous fermentation or unfermented flour, possibly due to microbial consumption outweighing synthesis [141]. Similarly, decreases in vitamin E (tocopherols/tocotrienols) [22,160] ] and carotenoids [138] have been observed, likely due to factors such as oxidative losses that occur during the fermentation process itself and further degradation during subsequent processing steps. Thiamine (B1) levels also show variability during fermentation, sometimes decreasing or remaining constant [22]. These contrasting outcomes, ranging from microbial synthesis to oxidative loss, underscore the complexity of fermentation’s net effect on vitamin content.

Table 6. Summary of key studies on the impact of fermentation on vitamin content.

Vitamin	Model/Substrate	Typical Effect	Mechanism and Key Factors	Comments/Key Findings	Reference
Active Vitamin B12 (Cobalamin)	In vitro (whey-based medium)	Increase (up to 4-fold)	Mechanism: enhanced biosynthesis of limiting precursor (DMBI). Factors: precursor supplementation, bacterial strain, timing.	Supplementation with safe precursors (RF + NAM) yielded comparable or superior B12 levels compared to direct precursor (DMBI) addition in most strains.	[161]
Vitamin B12 (Stability Study)	In vitro (wheat bread vs. sourdough rye bread)	Decrease (loss of 22–44%)	Mechanism: thermal degradation during baking. Factors: B12 form (CNCbl most stable), baking process, duration, pH	Sourdough process (longer baking, low pH) caused greater B12 loss compared to wheat bread processes.	[159]
Active Vitamin B12	In vitro and sensory (composite bread with fermented soya flour/rice bran)	Increase (3.0–4.9 µg/100 g in bread); high stability (~4% loss).	Mechanism: in situ biosynthesis by P. freudenreichii. Factors: starter culture combination, fermentation matrix.	Simultaneous production of B12 (for nutrition) and dextran (for improving texture and masking off-flavors).	[162]
Vitamin B12	Review article (various plant-based matrices)	Increase (significant)	Mechanism: in situ biosynthesis. Factors: microorganism selection (propionibacterium, LAB), matrix, conditions.	In situ fermentation is a promising, sustainable “Green Chemistry” approach to fortify plant-based foods with B12.	[163]
Primarily Vitamin B12	Review article (fermented vegetables, fruits, cereals and legumes)	Increase (significant amounts)	Mechanism: in situ biosynthesis. Factors: microorganism selection (propionibacterium), food matrix, post-fermentation processing.	Concludes fermented plants are viable B12 sources but highlights a major data gap regarding bioavailability.	[164]
Folic acid, Pantothenic acid	In vitro (multigrain sourdoughs: wheat, rye, barley)	Folic acid: Decrease (slight).	For Folic Acid loss: microbial consumption. Factors: flour composition, fermentation type (spontaneous vs. with starter culture).	While fermentation caused a slight decrease in folic acid, it led to increased antioxidant activity. Higher levels of pantothenic acid were also observed post-fermentation.	[141]
Vitamin E (all 8 vitamers)	In vitro (whole-meal wheat and rye bread with/without red palm oil)	Decrease (up to 90% loss); increased with oil addition.	Mechanism: loss via oxidation; retention via antioxidants from added oil. Factors: oil incorporation, fermentation type.	Breadmaking itself can destroy up to 90% of native vitamin E; fortification protects against these losses.	[117]
Various (Folate, K, B2, B12, etc.)	Review article (various matrices, incl. sourdough)	Variable: generally, folate, K, B2 increase; B12 often decreases.	Mechanism: balance between microbial synthesis and consumption. Factors: microorganism strain (primary factor).	The effect is highly strain-specific (e.g., some strains produce folate while others consume it). Wise strain selection is key to bio-fortification.	[156]
Riboflavin (B2), Folate (B9)	In vitro (ALE in defined medium); phenotype stability tested in milk	Increase (up to 10-fold for B2, 3-fold for B9)	Mechanism: adaptive laboratory evolution (ALE) of a LAB-yeast co-culture. Factors: selective pressure from obligatory mutualism.	Key innovation: A non-GMO method to enhance vitamin secretion. Evolved strains retained high-yield properties in milk.	[165]

Notes: This table summarizes findings from both primary research articles and comprehensive scientific reviews. The ‘Typical Effect’ column describes the main outcome, including quantitative data (e.g., % change, final concentration) where available in the source publication. For full experimental details and context, readers are encouraged to consult the original references cited. Abbreviations: ALE, adaptive laboratory evolution; DMBI, 5,6-dimethylbenzimidazole; LAB, lactic acid bacteria; NAM, nicotinamide; RF, riboflavin.

presents a comprehensive summary of the findings from the literature.

