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Scientific Evidence and Application Limits Chapter IV

by luciano

1. Scope and operational definitions

In technical language it is essential to separate three concepts that are often confused:

1. Gluten hydrolysis/proteolysis
→ fragmentation of proteins (gliadins and glutenins) into smaller peptides.

  1. Reduction of immunogenic peptides/epitopes for celiac disease→ degradation of specific sequences rich in proline and glutamine (e.g. “Pro-rich” peptides) that resist digestion and activate immune responses in celiac patients.

  1. “Elimination” of gluten→ a much more ambitious objective, achievable only under controlled technological conditions (selected strains, often enzymatic co-adjuvants, long fermentation times), and not equivalent to normal baking with traditional sourdough.

2. Evidence: what studies show

2.1 Fermentation with selected lactic acid bacteria: targeted degradation of immunogenic peptides

Di Cagno et al., 2004 (Applied and Environmental Microbiology) demonstrate that the use of selected lactobacilli with specialized peptidases is able to hydrolyze proline-rich peptides, including peptides with high immunogenicity (the work explicitly discusses the hydrolysis of “Pro-rich” peptides and the application to an experimental baked product).
The study also includes an acute clinical challenge test in subjects with celiac disease within the described experimental protocol. (PubMed)

Key technical points (what is “demonstrated”)

  • The ability to degrade prolamin fractions critically depends on strain selection (it is not an automatic effect of any sourdough). (PubMed)

  • Degradation involves peptides known to resist gastrointestinal digestion thanks to enzymatic systems (peptidases) not typical of baker’s yeast alone. (PubMed)

Immediate applicative limit

The protocol is not “generic sourdough”: it is a biotechnology using selected strains and defined conditions; it is not automatically transferable to any artisanal process. (PubMed)

2.2 “Enhanced” fermentation: selected lactobacilli + fungal proteases (extensive detoxification)

Rizzello et al., 2007 (Applied and Environmental Microbiology) show an even more “engineered” approach: a mixture of selected lactobacilli + fungal proteases during prolonged fermentation.

The study uses several analytical techniques (immunological and instrumental) to estimate residual gluten and the persistence of different protein fractions. (PubMed)

Key technical points

  • Complete hydrolysis of gliadins and other soluble fractions reported in the experimental process; partial persistence of a fraction of glutenins (not all structural fractions are necessarily “eliminated”). (PubMed)

  • Measurement of residual gluten through immunological tests (R5-ELISA) and confirmation through proteomic/spectrometric analyses in the protocol. (PubMed)

  • Biological evaluation of immunoreactivity (tests on immune cell lines) to estimate the “toxicity” of the pepsin-trypsin digest of the fermented product. (PubMed)

Applicative limit

This scenario requires enzymatic co-adjuvants (fungal proteases) and a controlled setup: it is an industrial/biotechnological process, not the equivalent of standard sourdough management in a bakery. (PubMed)

2.3 Selected lactic fermentation on different cereals: role of pH and endogenous enzymes

De Angelis et al., 2006 (Journal of Cereal Science) study the fermentation of rye flours with selected lactic acid bacteria, showing extensive hydrolysis of ethanol-soluble polypeptides and a reduction of immunochemical detectability (R5-Western), also discussing the role of pH in activating hydrolysis through endogenous flour enzymes. (ScienceDirect)

Key technical points

The observed degradation results from a combination of:

  • microbial proteolytic activity (selected strains)

  • pH-dependent hydrolysis (activation of endogenous cereal enzyme systems) (ScienceDirect)

The work supports the “biotechnological” logic of controlled fermentation as a tool to reduce contamination/reactivity risk in specific contexts (in experimental terms). (ScienceDirect)

Applicative limit

Again: selected strains + defined process conditions; this is not an automatic generalization for “any sourdough.” (ScienceDirect)

3. Where baker’s yeast and “traditional” sourdough fit

3.1 Baker’s yeast (Saccharomyces cerevisiae)

Within the framework of the above studies, the effect of baker’s yeast is mainly:

  • fermentative kinetics (CO₂, volumetric development)

  • indirect influence on maturation (time/temperature)

but not a proteolytic activity comparable to that of selected lactic bacteria and/or added proteases.

In other words: with baker’s yeast the “improved digestive management” (when observed) is more related to maturation time and transformations of the starch-protein matrix, not to extensive degradation of immunogenic gluten sequences (in the terms used in the cited studies).

(This is a conclusion derived by comparing the mechanisms reported in studies on selected LAB and proteases.) (PubMed)

3.2 “Non-selected” sourdough (spontaneous sourdough starter)

Spontaneous sourdough can determine:

  • acidification

  • partial proteolysis

  • rheological modifications

However, the literature showing “almost total” degradation or marked reduction of immunogenic epitopes typically uses:

  • selected lactic strains with specific peptidases (PubMed)

and/or

  • fungal proteases in combination (PubMed)

Therefore, at a manualistic level, the correct formulation is:

Fermentation with sourdough can increase gluten proteolysis; extensive degradation of immunogenic sequences requires controlled biotechnological protocols (selected strains and, in some cases, enzymatic co-adjuvants).

4. Applicative limits

Protocols aiming to drastically reduce the immunogenic fraction of gluten do not coincide with the standard production of sourdough bread/pizza. (PubMed)

The result depends on:

  • microbial species/strains used (selection) (PubMed)

  • fermentation time

  • acidity/pH (and relative enzymatic activation) (ScienceDirect)

  • possible use of technological proteases (PubMed)

Even when very extensive degradation is observed, some studies report possible persistence of certain fractions (e.g. part of the glutenins) depending on the protocol. (PubMed)

5. Cited studies

Di Cagno, R. et al. (2004). Sourdough bread made from wheat and nontoxic flours and started with selected lactobacilli is tolerated in celiac sprue patients. Applied and Environmental Microbiology, 70(2), 1088–1096. DOI: 10.1128/AEM.70.2.1088-1096.2004 (PubMed)

Rizzello, C.G. et al. (2007). Highly efficient gluten degradation by lactobacilli and fungal proteases during food processing: new perspectives for celiac disease. Applied and Environmental Microbiology, 73(14), 4499–4507. DOI: 10.1128/AEM.00260-07 (PubMed)

De Angelis, M. et al. (2006). Fermentation by selected sourdough lactic acid bacteria to decrease coeliac intolerance to rye flour. Journal of Cereal Science, 43(3), 301–314. DOI: 10.1016/j.jcs.2005.12.008 (ScienceDirect)

In-depth analysis

Effects of sourdough and/or yeast use in gluten fermentation: scientific evidence

Primary studies (main evidence)

1. Effects of LAB + yeast co-fermentation on gluten degradation

Title: Effects of Co-Fermentation with Lactic Acid Bacteria and Yeast on Gliadin Degradation in Whole-Wheat Sourdough

Summary: The study evaluates how selected strains of Lactic Acid Bacteria (LAB) and baker’s yeast (Saccharomyces cerevisiae) co-ferment gluten in whole-wheat sourdough. The combined fermentation leads to significant degradation of gliadin and glutenin fractions, with reduction of gluten content. Strains such as Lactobacillus brevis and Pediococcus pentosaceus show high proteolytic activity. (MDPI)

2. Reduction of gluten allergenicity in fermented products

Title: From gluten structure to immunogenicity: Investigating the effects of lactic acid bacteria and yeast co-fermentation on wheat allergenicity in steamed buns

Summary: LAB + baker’s yeast co-fermentation induces depolymerization of gluten macromolecules and reduces total immunoreactivity compared with non-fermented controls. Significant decreases in α/γ-gliadins and glutenins associated with celiac disease are observed. (PubMed)

3. Immunogenic peptides and sourdough

Title: A Case Study of the Response of Immunogenic Gluten Peptides to Sourdough Proteolysis

Summary: Fermentation with sourdough modifies gluten structure and the release profile of immunogenic peptides during in vitro digestion, without necessarily eliminating them completely. Comparative study between sourdough bread and rapid-leavened bread. (PubMed)

4. Bacillus spp. isolated from sourdough and gluten hydrolysis

Title: Gluten hydrolyzing activity of Bacillus spp isolated from sourdough

Summary: Bacillus strains isolated from sourdough degrade the immunogenic 33-mer peptide and gliadin sequences, reducing gluten below 110 mg/kg. Potential application in reduced-gluten products. (SpringerLink)

5. Pilot clinical study on fermented products

Title: Gluten-free sourdough wheat baked goods appear safe for young celiac patients: a pilot study

Summary: Fermentation with selected lactobacilli and fungal proteases reduces gluten below 10 ppm. Products tested on children with celiac disease in remission show good clinical tolerability. (PubMed)

6. Recent review on the role of fermentation (2025)

Title: Sourdough Fermentation and Gluten Reduction: A Biotechnological Approach for Gluten-Related Disorders

Summary: LAB fermentation contributes to the reduction of gluten peptides but is not sufficient alone to eliminate all immunogenic sequences. Combined processes with exogenous proteases are more effective. (MDPI)

Previously cited studies, with greater detail

A. Bacillus spp isolated from sourdough
DOI: 10.1186/s12934-020-01388-z

Further detail: The study demonstrates the high proteolytic activity of Bacillus strains against gliadin substrates and the 33-mer peptide. Extensive hydrolysis leads to gluten levels <110 mg/kg in fermented sourdough.