Ultimately, the final vitamin profile is strongly influenced by the specific microbial strains involved (their synthesis vs. consumption capabilities), the initial vitamin content and nature of the flour and fermentation conditions [22,156]. This high degree of variability, clearly illustrated by the range of outcomes in Table 6, underscores the importance of optimizing microbial selection and fermentation conditions to potentially maximize vitamin yields in sourdough products [166]. Subsequent losses during baking and storage also play a critical role; vitamin B12 stability, for instance, can vary significantly, with losses of around 29% reported for in situ-produced B12 during rye sourdough baking [159], a stability level found comparable to that of added cyanocobalamin in the same study. This highlights that net outcomes are often vitamin-specific [141,156]. Therefore, a specific final product analysis is necessary rather than assuming general enrichment.

Beyond quantifying these net changes, interpreting the nutritional significance of these observed vitamin changes in sourdough is crucial. The contribution of typical sourdough bread to the Recommended Dietary Allowances (RDA) for vitamins can be highly variable and is not always guaranteed, owing to the multiple factors previously discussed (e.g., strain, flour, fermentation conditions). For instance, regarding vitamin B12, standard sourdough bread is generally considered insufficient as a reliable source to meet the needs of individuals on strict plant-based diets [163,164]; this contrasts with the potential of products specifically enriched through advanced in situ biofortification strategies using fermentation [163]. Thus, further research is still needed to more clearly link measured vitamin levels in diverse sourdough breads to their practical dietary impact [164].

In light of these nutritional limitations, several innovative strategies have been explored to enhance vitamin content in a controlled and sustainable manner. Metabolic engineering of lactic acid bacteria (LAB), for instance, has shown promise in significantly increasing folate and riboflavin content in vitro [167,168], though challenges related to in vivo bioavailability and stability persist [166]. Further strategies include adaptive laboratory evolution (ALE), an evolutionary engineering technique that uses prolonged cultivation under specific selective pressures to enhance desired microbial traits without direct genetic modification. For example, a study by Konstantinidis et al. [165] applied this approach to LAB–yeast co-cultures in an obligatory mutualistic system, achieving significant, non-GMO increases in B-vitamins (e.g., riboflavin up to 10-fold; folate up to 3-fold). This approach shows potential for developing highly productive starter cultures by guiding microbial interactions. However, when developing strategies for vitamin fortification or enhancement in bread, achieving a careful balance is crucial for ensuring that the sensory properties of the final product (such as texture, flavor, and aroma) are not negatively impacted. Consumer acceptance, which is heavily influenced by these sensory attributes, remains paramount for the success of any functional food product. Notably, some innovative research [162] actively addresses this challenge by aiming to enhance vitamin value and improve sensory characteristics concurrently.

In conclusion, the impact of sourdough fermentation on vitamin content is a double-edged sword. While specific LAB strains can synthesize key B-vitamins like folate and riboflavin, this potential is often offset by significant processing losses and the consumption of vitamins by the microbiota itself. Consequently, relying on standard sourdough fermentation for consistent vitamin enrichment is unreliable. The ‘nutritional reality’ is that targeted strategies, such as the use of high-producing adjunct cultures or advanced evolutionary engineering, are necessary to overcome these limitations and achieve meaningful biofortification.

3.7. Sourdough Fermentation for FODMAP Reduction: Mechanisms, Challenges, and Relevance to IBS

In cereal-based products, sourdough fermentation is a promising method for reducing FODMAPs—especially fructans and galacto-oligosaccharides (GOS) [8]. These compounds are major triggers of gastrointestinal symptoms in individuals with irritable bowel syndrome (IBS) [8,169]. Indeed, clinical trials have demonstrated that a carefully managed low-FODMAP diet can significantly improve symptoms and quality of life in IBS patients, particularly those with diarrhea-predominant IBS, compared to traditional dietary advice [170].

3.7.1. Mechanisms, Efficacy, and Influencing Factors in FODMAP Modulation

One of the main mechanisms is microbial enzymatic hydrolysis. Lactic acid bacteria (LAB) and yeasts produce enzymes like fructanases (e.g., β-fructofuranosidases) and invertases, which break down wheat and rye fructans into fructose and glucose [9,171]. These enzymes act outside the cell, at the cell wall, or inside the cell after uptake [171]. Similarly, the metabolism of GOS, such as raffinose, can be mediated by α-galactosidase activity, an enzymatic capability present in some, but not all, sourdough-relevant lactic acid bacteria [9,172].

The efficacy of FODMAP reduction in sourdough is significantly influenced by the specific fermentation type and the metabolic activities of the microbial strains employed. While various sourdough types exist, the most pronounced effects on FODMAP load and mannitol accumulation, particularly those driven by active microbial metabolism, are primarily observed in Type I and Type II sourdoughs. Specifically, traditional Type I sourdoughs are often dominated by heterofermentative LAB species, such as Fr. sanfranciscensis, which possess the metabolic pathways to convert fructose into mannitol—a polyol FODMAP [169,173,174]. This can lead to substantial increases in mannitol levels (e.g., more than 500%), potentially offsetting or even counteracting the net FODMAP reduction achieved through the degradation of fructans and GOS [173,174]. In contrast, controlled Type II sourdough fermentations, particularly those utilizing selected homofermentative LAB strains (e.g., Lp. plantarum, Lacticaseibacillus paracasei), are optimized to minimize mannitol accumulation while maximizing fructan hydrolysis [9,171]. Such optimized Type II fermentations have consistently demonstrated significant fructan degradation, often exceeding 90%, resulting in breads compliant with low-FODMAP thresholds (<0.3 g of oligosaccharides per serving) [175]. Overall, fructan levels in sourdough bread can decrease by 42–82% depending significantly on the fermentation process employed, with specific examples and comparative data summarized in

Table 7. Comparative effects of sourdough vs. yeast fermentation on fructan, polyol, and GOS content in cereal breads.

FODMAP Component	Fermentation Type/Key Condition(s) and Comparison	Observed Change (% Reduction/Increase)	Statistical Significance	Reference(s)
Fructans	SD (Rye or Wheat starter), Whole HRSW flour, 12 h@ ~26 °C (vs. Flour)	~60% Reduction	p < 0.05	[177]
Fructans	Yeast Control (Rye/Wheat)	~50% Reduction	NR	[178]
Fructans	SD (Lm. reuteri, no FruA), Rye/Wheat, 16 h@37 °C	~65–75% Reduction	p < 0.05 (vs. Control)	[178]
Fructans	SD (L. crispatus, FruA+), Rye/Wheat, 16 h@37 °C	>90% Reduction	p < 0.05 (vs. Others)	[178]
Mannitol	SD (Lm. reuteri, no FruA), Rye/Wheat, 16 h@37 °C	Increased/Accumulated	p < 0.05 (vs. Lc/Yeast)	[178]
Mannitol	SD (L. crispatus, FruA+), Rye/Wheat, 16 h@37 °C	Lower levels (vs. Lm. reuteri)	p < 0.05	[178]
Fructans	SD (Type I Spont., Wheat, 10 + 2 h proof) vs. Yeast Control	Sig. Lower levels (e.g., 0.19–0.29 vs. 0.40–0.49 g/100 g)	p < 0.05	[179]
Polyols	SD (Type I Spont., Wheat, 10 + 2 h proof) vs. Yeast Control	Sig. Increased levels (e.g., ~0.5–1.0 vs. ~0.05mg/100 g)	p < 0.05	[179]
GOS	SD (Type I Spont., Wheat) vs. Yeast Control (Final Bread)	No Significant Difference	NS	[179]
Fructans	Yeast (Short/Long Ferm.), Whole Wheat (various cultivars) (vs. Flour)	>65% Reduction (Average)	N/A	[180]
Fructans	Yeast (Wheat/Spelt), 1 h proof@30 °C (vs. Flour)	NS change (Fructan decrease was offset by fructose increase.)	NS	[181]
Mannitol	Yeast (Wheat/Spelt), various proof times	Trace amounts only	N/A	[181]
Fructans	SD Type II (Selected LAB consortia), White Wheat (vs. Yeast Control)	~70–93% Reduction (Low-FODMAP levels achieved)	p < 0.05	[175]
Polyols	SD Type II (Selected LAB consortia), White Wheat (vs. Yeast Control)	Sig. Increased (but low absolute levels, ~14–32 mg/100 g)	p < 0.05	[175]
Fructans	SD + Yeast vs. Yeast only (Refined Wheat, 60 min proof)	Reduced (e.g., ~340 → ~240 mg/100 g DM)	p < 0.05	[169]
Mannitol	SD + Yeast vs. Yeast only (Whole Wheat)	Massively Increased (e.g., ~30 → ~515 mg/100 g DM)	p < 0.05	[169]
Mannitol	SD + Yeast (Rye—Refined or Whole)	Very High levels remaining (e.g., ~900–1500 mg/100 g DM)	N/A	[169]
Fructans	SD + Yeast (Rye—Refined or Whole)	High levels remaining (e.g., ~1700–1950 mg/100 g DM)	N/A	[169]
Fructans	SD Type II (Selected Homoferm. LAB + Yeast), WW Flour (vs. Yeast Control)	~69–73% Red. vs. Flour (Sig. lower than Yeast); Low-FODMAP levels	p < 0.05	[171]
GOS	SD Type II (Homoferm. LAB + Yeast), WW Flour (vs. Yeast Control)	Sig. lower than Yeast Control; ~50–60% Red. vs. Flou	p < 0.05	[171]
Polyols (Mannitol + Sorbitol)	SD Type II (Homoferm. LAB + Yeast), WW Flour (vs. Yeast Control)	Remained Very Low (<0.06% DM); Sig. lower than Yeast Control	p < 0.05	[171]