B. Label-free quantitative proteomics and sourdough fermentation
DOI: 10.1016/j.foodchem.2023.137037

Further detail: Proteomic analysis identifies 85 allergenic proteins modulated by fermentation. Some microbial combinations show reduction of gliadins containing immunogenic sequences, suggesting a selective effect of fermentation on the wheat protein fraction.

C. Yeast–bacteria interactions and immunogenicity
DOI: 10.1016/j.ifset.2023.103281

Further detail: Co-cultures of yeasts (Saccharomyces, Torulaspora) with Pediococcus acidilactici show greater gluten depolymerization and reduced immunogenicity compared with single-yeast fermentations.

General conclusions

Sourdough fermentation can partially degrade gluten and reduce specific immunogenic peptides. The reduction does not equal complete elimination: without exogenous proteases, residual gluten often remains. Effectiveness strongly depends on microbial strains and fermentation conditions.

What does all this mean for those seeking gluten-light products?

Products made with sourdough (sourdough fermentation) generally present technological and biochemical characteristics superior to products obtained with rapid leavening, especially regarding tolerability and overall quality.

In particular:

Partial gluten degradation

Prolonged fermentation promotes hydrolysis of some gliadin and glutenin fractions, reducing protein complexity compared with non-fermented doughs.

Modified peptide profile

Even when gluten is not eliminated, its structure changes, with a potential reduction of specific immunogenic peptides.

Perceived improved digestibility

Many non-celiac consumers report better gastrointestinal tolerance compared with industrial baked products produced with rapid fermentation.

Reduction of other critical factors

Sourdough fermentation also contributes to decreasing FODMAPs and some antinutritional compounds.

⚠️ Important note: gluten-light products are not automatically safe for people with celiac disease. Traditional fermentation improves quality and tolerability, but only controlled and validated processes can lead to gluten levels compatible with a gluten-free diet.

For those who are not celiac but seek products that are more digestible, less stressful for the intestine and based on natural fermentation processes, sourdough currently represents one of the most interesting solutions supported by scientific literature.

The Science Behind Bread and Pizza

Chapter I – Gliadins and Glutenins: the essential building blocks
Chapter II – Fermentation in professional baking and pizzeria production
Chapter III – Gluten degradation during fermentation
Chapter IV – Scientific evidence and application limits

Gluten Degradation During Fermentation (Chapter III)

by luciano

Fermentation, Proteolysis and Potential Modulation of Mucosal Stimuli

1. Technical premise

Physiological evidence shows that some protein peptides resistant to digestion can:

  • modulate paracellular permeability

  • activate innate immunity pathways

  • interact with the intestinal microbial ecosystem

In the context of professional baking, the technological interest is not clinical but biochemical and structural: reducing the fraction of peptides relatively resistant to enzymatic digestion and modifying digestive kinetics through appropriately designed fermentation.

It should be emphasized that the primary function of protein digestion is the hydrolysis of dietary proteins — including gluten — into free amino acids and small peptides (mainly di- and tripeptides), which can cross the intestinal epithelium via specific transport systems and be used as metabolic substrates for the various metabolic functions of the body.

The peptide fraction that is not completely hydrolyzed, consisting of larger peptides, nor absorbed at the level of the small intestine, reaches the colon where it is partially metabolized by the intestinal microbiota through fermentative processes; the unused portion is eliminated with feces.

Enzymatic hydrolysis therefore represents the key step for making proteins nutritionally available and limiting the presence of peptide fractions relatively resistant to digestion.

2. How fermentation can act on resistant peptides

2.1 Acidification and enzymatic activation

Sourdough fermentation leads to:

  • pH reduction (≈ 3.8–4.8 depending on the system)

  • activation/modulation of endogenous flour proteases

  • production of microbial peptidases

Resulting effect:

  • reduction of the average molecular weight of protein fractions

  • increase in the pool of short peptides and free amino acids

  • remodeling of the peptide profile

This does not correspond to “elimination of gluten,” but to modification of the distribution of protein fragments (greater quantity of short peptides).

2.2 Depolymerization of the gluten network

Prolonged fermentation can:

  • reduce the gluten macropolymer

  • modify the secondary structure of proteins

  • make the network less compact and more accessible to digestive enzymes

Potential physiological consequence:

  • improved accessibility to gastric/pancreatic proteolysis

  • reduction of the fraction of persistent long peptides

2.3 Time as a critical variable

The maturation time is determinant:

Short time

Prolonged time

Prevalence of gas development

Greater proteolysis

Network still compact

Greater protein reorganization

Peptide profile little modified

Distribution toward shorter peptides

In professional practice, fermentations of 24–72 h at controlled temperature increase the probability of significant but structurally controlled proteolysis.

3. Baker’s yeast vs sourdough

Baker’s yeast (Saccharomyces cerevisiae)

  • limited proteolytic activity

  • mainly indirect effect (time, hydration, activation of flour enzymes)

  • reduction of resistant peptides mainly dependent on maturation time

Sourdough (LAB + yeasts)

  • direct peptidase activity

  • structuring acidification

  • greater protein remodeling at equal time

4. Interaction with microbiota and intestinal barrier

In light of physiological and experimental evidence, there is in vitro and murine model evidence suggesting a possible systemic impact of gluten on intestinal permeability and inflammatory balance, particularly in subjects with genetic predisposition, immunological vulnerability or pre-existing clinical conditions.

In this context, long and resistant peptides derived from gluten may interact with the intestinal barrier and innate immunity, influencing their functionality. Such interaction may translate into modifications of intestinal permeability, variations in microbiota composition and modulation of immune responses.

Therefore, a criterion of nutritional prudence does not represent excessive caution but rather an act of preventive responsibility.

In healthy individuals there are currently no solid and conclusive clinical data demonstrating a significant systemic impact of gluten on intestinal permeability or inflammatory balance.

The real effect also depends on:

  • the state of the intestinal mucosa

  • the composition of the microbiota

  • the overall composition of the meal

  • stress level and lifestyle

  • exposure to environmental contaminants

* By “possible impact” it is meant that the interaction between gluten and the organism is closely related to the state of the subject and to their overall biological context. Numerous factors — diet, stress, lifestyle habits and environment — may influence the outcome.

** Finally, it must be specified that by “healthy subject” we do not simply mean an individual without clinically manifest diseases, but a person without ongoing pathologies and without a state of chronic low-grade inflammation. This distinction is fundamental, since in clinical practice the term “healthy” is often used in a limited sense, coinciding only with the absence of formal diagnoses.

5. Digestibility as a property of the food matrix

It is essential to reiterate:

Digestibility is not a property of the protein or starch fraction alone, but of the entire food matrix.

Factors influencing the real digestion of the finished product include:

  • fibers (bran, arabinoxylans)

  • lipids

  • final hydration

  • alveolar structure

  • protein–starch interaction

  • baking method

The presence of fibers, for example, modifies the digestive kinetics of starch and proteins much more than a simple variation in protein content would.