Note: Summary of quantitative changes in key FODMAP components during sourdough (SD) or yeast control fermentation under various conditions, extracted from cited primary studies. Changes are reported as % reduction/increase or levels (e.g., Q DM). Statistical significance is reported as p < 0.05 where available. The results highlight considerable variability depending on fermentation strategy (e.g., Type I vs. Type II sourdough), microbial strains (e.g., heterofermentative vs. homofermentative), and substrate (e.g., wheat vs. rye). A significant increase in mannitol is primarily observed with Type I/presumed heterofermentative sourdoughs, whereas levels remain very low with selected homofermentative strains. Abbreviations: GOS, galacto-oligosaccharides; WW, whole wheat; DM, dry matter; LAB, lactic acid bacteria; NS, non-significant (p > 0.05); NR, not reported; N/A, not applicable (comparison not the focus of the analysis).

[171,173,176].

Given these intricate microbial metabolic pathways and their varying impacts on FODMAP profiles, the reproducible production of low-FODMAP sourdough products necessitates strict standardization of fermentation parameters and precise control over raw material characteristics. Several key parameters profoundly shape the final FODMAP load. Fermentation time is critical, with prolonged phases (e.g., 48 h) often required for significant degradation with selected strains [9,171,176]. Temperature also plays a crucial role by influencing microbial selection and polyol dynamics [8,179]. Finally, flour composition, such as high initial fructan content in rye or elevated levels of damaged starch, can significantly impede FODMAP reduction [17,176]. Therefore, rigorous control and harmonization of these variables are crucial to ensure consistent and reliable low-FODMAP outcomes in sourdough production for individuals with IBS.

3.7.2. Clinical Relevance, Broader Implications, Challenges, and Future Directions

The relevance of understanding and controlling these factors lies primarily in the potential application of low-FODMAP sourdough for managing irritable bowel syndrome (IBS) [8]. The implications for IBS management are supported by in vitro evidence, with sourdough bread eliciting significantly lower gas production in fecal fermentation models than conventional yeast bread, suggesting improved digestive tolerance [182]. Indeed, in vivo, a specifically formulated low-FODMAP rye sourdough consumed by a small group of IBS patients (n = 7) reduced colonic fermentation and suggested less flatulence, although average overall GI symptoms remained unchanged [183]. Similarly, a study by Laatikainen et al. (2017) [184] investigated sourdough wheat bread, which had a significantly lower fructan content compared to its yeast-leavened counterpart. Despite this reduction, no improvement in overall gastrointestinal symptoms was observed in the IBS patients (N = 26) consuming the low-fructan sourdough bread; in fact, some extra-intestinal symptoms reportedly worsened. These findings highlight the complex interplay of factors influencing symptom response in IBS, extending beyond FODMAP levels alone. Additionally, the trade-offs between FODMAP reduction and nutritional quality warrant consideration, as prebiotic fructans may confer gut health benefits absent in their degraded forms [9]. Illustrating this concern, clinical trials have shown that adherence to a low-FODMAP diet can lead to potentially undesirable changes in the gut microbiota. These changes include reductions in beneficial *Bifidobacterium* populations and lower fecal butyrate levels [185]. While a low-FODMAP diet is often effective for symptom management, these microbial impacts raise questions about its sustainability as a strict, long-term solution for all individuals. This emphasizes the critical importance of a carefully guided FODMAP reintroduction phase.