6. Practical implications for the professional

If the goal is to obtain a product with:

  • high biochemical maturation

  • more evolved protein profile

  • lower fraction of peptides relatively resistant to digestion

the design levers are:

  1. reduction of yeast dosage

  2. controlled extension of fermentation

  3. use of well-managed sourdough

  4. control of temperature and pH

  5. balance between proteolysis and structural stability

7. Technical conclusion

In traditional baking:

Prolonged fermentation and controlled acidification can remodel the peptide profile of gluten. This remodeling may reduce the fraction of protein fragments relatively resistant to digestion. Physiological evidence shows that such fragments, in experimental models, can modulate barrier function and innate immunity. Direct transfer of these results to healthy humans requires interpretative caution.

Chapter IV – Scientific Evidence and Applicative Limits

1. Scope and operational definitions

In technical language it is essential to separate three concepts that are often confused:

1. Gluten hydrolysis/proteolysis
→ fragmentation of proteins (gliadins and glutenins) into smaller peptides.

  1. Reduction of immunogenic peptides/epitopes for celiac disease→ degradation of specific sequences rich in proline and glutamine (e.g. “Pro-rich” peptides) that resist digestion and activate immune responses in celiac patients.

  1. “Elimination” of gluten→ a much more ambitious objective, achievable only under controlled technological conditions (selected strains, often enzymatic co-adjuvants, long fermentation times), and not equivalent to normal baking with traditional sourdough.

2. Evidence: what studies show

2.1 Fermentation with selected lactic acid bacteria: targeted degradation of immunogenic peptides

Di Cagno et al., 2004 (Applied and Environmental Microbiology) demonstrate that the use of selected lactobacilli with specialized peptidases is able to hydrolyze proline-rich peptides, including peptides with high immunogenicity (the work explicitly discusses the hydrolysis of “Pro-rich” peptides and the application to an experimental baked product).
The study also includes an acute clinical challenge test in subjects with celiac disease within the described experimental protocol. (PubMed)

Key technical points (what is “demonstrated”)

  • The ability to degrade prolamin fractions critically depends on strain selection (it is not an automatic effect of any sourdough). (PubMed)

  • Degradation involves peptides known to resist gastrointestinal digestion thanks to enzymatic systems (peptidases) not typical of baker’s yeast alone. (PubMed)

Immediate applicative limit

The protocol is not “generic sourdough”: it is a biotechnology using selected strains and defined conditions; it is not automatically transferable to any artisanal process. (PubMed)

2.2 “Enhanced” fermentation: selected lactobacilli + fungal proteases (extensive detoxification)

Rizzello et al., 2007 (Applied and Environmental Microbiology) show an even more “engineered” approach: a mixture of selected lactobacilli + fungal proteases during prolonged fermentation.

The study uses several analytical techniques (immunological and instrumental) to estimate residual gluten and the persistence of different protein fractions. (PubMed)

Key technical points

  • Complete hydrolysis of gliadins and other soluble fractions reported in the experimental process; partial persistence of a fraction of glutenins (not all structural fractions are necessarily “eliminated”). (PubMed)

  • Measurement of residual gluten through immunological tests (R5-ELISA) and confirmation through proteomic/spectrometric analyses in the protocol. (PubMed)

  • Biological evaluation of immunoreactivity (tests on immune cell lines) to estimate the “toxicity” of the pepsin-trypsin digest of the fermented product. (PubMed)

Applicative limit

This scenario requires enzymatic co-adjuvants (fungal proteases) and a controlled setup: it is an industrial/biotechnological process, not the equivalent of standard sourdough management in a bakery. (PubMed)

2.3 Selected lactic fermentation on different cereals: role of pH and endogenous enzymes

De Angelis et al., 2006 (Journal of Cereal Science) study the fermentation of rye flours with selected lactic acid bacteria, showing extensive hydrolysis of ethanol-soluble polypeptides and a reduction of immunochemical detectability (R5-Western), also discussing the role of pH in activating hydrolysis through endogenous flour enzymes. (ScienceDirect)

Key technical points

The observed degradation results from a combination of:

  • microbial proteolytic activity (selected strains)

  • pH-dependent hydrolysis (activation of endogenous cereal enzyme systems) (ScienceDirect)

The work supports the “biotechnological” logic of controlled fermentation as a tool to reduce contamination/reactivity risk in specific contexts (in experimental terms). (ScienceDirect)

Applicative limit

Again: selected strains + defined process conditions; this is not an automatic generalization for “any sourdough.” (ScienceDirect)

3. Where baker’s yeast and “traditional” sourdough fit

3.1 Baker’s yeast (Saccharomyces cerevisiae)

Within the framework of the above studies, the effect of baker’s yeast is mainly:

  • fermentative kinetics (CO₂, volumetric development)

  • indirect influence on maturation (time/temperature)

but not a proteolytic activity comparable to that of selected lactic bacteria and/or added proteases.

In other words: with baker’s yeast the “improved digestive management” (when observed) is more related to maturation time and transformations of the starch-protein matrix, not to extensive degradation of immunogenic gluten sequences (in the terms used in the cited studies).

(This is a conclusion derived by comparing the mechanisms reported in studies on selected LAB and proteases.) (PubMed)

3.2 “Non-selected” sourdough (spontaneous sourdough starter)

Spontaneous sourdough can determine:

  • acidification

  • partial proteolysis

  • rheological modifications

However, the literature showing “almost total” degradation or marked reduction of immunogenic epitopes typically uses:

  • selected lactic strains with specific peptidases (PubMed)

and/or

  • fungal proteases in combination (PubMed)

Therefore, at a manualistic level, the correct formulation is:

Fermentation with sourdough can increase gluten proteolysis; extensive degradation of immunogenic sequences requires controlled biotechnological protocols (selected strains and, in some cases, enzymatic co-adjuvants).

4. Applicative limits

Protocols aiming to drastically reduce the immunogenic fraction of gluten do not coincide with the standard production of sourdough bread/pizza. (PubMed)

The result depends on:

  • microbial species/strains used (selection) (PubMed)

  • fermentation time

  • acidity/pH (and relative enzymatic activation) (ScienceDirect)

  • possible use of technological proteases (PubMed)

Even when very extensive degradation is observed, some studies report possible persistence of certain fractions (e.g. part of the glutenins) depending on the protocol. (PubMed)

5. Cited studies

Di Cagno, R. et al. (2004). Sourdough bread made from wheat and nontoxic flours and started with selected lactobacilli is tolerated in celiac sprue patients. Applied and Environmental Microbiology, 70(2), 1088–1096. DOI: 10.1128/AEM.70.2.1088-1096.2004 (PubMed)

Rizzello, C.G. et al. (2007). Highly efficient gluten degradation by lactobacilli and fungal proteases during food processing: new perspectives for celiac disease. Applied and Environmental Microbiology, 73(14), 4499–4507. DOI: 10.1128/AEM.00260-07 (PubMed)

De Angelis, M. et al. (2006). Fermentation by selected sourdough lactic acid bacteria to decrease coeliac intolerance to rye flour. Journal of Cereal Science, 43(3), 301–314. DOI: 10.1016/j.jcs.2005.12.008 (ScienceDirect)

In-depth analysis

Effects of sourdough and/or yeast use in gluten fermentation: scientific evidence

Primary studies (main evidence)

1. Effects of LAB + yeast co-fermentation on gluten degradation

Title: Effects of Co-Fermentation with Lactic Acid Bacteria and Yeast on Gliadin Degradation in Whole-Wheat Sourdough

Summary: The study evaluates how selected strains of Lactic Acid Bacteria (LAB) and baker’s yeast (Saccharomyces cerevisiae) co-ferment gluten in whole-wheat sourdough. The combined fermentation leads to significant degradation of gliadin and glutenin fractions, with reduction of gluten content. Strains such as Lactobacillus brevis and Pediococcus pentosaceus show high proteolytic activity. (MDPI)

2. Reduction of gluten allergenicity in fermented products

Title: From gluten structure to immunogenicity: Investigating the effects of lactic acid bacteria and yeast co-fermentation on wheat allergenicity in steamed buns

Summary: LAB + baker’s yeast co-fermentation induces depolymerization of gluten macromolecules and reduces total immunoreactivity compared with non-fermented controls. Significant decreases in α/γ-gliadins and glutenins associated with celiac disease are observed. (PubMed)

3. Immunogenic peptides and sourdough

Title: A Case Study of the Response of Immunogenic Gluten Peptides to Sourdough Proteolysis

Summary: Fermentation with sourdough modifies gluten structure and the release profile of immunogenic peptides during in vitro digestion, without necessarily eliminating them completely. Comparative study between sourdough bread and rapid-leavened bread. (PubMed)

4. Bacillus spp. isolated from sourdough and gluten hydrolysis

Title: Gluten hydrolyzing activity of Bacillus spp isolated from sourdough

Summary: Bacillus strains isolated from sourdough degrade the immunogenic 33-mer peptide and gliadin sequences, reducing gluten below 110 mg/kg. Potential application in reduced-gluten products. (SpringerLink)

5. Pilot clinical study on fermented products

Title: Gluten-free sourdough wheat baked goods appear safe for young celiac patients: a pilot study

Summary: Fermentation with selected lactobacilli and fungal proteases reduces gluten below 10 ppm. Products tested on children with celiac disease in remission show good clinical tolerability. (PubMed)

6. Recent review on the role of fermentation (2025)

Title: Sourdough Fermentation and Gluten Reduction: A Biotechnological Approach for Gluten-Related Disorders

Summary: LAB fermentation contributes to the reduction of gluten peptides but is not sufficient alone to eliminate all immunogenic sequences. Combined processes with exogenous proteases are more effective. (MDPI)

Previously cited studies, with greater detail

A. Bacillus spp isolated from sourdough
DOI: 10.1186/s12934-020-01388-z

Further detail: The study demonstrates the high proteolytic activity of Bacillus strains against gliadin substrates and the 33-mer peptide. Extensive hydrolysis leads to gluten levels <110 mg/kg in fermented sourdough.

B. Label-free quantitative proteomics and sourdough fermentation
DOI: 10.1016/j.foodchem.2023.137037

Further detail: Proteomic analysis identifies 85 allergenic proteins modulated by fermentation. Some microbial combinations show reduction of gliadins containing immunogenic sequences, suggesting a selective effect of fermentation on the wheat protein fraction.

C. Yeast–bacteria interactions and immunogenicity
DOI: 10.1016/j.ifset.2023.103281

Further detail: Co-cultures of yeasts (Saccharomyces, Torulaspora) with Pediococcus acidilactici show greater gluten depolymerization and reduced immunogenicity compared with single-yeast fermentations.

General conclusions

Sourdough fermentation can partially degrade gluten and reduce specific immunogenic peptides. The reduction does not equal complete elimination: without exogenous proteases, residual gluten often remains. Effectiveness strongly depends on microbial strains and fermentation conditions.

What does all this mean for those seeking gluten-light products?

Products made with sourdough (sourdough fermentation) generally present technological and biochemical characteristics superior to products obtained with rapid leavening, especially regarding tolerability and overall quality.

In particular:

Partial gluten degradation

Prolonged fermentation promotes hydrolysis of some gliadin and glutenin fractions, reducing protein complexity compared with non-fermented doughs.

Modified peptide profile

Even when gluten is not eliminated, its structure changes, with a potential reduction of specific immunogenic peptides.

Perceived improved digestibility

Many non-celiac consumers report better gastrointestinal tolerance compared with industrial baked products produced with rapid fermentation.

Reduction of other critical factors

Sourdough fermentation also contributes to decreasing FODMAPs and some antinutritional compounds.

⚠️ Important note: gluten-light products are not automatically safe for people with celiac disease. Traditional fermentation improves quality and tolerability, but only controlled and validated processes can lead to gluten levels compatible with a gluten-free diet.

For those who are not celiac but seek products that are more digestible, less stressful for the intestine and based on natural fermentation processes, sourdough currently represents one of the most interesting solutions supported by scientific literature.

The Science Behind Bread and Pizza

Chapter I – Gliadins and Glutenins: the essential building blocks
Chapter II – Fermentation in professional baking and pizzeria production

Chapter III – Gluten degradation during fermentation
Chapter IV – Scientific evidence and application limits

The Science Behind Bread and Pizza (Chapter I and II)

by luciano

sangiorgio.l@libero.it

Biochemistry, Rheology and Microbiology of Fermentation and the Starch–Protein Matrix

The present text analyzes the biochemical, rheological and microbiological foundations underlying the production of bread and pizza. The role of gluten proteins (gliadins and glutenins), fermentative systems (baker’s yeast and sourdough), dosage and time variables, and direct and indirect dough-making methods are examined. The approach adopted is technological-functional, with particular attention to the structural, aromatic, digestive and shelf-life implications of the finished product.

Chapter I – Protein Architecture of Dough: Gliadins, Glutenins and the Gluten Network

When we mix flour and water, we are not simply combining ingredients: we are activating a complex protein system that determines structure, consistency and the final result.
At the base of everything is gluten, a three-dimensional network created by the interaction between two families of wheat proteins: gliadins and glutenins. Understanding their balance means understanding why a pizza dough stretches easily while bread dough must sustain a tall, aerated structure.

1️⃣ The Gluten Network: A Dynamic Balance

Gluten does not exist “already formed” in flour. It is created when:

Glutenin + Gliadin + Water + Mixing = Gluten network

Water hydrates the proteins, the mechanical energy of mixing makes them interact, and an elastic network capable of trapping fermentation gases is formed. But the two proteins perform different and complementary roles.

2️⃣ The Role of Glutenins: Strength and Elasticity

Glutenins – Structural Effects

Glutenins provide:

Elasticity (ability to return to the original shape)
Tenacity (resistance to deformation)
Structure

A dough rich in glutenins:

Is more resistant
Retains gases better
Develops vertical volume

If excessive:

Too tenacious
Difficult to stretch
“Spring-back” effect

3️⃣ The Role of Gliadins: Extensibility and Viscosity

Gliadins are responsible for:

Extensibility (ability to stretch without tearing)
Malleability
Viscosity

Thanks to gliadins, the dough:

Stretches easily
Does not tear during handling
Maintains good workability

If they dominate excessively, however, the dough:

Becomes soft
“Spreads”
Struggles to maintain shape

4️⃣ Pizza: Extensibility Is Required

In the case of pizza, the goal is to obtain a thin disk that:

Stretches easily
Does not tear
Does not retract during shaping

Extensibility is therefore fundamental. A dough too rich in glutenins would be “rubbery” and difficult to open.

For this reason, pizza flours (often soft wheat) are designed to have:

A good balance between strength and extensibility
A P/L ratio (tenacity/extensibility) balanced or slightly shifted toward extensibility

If the dough is too tenacious, it is possible to intervene with:

Longer maturation (longer resting time)
Increased hydration
Choosing a flour with a lower P/L ratio

In summary: more extensibility = easy stretching and good alveolation.

5️⃣ Bread: Structural Strength Is Required

Why does bread need more glutenins?

Bread has a different goal: to develop vertically and sustain an internal structure rich in alveoli.

Here the following come into play:

Elasticity
Structural strength
Capacity to retain fermentation gases

Bread dough therefore requires a stronger gluten network, with a higher glutenin component.

If gliadins dominate too much:

The dough becomes weak
It spreads instead of rising
The bread results low and poorly structured

In summary: more glutenins = more strength and vertical development.

6️⃣ Balance Is the Key

The fundamental point is not “which protein is better”, but their ratio.

Too much glutenin → tenacious dough, hard to stretch
Too much gliadin → soft and unstable dough
Correct balance → elastic and extensible structure

The difference between pizza and bread lies precisely in this balance:

Product

Dominant characteristic

Protein ratio

Pizza

Extensibility

Good presence of gliadins

Bread

Strength and elasticity

Greater glutenin component

7️⃣ Conclusion

Pizza → more extensibility (gliadins)
Bread → more strength and elasticity (glutenins)

The quality of a dough does not depend only on the quantity of proteins, but on their interaction, processing, hydration and maturation time. Every time we stretch a pizza or shape a loaf of bread, we are working with a delicate molecular balance: a true protein architecture that transforms flour and water into a living, elastic and extensible structure.