In conclusion, sourdough fermentation offers a promising strategy for reducing FODMAPs, particularly fructans, through microbial enzymatic hydrolysis, potentially benefiting IBS patients [8,169,170]. However, its efficacy is highly dependent on specific LAB strains (e.g., homofermentative vs. heterofermentative to minimize mannitol accumulation), precise fermentation conditions (e.g., time, temperature), and flour type [9,17,171,176]. Despite promising in vitro and some in vivo evidence for reduced colonic fermentation, the broader clinical picture regarding overall GI symptom improvement in IBS remains inconsistent [183,184]. Furthermore, significant challenges remain, including nutritional trade-offs, such as the reduction in beneficial prebiotics [9] and hurdles in scalability [8], and the impact of individual microbiome variability, which may complicate the predictability of clinical outcomes [186]. Overcoming these obstacles will require targeted strain selection and robust, long-term clinical trials to definitively validate the therapeutic utility of low-FODMAP sourdough in IBS management [8,9,184].

4. Conclusions, Challenges, and Future Directions

Lactic acid bacteria (LAB) play a pivotal role in sourdough fermentation, utilizing their metabolic versatility to enhance nutritional attributes, including mineral bioavailability, the release of bioactive peptides, and glycemic modulation. While these biochemical transformations underscore sourdough’s potential as a functional food, this review highlights a critical finding: translating such in vitro promise into consistent, measurable human health benefits remains a complex challenge. Specifically, while some benefits, notably a decrease in the glycemic index, have been more consistently verified in human studies, others, such as significant improvements in mineral absorption and clear benefits for individuals with celiac disease, largely await more robust clinical validation. This challenge arises from interacting variables, including microbial strain specificity, fermentation parameters, matrix composition, and host physiological differences, collectively modulating LAB efficacy. The current evidence base reveals a significant gap between compositional improvements observed in controlled settings and validated in vivo or clinical outcomes, underscoring the context-dependent nature of LAB-driven nutritional enhancements.

Table 8. The hierarchy of evidence: a comparative framework of experimental models for assessing nutritional outcomes in sourdough research.