Chapter II – Fermentation in Professional Baking and Pizzeria Production

Role of baker’s yeast and sourdough, quantities, time and dough-making methods

1. Introduction

Fermentation represents the biological and technological core of professional baking and pizza production. It is not limited to the production of gas for dough volume increase, but profoundly determines:

Mechanical structure
Extensibility
Alveolation
Aromatic profile
Digestibility
Shelf life

The professional does not simply manage a “leavening”, but a complex biochemical process in which interact:

Microorganisms
Endogenous flour enzymes
Gluten proteins
Starches
Time
Temperature

This chapter systematically analyzes the role of baker’s yeast and sourdough, the influence of dosage and fermentation time, and the impact of processing methods (direct dough and indirect dough with biga) on the finished product.

2. The Role of Baker’s Yeast

2.1 Microbiological Nature

Baker’s yeast consists predominantly of Saccharomyces cerevisiae, a unicellular microorganism capable of metabolizing simple sugars present in dough.

Alcoholic fermentation produces:

Carbon dioxide (CO₂)
Ethanol
Secondary metabolites (esters, higher alcohols, aldehydes)

CO₂ is retained by the gluten network and generates the increase in volume.

2.2 Technological Effects

Baker’s yeast:

Provides gas for structural development
Indirectly stimulates enzymatic activity
Influences fermentation rate
Determines part of the aromatic profile

It does not significantly modify dough pH (limited acidity), therefore the effect on the protein structure is mainly mechanical and fermentative, not acidifying.

3. The Role of Sourdough

3.1 Microbiological Nature

Sourdough is an ecosystem composed of:

Wild yeasts
Lactic acid bacteria (homo- and hetero-fermentative)

These microorganisms produce:

CO₂
Lactic acid
Acetic acid
Proteolytic enzymes
Complex aromatic compounds

3.2 Technological Effects

The combined activity of yeasts and bacteria

The combined activity of yeasts and bacteria determines

The controlled acidity of yeasts and bacteria directly influences

Progressive acidification (pH reduction)
Elasticity
Modification of gluten structure
Extensibility
Activation of proteases
Shelf life
Improved microbiological stability
Aromatic depth

4. Quantity and Time: General Principles

4.1 Relationship Between Dosage and Speed

The quantity of fermenting agent regulates:

CO₂ production speed
Metabolic intensity
Process duration

Fundamental principle:

More yeast → rapid fermentation
Less yeast → slow fermentation

However, speed does not coincide with maturation.

4.2 Time as a Key Variable

Time allows:

Enzymatic degradation of starches (amylases)
Partial protein hydrolysis
Reorganization of the gluten network
Formation of aromatic metabolites

A short fermentation may produce volume, but not necessarily structural and biochemical maturation.

5. Effects on Digestibility

5.1 Technical Definition

Digestibility refers to:

Reduction of intestinal fermentable load
Partial pre-digestion of starches and proteins
Better structural organization of the crumb

It does not imply absence of gluten, but a more advanced biochemical transformation.

5.2 Baker’s Yeast

Baker’s Yeast Dosage

High dosage + short time
Low dosage + long time

Limited maturation
Greater maturation

Lower enzymatic activity
Better enzymatic degradation

Higher presence of residual sugars
Biochemically evolved dough

Possible sensation of heaviness
Greater sensation of lightness

5.3 Sourdough

Fermentation with sourdough determines:

Progressive reduction of pH (controlled acidification)
Increase of proteolytic activity (endogenous enzymes + microbial activity)
Partial hydrolysis of gluten proteins
Greater degradation of fermentable sugars
Modification of the rheological properties of the gluten network

Measurable technological and physiological effects

Prolonged sourdough fermentations involve:

Reduction of residual fermentable carbohydrates
Partial protein pre-digestion
Better structural organization of the crumb
Slower glycemic response compared to short fermentations
Greater microbiological stability of the product

The combination of these factors may determine:

Reduction of intestinal fermentative load
Lower intestinal gas production compared to rapidly fermented doughs

Individual physiological response may vary depending on personal conditions, but the biochemical mechanisms described above are objectively measurable.

6. Effects on Pizza and Bread

6.1 Pizza

Structural objectives

High extensibility
Absence of “spring-back” effect
Aerated cornicione
Melt-in-the-mouth texture

Typical strategy

Very low yeast dosage
Long maturation (24–72 hours)
Temperature control

Results

Greater extensibility
More complex aroma
Lower sensation of bloating

6.2 Bread

Structural objectives

Vertical development
Crumb stability
Shelf life

Objectives with baker’s yeast

Regular structure
Delicate aroma

Objectives with sourdough

Irregular alveolation
Thick crust
Deep aroma
Longer shelf life

7. Dough-Making Methods

7.1 Direct Dough

7.1.1 Definition

All ingredients are mixed in a single phase.

7.1.2 Fermentation Dynamics

Immediate complete hydration
Single fermentation
Structure progressively built

7.1.3 Effects on the Product

Effects on the product

Texture
Homogeneous crumb
Regular alveolation

Aroma
Linear profile
Lower complexity

Digestibility
Good if accompanied by long fermentation times
Lower if fermentation is short

Shelf life
Faster staling
Lower protective acidification

7.2 Indirect Dough with Biga

7.2.1 Definition

Solid preferment (45–50% hydration) with:

Flour
Water
Small quantity of yeast

Fermentation 16–24 hours before the final dough.

7.2.2 Biochemical Dynamics

During biga maturation:

Early enzymatic activation
Production of light organic acids
Pre-maturation of gluten
Development of aromatic precursors

7.2.3 Effects on the Product

Effects on the product

Texture
Large and irregular alveolation
Greater lightness
Crispier crust

Aroma
Greater complexity
Light lactic aromas
Intensification of toasted notes

Digestibility
Dough already partially matured
Lower residual fermentable load

Shelf life
Better moisture retention
Slower staling
Greater aromatic persistence

8. Systemic Comparison

Variable

Direct

Biga

Structure

Regular

Airy and light

Aroma

Linear

Complex

Digestibility

Depends on time

Generally higher

Shelf life

Medium

Higher

Management complexity

Low

Medium/High

9. Integrated Design Principle

In professional contexts, dough design simultaneously considers:

Type of fermenting agent
Dosage
Time
Temperature
Method (direct or indirect)

There is no universally superior solution, but rather a balance consistent with:

Product identity
Sensory objectives
Desired structure
Production organization

10. Conclusion

Fermentation is not an accessory step, but a process of structural and biochemical transformation. The quantity of yeast, the choice between baker’s yeast and sourdough, the maturation time and the adopted method (direct or biga) constitute tools of applied food engineering. The professional does not simply “let a dough rise”: they design the behavior of matter over time in order to obtain a structural, aromatic and functional result consistent with the identity of the final product.

Essential Bibliography

Gluten, Protein Structure and Rheology

  1. Wieser, H. (2007).
    Chemistry of gluten proteins.
    Food Microbiology, 24(2), 115–119.
    DOI: 10.1016/j.fm.2006.07.004

  2. Shewry, P. R., & Tatham, A. S. (1997).
    Disulphide bonds in wheat gluten proteins.
    Journal of Cereal Science, 25(3), 207–227.
    DOI: 10.1006/jcrs.1996.0100

  3. Belton, P. S. (1999).
    On the elasticity of wheat gluten.
    Journal of Cereal Science, 29(2), 103–107.
    DOI: 10.1006/jcrs.1998.0233

  4. Dobraszczyk, B. J., & Morgenstern, M. P. (2003).
    Rheology and the breadmaking process.
    Journal of Cereal Science, 38(3), 229–245.
    DOI: 10.1016/S0733-5210(03)00059-6

Baker’s Yeast and Alcoholic Fermentation

  1. Fleet, G. H. (2007).
    Yeasts in foods and beverages: impact on product quality and safety.
    Food Microbiology, 24(2), 103–112.
    DOI: 10.1016/j.fm.2006.07.002

  2. Walker, G. M. (1998).
    Yeast Physiology and Biotechnology.
    John Wiley & Sons.
    ISBN: 978-0471964467

Sourdough and Sourdough Microbiology

  1. De Vuyst, L., & Neysens, P. (2005).
    The sourdough microflora: biodiversity and metabolic interactions.
    Trends in Food Science & Technology, 16(1–3), 43–56.
    DOI: 10.1016/j.tifs.2004.02.012

  2. Gobbetti, M., De Angelis, M., Di Cagno, R., Calasso, M., & Archetti, G. (2019).
    Novel insights on the functional/nutritional features of sourdough fermentation.
    International Journal of Food Microbiology, 302, 103–113.
    DOI: 10.1016/j.ijfoodmicro.2018.05.018

  3. Poutanen, K., Flander, L., & Katina, K. (2009).
    Sourdough and cereal fermentation in a nutritional perspective.
    Food Microbiology, 26(7), 693–699.
    DOI: 10.1016/j.fm.2009.07.011

  4. Hammes, W. P., & Gänzle, M. G. (1998).
    Sourdough breads and related products.
    Food Microbiology, 15(5), 487–495.
    DOI: 10.1006/fmic.1998.0191

Focus on Digestibility

1️⃣ Arendt et al., 2007

Impact of sourdough on the texture of bread
Food Microbiology, 24(2), 165–174.