Experimental Model	Strengths	Limitations	Translational Value
In Vitro Models	Enables precise, reproducible, and high-throughput screening of multiple variables (e.g., starter cultures, processing parameters, flour types) to assess their impact on specific outcomes like anti-nutrient degradation, FODMAP levels, or bioactive compound synthesis [62,64,72,138,186,187]. Permits the use of advanced dynamic gut simulators (e.g., Colon-on-a-plate™ and MicroMatrix) to model regional colon fermentation and track temporal changes in microbial community composition and metabolic output (e.g., SCFAs) in response to sourdough-derived fibers like AXOS [68,98,186]. Isolates the metabolic impact of specific microbial strains from the effects of general processing parameters (e.g., pH, fermentation time) [175,186].	Lacks key physiological context by omitting integrated systemic responses such as hormonal feedback and the effect of sourdough-derived organic acids on gastric emptying, which are crucial for determining the true postprandial glycemic response [20,119,127,188]. Often relies on simplified experimental systems, such as using purified substrates (e.g., extracted fibers) or pure microbial cultures, which do not reflect the complexity of a whole food matrix or a complete LAByeast ecosystem [20,86,98,99,189]. Fails to account for the physical properties of the final baked bread, where the gluten–starch–fiber matrix and its altered water mobility significantly impact enzyme accessibility and overall digestibility [95,117,190,191]. Utilizes simplistic absorption models (e.g., dialysis, Caco-2 cells) that primarily measure passive diffusion and cannot fully replicate the complex, carrier-mediated transport of nutrients or the influence of a protective mucus layer in the human intestine [62,68,72,188].	Low to Moderate. Acts as a powerful and essential pre-clinical screening platform to understand mechanisms, optimize formulations (e.g., select specific phytase-producing LAB strains), and generate targeted hypotheses before undertaking expensive and ethically complex animal or human trials
Animal (In Vivo) Models	Provides an integrated, whole-organism system to confirm if bioavailability improvements observed in vitro (e.g., increased soluble iron) translate to a systemic physiological change (e.g., increased serum ferritin) [64]. Allows for the study of complex metabolic pathways and tissue-level responses that are impossible to model in vitro, offering a more holistic view of the nutritional impact of sourdough bread consumption [20,191].	Physiological, metabolic, and gut microbial differences between animal models (especially rodents) and humans mean that results on protein quality are not directly translatable [20,191,192]. The use of animal bioassays is a significant ethical and regulatory hurdle, preventing many food producers from substantiating protein quality claims (e.g., PDCAAS) for novel sourdough products, thereby stifling innovation [188,191,192]. It is difficult to assess subjective or complex human responses, such as satiety or specific gastrointestinal symptoms related to wheat sensitivity, in an animal model [20,127].	Moderate to High. Serves as a crucial intermediate step to validate systemic bioavailability and safety before moving to human trials, but its relevance is decreasing due to ethical concerns and the development of more sophisticated in vitro alternatives
Human (In Vivo) Models	Represents the definitive “gold standard” for determining if a sourdough intervention provides a clinically relevant health benefit in the target population [20,66]. Allows for the direct assessment of complex endpoints, including the impact of a low-FODMAP sourdough bread on functional gut symptoms in IBS patients or subjective measures like appetite and satiety [127,183,184]. Can utilize objective biomarkers, such as plasma alkylresorcinols, to verify compliance with the consumption of specific whole-grain sourdough breads, increasing the reliability of the study’s findings [66]. Enables the investigation of systemic and nuanced biological interactions, including sourdough’s effect on the gut microbiome, specific immune markers (e.g., IL-8), and gut-brain axis endpoints like fatigue and alertness [20,184]	Extremely difficult to design an appropriate control; comparing a sourdough bread to a commercial yeast bread introduces multiple confounding variables (acidity, flour type, texture, organic acid profile), making it hard to isolate the effect of the fermentation process itself [20,117,190]. The effects of the intervention bread can be masked or confounded by the subjects’ uncontrolled background diet, which may contain other inhibitors or enhancers (e.g., phytates or polyphenols from other foods) [66]. Often show high inter-individual variability in outcomes (e.g., glycemic response), suggesting that personal physiology and baseline gut microbiota may have a stronger effect than the type of bread consumed [20]. These trials are expensive, logistically complex. They can suffer from high drop-out rates, especially when the intervention food (e.g., a specific type of bread) must be consumed daily for long periods [66,188]. Prone to significant placebo/nocebo effects driven by consumer beliefs [127,184] and challenged by a lack of product standardization (e.g., starter cultures, fermentation times), which complicates the comparison of findings across studies [20] It is difficult to establish a clear dose-response relationship for sourdough interventions (e.g., if the daily reduction in FODMAPs is clinically meaningful), and most trials are too short-term to understand the long-term effects of daily sourdough consumption, representing a major knowledge gap [184]	High Provides the only definitive evidence required for substantiating health claims and changing dietary guidelines. However, due to their complexity and cost, they are best employed to confirm strong hypotheses that have already been well-supported by robust in vitro and, where appropriate, animal studies

summarizes the hierarchy of evidence in this field, contextualizing the strengths and limitations of the primary experimental models used to generate these findings.

The methodological framework summarized in Table 8 reveals that key limitations hinder the realization of LAB’s nutritional potential. Bioavailability barriers for LAB-generated compounds, strain-specific functional variability, and process-dependent trade-offs in compound stability [193] complicate the predictability of these compounds. Methodological shortcomings further exacerbate these challenges, including overreliance on in vitro models, a paucity of robust randomized controlled trials [20], and limited alignment between laboratory studies and real-world sourdough products [40]. Addressing these gaps necessitates a systems-level approach prioritizing three fronts: (1) high-quality human trials with clinically relevant endpoints to rigorously assess the net health benefits of sourdough bread, not only in comparison to yeast-leavened bread but also in the context of other functional fermented foods; (2) integration of omics and computational modeling to elucidate strain–matrix–host interactions and in vivo compound fate [194]; and (3) methodological advancements, including standardized protocols and optimization frameworks for real-world applicability [40,193].

Emerging trends position LAB-engineered sourdoughs at the intersection of sustainable food systems and personalized nutrition. Innovations in strain selection, guided by genomic insights [194], could tailor sourdoughs to individual dietary needs, while synergistic microbial consortia may amplify nutritional outcomes [38]. Despite persistent challenges in clinical validation, the field must strike a balance between cautious optimism and rigorous science, ensuring that LAB’s biochemical promise is grounded in translational evidence. Future progress hinges on interdisciplinary collaboration to harmonize microbial potential with human health imperatives. It is also crucial for researchers in this field to acknowledge that many beneficial properties of LAB are well-established in other ecosystems, such as fermented dairy. Therefore, a key future challenge will be to delineate the unique, matrix-dependent benefits conferred by sourdough fermentation itself.