Study Objective

Analyze the effect of sourdough fermentation on:

Crumb structure
Texture
Starch retrogradation
Shelf life

Key Points Relevant to Digestibility

  1. Controlled acidification

Reduction of pH
Influence on starch gelatinization and retrogradation

  1. Modification of starch structure

Acidity slows retrogradation
Better water retention
Greater crumb stability

  1. Gluten–starch interaction

Acid fermentation modifies the protein matrix
Better starch distribution within the gluten network

Implications for Digestibility

Digestibility is influenced through:

Greater enzymatic accessibility to starch
Less compact and less collapsed structure
More modulated carbohydrate release

In technical terms: acid fermentation modifies the microstructure of the starch–protein matrix, influencing digestive kinetics.

2️⃣ Liljeberg & Björck, 1998

Delayed gastric emptying rate may explain improved glycaemia…
European Journal of Clinical Nutrition, 52(5), 368–371.

Study Objective

Evaluate the effect of food acidity on post-prandial glycemic response.

Key Points

  1. Reduction of meal pH

Slows gastric emptying

  1. More gradual glycemic response

Lower glycemic peak
Better control of glucose absorption

  1. Physiological mechanism

A more acidic environment modifies digestion and absorption rate.

Implications for Bread and Pizza

In sourdough bread:

Lower pH
Presence of organic acids

may contribute to:

Slowing digestive kinetics
Reducing the rate of glucose release

Digestibility does not mean “fewer calories”, but a more modulated metabolic release.

3️⃣ Katina et al., 2006

Effects of sourdough and enzymes on staling of high-fibre wheat bread
LWT – Food Science and Technology, 39(5), 479–491.

Study Objective

Analyze the effect of:

Sourdough
Enzymatic activity
Fiber structure

on:

Staling
Retrogradation
Texture

Key Points

  1. Prolonged enzymatic activity

Greater degradation of starches
Partial hydrolysis of polysaccharide structures

  1. Slower retrogradation

Better crumb stability over time

  1. Enzyme–structure interaction

Greater modification of the structural matrix

Implications for Digestibility

Partially modified starch → different digestive enzymatic response
Greater enzymatic availability
Reduction of residual fermentable substrates

Long fermentation alters carbohydrate structure before baking.

What Is Scientifically Meant by “Digestibility” in Long-Fermented Bread and Pizza

Based on the three studies, it can be defined as:

1️⃣ Structural modification of the matrix

Gluten–starch reorganization
Greater accessibility to digestive enzymes

2️⃣ Reduction of residual fermentable load

Lower presence of rapidly fermentable sugars

3️⃣ Modulation of glycemic response

Slower glucose release
Buffering effect of acidity

4️⃣ Influence on gastric emptying

Lower pH → slower emptying
More gradual absorption

Final Technical Synthesis

Digestibility in long-fermented bread and pizza is obtained through:

Time (enzymatic maturation)
Acidification (sourdough)
Structural modification of starch and proteins
Reduction of residual fermentable load
Modulation of glycemic response

It is a structural biochemical effect, not a merely “perceived” property.

Enzymatic and microbial transformations do not concern only starch and aromas: under specific conditions, they also involve the protein fraction of gluten, reshaping its peptide profile. This topic is addressed in Chapter III.

The Science Behind Bread and Pizza

Chapter I – Protein Architecture of Dough: Gliadins, Glutenins and the Gluten Network
Chapter II – Fermentation in professional baking and pizzeria production

Chapter III – Gluten degradation during fermentation
Chapter IV – Scientific evidence and application limits

Experimental application of the advanced method for the production of bread doughs with flours with limited gluten development capacity: analysis of the results. (Analysis carried out by ChatGPT)

by luciano

sangiorgio.l@libero.it

Related article: Advanced method for making bread dough with flours with limited gluten development capacity

(Dynamics of the protein network in Triticum monococcum bread subjected to prolonged cold maturation and controlled thermal reactivation. Physical-dynamic model, qualitative rheological interpretation, and experimental validation through photographic documentation and evaluation of the finished product).

Technical-scientific synthesis. The present study analyzes the structural and rheological behavior of an einkorn dough (Triticum monococcum L.) subjected to a process structured into biga, dispersion in the liquid phase, prolonged cold maturation, thermal reactivation on a warm surface, intermediate manipulations, final proofing, and baking. The objective is to verify whether the protein network of einkorn follows a linear development dynamic or a non-linear dynamic, characterized by a phase of transient instability followed by possible reorganization. The experiment compared two dough series identical in their general scheme but differing in maturation duration and, above all, in temperature control during the post-retard phase. Series I, conducted with more coherent thermal control, showed recovery of surface continuity, satisfactory vertical development, guided opening of the score, and a structurally functional crumb. Series II, subjected to longer maturation and compromised thermal control, showed a more fragile surface, greater morphological irregularity, less controlled expansion, and a more heterogeneous crumb, though still fully functional from a food standpoint. Based on the images and process data, a six-stage model is proposed: 1)initial aggregation of the preferment; 2)controlled dispersion; 3)cold maturation with biochemical relaxation; 4)critical post-retard instability window; 5)protein reorganization assisted by rest and manipulation; 6)stabilization of the functional network. The qualitative rheological interpretation suggests that the decisive parameter in einkorn is not merely the intrinsic strength of the flour, but the synchronism between reorganization of the protein matrix and gaseous development [1][2]. The theoretical framework is consistent with the literature on wheat gluten, which assigns a central role in dough viscoelasticity to glutenins, gliadins, and glutenin macropolymer, and with the literature on einkorn, which generally describes softer, less elastic, and more extensible doughs than common wheat, albeit with strong dependence on genotype and process [1][3][4]

1. Introduction

Einkorn is one of the oldest domesticated wheat species and is today being rediscovered for its nutritional, agronomic, and sensory interest. However, its breadmaking remains problematic, because its high protein content does not automatically translate into a strong gluten network in the technological sense typical of modern bread wheat. The literature indicates, in fact, that the breadmaking quality of wheat depends not only on total protein quantity, but also on protein composition, the gliadin/glutenin ratio, the presence and functionality of glutenin subunits, and the ability to form high-molecular-weight aggregates capable of conferring elasticity, cohesion, viscosity, and gas retention. Wieser recalls that gliadins are predominantly monomeric and associated mainly with viscosity and extensibility, whereas glutenins are polymeric, aggregated through interchain disulfide bonds, and constitute the component most directly associated with elasticity and the structure of the dough network [1].

In the case of einkorn, the situation is more delicate. Recent literature reports that, despite its high protein content, einkorn flours tend on average to produce softer, less elastic, and more extensible doughs than modern wheats, due to a generally lower gluten quality and a different balance between protein fractions. Brandolini and co-workers observe that einkorn “generally has poor breadmaking value,” although some elite lines show very interesting technological performance; Mefleh and co-workers, in a holistic approach on Italian genotypes, show marked differences in breadmaking behavior between ancient and improved varieties [3][4]. The overall picture suggests that einkorn should not be judged as a species intrinsically incapable of making bread, but as a system highly sensitive to genotype and process.

The present study is situated in this area of interest. It does not aim to generically demonstrate whether einkorn “does” or “does not” make bread, but rather to describe the structural dynamics of its dough during a complex protocol of cold maturation and thermal reactivation. The starting hypothesis is that the protein network of einkorn does not evolve in a linear and monotonic way, but rather passes through a phase of transient instability in which the apparent rupture of the surface may precede a useful reorganization of the matrix. The literature on glutenin macropolymer offers theoretical support for this possibility: Feng and co-workers show that mixing reduces the apparent content of GMP, whereas resting can restore part of it; at the same time, excessive resting can worsen the structural framework again [2].

This scientific framework makes particularly significant the empirical observation that guides the entire work: bread must be judged not only as a form to admire, but as a food material to be eaten. From a technological point of view, the distinction is crucial, because a bread with irregular morphology may still possess a continuous, non-sticky, sufficiently elastic, and sensorily valid crumb. This dual perspective, morphological and functional, guides the entire following analysis.

2. Objectives of the study

The main objective is to verify whether, in einkorn, the protein network during a breadmaking process with cold maturation follows a stage-based dynamic, in which a phase of transient fragility may be followed by a phase of structural reorganization.
The secondary objective is to identify the role of thermal control in the post-retard phase as a possible bifurcation variable between two outcomes: 1)a relatively stable functional network; 2)a still edible but more irregular network.

The third objective is to propose a qualitative rheological interpretation of the images and the behavior of the finished bread, without resorting to direct instrumental tests, while remaining consistent with the literature on the relationships between GMP, viscoelasticity, gas retention, and bread quality [1][2].

3. Materials, experimental setup, and data sources

The study is based on experimental documentation consisting of a written protocol, temperature and pH measurements, pre- and post-baking weights, and a photographic sequence of the main processing stages and the final product.
The raw material is stone-ground whole einkorn flour.

The process includes: 1)initial biga; 2)dissolution/dispersion; 3)cold maturation; 4)reactivation on a warm surface; 5)intermediate manipulations; 6)division; 7)shaping; 8)proofing in a basket; 9)scoring; 10)baking.

Two series were compared.

Series I: 24-hour maturation, ambient temperature approximately 22 °C, surface temperature on removal approximately 6 °C, more coherent management of the warm surface.
Series II: 28-hour maturation, ambient temperature approximately 23 °C, surface temperature on removal approximately 7.8 °C, lower initial pH, and compromised thermal control of the warm surface.
The temperature, pH, time, weight, surface description, and crust and crumb evaluations are those experimentally recorded in the protocol; the scientific interpretations of their meaning are related to the literature [2][3][4]. The images were read as morphological evidence of the mechanical and structural behavior of the dough. This is therefore not a study with standardized instrumental tests of alveography, farinography, dynamic oscillation, or texture profile analysis, but rather a high-resolution observational qualitative technical-scientific analysis.

4. Theoretical framework of reference

The breadmaking quality of wheat derives from a complex protein system in which the gliadin fraction and the glutenin fraction play complementary roles.
1)Gliadins, monomeric, contribute mainly to viscosity and extensibility.
2)Glutenins, aggregated through interchain disulfide bonds, constitute the high-molecular-weight polymers most involved in the elastic response of dough.
Glutenin macropolymer is considered a key component of this system, and several sources link it directly to viscoelastic properties and bread volume. Feng and co-workers report that GMP plays a prominent role in dough properties and breadmaking quality, that mixing reduces its apparent content through depolymerization, and that resting may favor a partial recovery through reformation of disulfide bonds and growth in the fraction of large particles [1][2].
In einkorn this balance proves on average less favorable to mechanical resistance. Brandolini and co-workers describe protein-rich einkorn flours with breadmaking value generally lower than that of the control soft wheat; Mefleh and co-workers highlight that the dough and bread performance of ancient and improved wheats depends on a set of compositional, rheological, and fermentative parameters [3][4]. The conclusion that emerges is therefore not a technological condemnation of einkorn, but the recognition of a strong dependence on genotype and process. This approach fits the present experiment well: two series very close in formulation, but diverging in structural outcome especially in relation to time, pH, and temperature of the post-retard phase.

The distinction between the two series is essential to correctly interpret the results.

Main process parameters distinguishing Series I and Series II during post-retard dough handling.

Parameter

Series I

Series II

Impact

Retard maturation

24 h

28 h

Greater enzymatic degradation in Series II

Temperature on removal from retard

6 °C

7.8 °C

More active dough network in Series II

Initial pH

5.2

4.9

Higher acidity in Series II

Warm surface control

Stable

Unstable

Less controlled dough development in Series II

Ambient temperature

22 °C

23 °C

Faster fermentation in Series II

Series II therefore started from a more degraded structural condition, characterized by higher acidity, increased enzymatic activity, and reduced thermal stability.

Series II therefore starts from a more degraded structural condition, characterized by greater acidity, greater enzymatic activity, and greater thermal instability. This explains the presence of a more degraded surface, widespread ruptures, and more irregular alveolation. The most important experimental finding is that the difference between the two series does not seem to depend primarily on the dough, formula, or fermentative agent, but on the thermal management of the post-retard network reactivation phase.

6. Exerimental results: phase-by-phase readin

6.1.Mature pre-dough

The In the initial images the pre-dough appears compact, fragmented, with sharp fractures and a load-bearing mass (Photo 1; Photo 2). This morphology is compatible with a mature pre-dough that has not collapsed. It is not yet a final bread network, but a pre-organized matrix. From the material point of view, the initial state is neither liquid nor completely plastic: it is a cohesive, locally rigid system, rich in structural potential. The visual characteristics are those of a correctly matured pre-dough (biga): hydration distributed, proteolysis present but not excessive, structure still load-bearing. This is important because the quality of the final network originates here.

6.2. Dissolution or dispersion phase

The dissolution phase shows a viscous, aerated system, with microbubbles and fluid continuity (Photo 3). In structural terms this step is equivalent to a controlled dispersion of the original matrix. It is here that the system loses the macroscopic continuity of the biga but gains mobility. The literature on GMP interprets mixing as a phase capable of partially depolymerizing glutenin aggregates and reducing the average particle size, with an increase in the proportion of smaller and more extractable forms [2]. Visually, medium viscosity, the presence of microbubbles, and a fluid but non-liquid structure are observed. This phase produces disaggregation of the gluten network, distribution of the proteins, and dispersion of GMP. This is exactly the principle of the proposed model.

6.3. Dough after cold maturation

After 24 hours at about 5 °C, in the images of the first series the surface appears relatively uniform and the bottom shows structural continuity (Photo 5; Photo 6). This phase does not suggest collapse, but relaxation. From a biochemical point of view it is reasonable to infer that hydration, acidification, and slow protein modifications continued in the retard. Cold slows but does not cancel such processes. In this phase, according to the model that will emerge later, the dough is not yet “ready” in a mechanical sense: it is a relaxed, biochemically modified system awaiting reactivation. Visual observations show a smooth surface, uniform structure, and signs of relaxation; the bottom presents slight adhesiveness but continuous structure. The most coherent interpretation is that of controlled enzymatic maturation, with active amylases, moderate proteases, and a weakened but not destroyed network.

6.4. Critical window after removal from retard

The images of Series I after about two hours on a warm surface show multiple surface ruptures, discontinuities, and loss of homogeneity of the skin (Photo 7, Series I). This is the most important phase of the entire work, because it suggests that einkorn enters a zone of temporary instability. The surface seems to worsen before improving. In a linear model this would be a sign of failure; in the protocol examined here, however, this fragility appears as a transient stage.
Here the most interesting datum is precisely the behavior of the gluten network. Photo 7 represents the phase of network rupture. The observed sequence is the following: 1)removal from retard with a fragile network; 2)manipulation with partial rupture; 3)warm rest with subsequent reaggregation; 4)more elastic structure. You have therefore demonstrated experimentally that the network can reorganize after rupture.

Series II shows in this same phase a more advanced and more vulnerable picture: greater surface porosity, more marked yielding lines, a more open texture already present, and faster fermentative activation (Photo 2, Series II). The whole is consistent with the process data: 1)4 more hours of maturation; 2)lower pH; 3)higher initial dough temperature; 4)less effective control of the warm surface. The literature does not provide a direct photographic relationship, but it makes the general mechanism plausible: in an einkorn dough, already more extensible and less elastic on average, an excessively rapid reactivation can unbalance the relationship between protein reorganization and gas development [3][4].

It is important to note that surface ruptures do not necessarily constitute an absolute defect. In einkorn they may indicate a more rigid surface network in the presence of active internal fermentation. If the internal network holds, the bread can still expand, as happens in this case.

6.5. Manipulation and reorganization

In Series I, after 2 hours and 30 minutes and manipulations, the surface becomes more continuous, smoother, more legible as a structured skin (Photo 9, Series I). The images indicate that the dough recovers cohesion instead of worsening further. The subsequent division also confirms greater stability of the mass (Photo 10, Series I).

Here lies the strongest theoretical point of the entire experiment. Photo 9 documents the reorganization of GMP. The protein system realigns the chains, reforms disulfide bonds, and builds a new, more elastic network. This is exactly the behavior predicted by the model of dispersion and reaggregation of the gluten network.

In Series II, after manipulation, only a partial improvement of the surface is observed, which nevertheless remains more fragile and porous than in Series I (Photo 3, Series II). This suggests that manipulation had a corrective effect, but not a fully refounding one. The connection with Feng et al. is particularly useful here: the resting following mixing can restore part of GMP, but the response depends strongly on the duration of the phase and on the conditions of the system [2].
When fermentation is already too advanced, manipulation does not completely regenerate the network but also tends to break already developed gas chambers. This leads to more irregular alveoli and a less stable structure.

6.6. Division, shaping, and final proofing

In Series I the divided and shaped doughs maintain shape, volume, and a fairly homogeneous surface (Photo 10, Series I). In the basket and at the end of proofing they show growth, the presence of small discontinuities, but good geometric legibility (Photo 13; Photo 14, Series I). The mass holds well, does not collapse, and maintains structure. This is a clear sign that the network has recovered elasticity.

The images of the proofing baskets show an increase in volume, surface microfractures, and still stable structure. The network appears extensible but not excessively weak. In einkorn this is a very good result.

In Series II, by contrast, already at the beginning of proofing in the basket, widespread points of weakness are observed (Photo 5, Series II); before baking the surface appears further compromised and much closer to the fracture threshold (Photo 6; Photo 7, Series II). This visual datum agrees with the hypothesis that the control of temperatures and times during proofing was compromised. In essence, Series II reaches baking with a surface already close to its own fracture threshold. The surface is already very tense, with small widespread rupture points and less elastic structure: a sign that the network is at the limit of its extension capacity.

6.7. Baking, crust, and final shape

In Series I the bread shows good vertical development, wide but legible opening, and orderly growth (Photo 16; Photo 17; Photo 18; Photo 19, Series I). The fracture follows the score coherently and no marked lateral spread is observed. Here we see good expansion, controlled opening, and uniform crust. The fissure follows the score: this indicates functional oven spring.
Vertical development is one of the most important signals of the entire test. It means that the network was extensible but also resistant and that fermentation did not destroy the structure. If the network had been too degraded, broad bread, flat bread, or collapse would have been observed. Instead, the experimental data show final heights between 7 and 7.5 cm for loaves of about 780 g: a very good result.

In Series II the bread instead shows more violent openings and more widespread fractures, with less control of expansion (Photo 8, Series II). However, and this is fundamental to emphasize, neither series shows the catastrophic collapse typical of a completely failed network. No flattened bread, fully slumped bread, or bread lacking internal crumb is observed. The difference is therefore one of control and uniformity, not of the existence or non-existence of structure.
In Series II no true progressive oven spring is observed, but rather a more explosive structural rupture. This is consistent with a situation in which the internal gas exceeds the resistance of the network.

6.8. Crumb

The crumb of Series I appears fine-medium, sufficiently distributed, with some irregularity also attributable to incorporated air, but without large anomalous cavities or massively compact zones (Photo 20; Photo 21, Series I). The bottom of the bread also confirms a well-supported structure (Photo 22, Series I). The bottom shows complete baking, the absence of compressed zones, and the absence of collapse, indicating a good balance between internal structure and hydration/baking ratio.
The description of the crumb is very clear: fine-medium alveoli, fairly homogeneous distribution, elastic crumb, slightly moist, absence of sticky crumb. This means that starch degradation was controlled, the malt is not excessive, and gelatinization occurred correctly. This is a very important finding for einkorn.

Series II presents a more irregular but still valid crumb (Photo 9, Series II). Medium and small alveoli remain predominant, the walls are in most cases continuous, and no sticky crumb or widespread collapse is observed. This implies that the network, although more disordered, retained gas sufficiently to generate a functional food structure. This point is extremely important: Series II is not aesthetically optimal, but it is not technologically null. It is an edible bread, endowed with crumb, structure, and sufficient integrity for food use.

The crumb of Series II deserves a specific evaluation. Despite the degradation of the network, the bread remains functional and edible. The alveolar structure shows predominantly medium-small alveoli, some isolated larger alveoli, non-uniform distribution, but absence of large empty cavities. This indicates that gas retention occurred, that the gluten network did not collapse, and that fermentation produced gas while still maintaining a certain structural cohesion. The crumb is granular but continuous; it does not appear gummy or sticky. The alveolar walls, although thin, are continuous and show good moisture distribution. This suggests an elastic and non-crumbly crumb.
The irregularity of the structure is the sign of the fermentative problem: less uniform alveoli, some isolated larger ones, less ordered structure compared with Series I. The probable causes are: 1)faster fermentation; 2)manipulation on already gassed dough; 3)partial rupture of gas chambers with random recombination of the bubbles.

From a technological point of view, Series II is therefore a valid but less controlled bread. In synthetic terms: good structure, good gas retention, elastic texture, medium homogeneity, irregular aesthetics. Series II demonstrates that even with excessively high temperature, accelerated fermentation, and a more fragile network, einkorn can still produce a stable and edible crumb. The protein system does not collapse completely, but loses part of its structural control.

7. Final quantitative parameters

The The quantitative parameters confirm the morphological and structural interpretation.
For Series I, the pre-baking weight of the loaves was approximately 780 g. The post-baking cold weight was around 652 g. Weight loss is therefore approximately 16-17%, a value fully consistent with a well-baked bread, with balanced internal moisture and a correctly formed crust. For rustic breads and ancient flours, a range of 15-18% can be considered very good.
The recorded final heights, between 7 and 7.5 cm, are also very positive for loaves of this mass.
The recorded final internal temperature was 93.6°C. This value falls within the ideal range of 92-96°C for breads with medium-high hydration and ancient flours, confirming the correctness of the baking.

8. Proposed physical-dynamic model

In light of the images, the data, and the theoretical framework, the behavior of einkorn in the protocol under examination is better described by a six-stage model than by a simple scheme of progressive development.

8.1. State A: initial aggregation of the pre-dought

The mature biga represents a fragmented but load-bearing protein pre-matrix (Photo 1; Photo 2). Water has not yet been redistributed in the way typical of the final dough, but a useful organization already exists. This state is locally stable, even if not yet suitable for final gas retention.

8.2. State B: controlled dispersion

The liquid or dissolution phase reduces macroscopic rigidity and increases the mobility of the components (Photo 3). From the GMP point of view, it is compatible with a partial depolymerization induced by shear and hydration. This does not constitute a definitive loss of function, but a temporary lowering of continuity [2].

8.3. State C: cold maturation and biochemical relaxation

During the stay in the retard, the matrix relaxes, acidifies, and hydrates more finely (Photo 5; Photo 6). The system does not strengthen mechanically in a simple sense, but predisposes itself to a new organization. The low temperature slows the processes but does not cancel them.

8.4. State D: critical post-retard instability window

This phase coincides with the emergence of surface ruptures and discontinuities (Photo 7, Series I; Photo 2, Series II). It is the point at which the system passes from a cold, relaxed, and biochemically modified network to a network once again mobile, fermentatively active, and mechanically stressed. In this window einkorn manifests its typical fragility: if reactivation is well synchronized, the instability is transient; if it is too rapid or too prolonged, the instability is amplified [3][4].

8.5. State E: assisted protein reorganization

Manipulation and rest, within the correct window, allow a recomposition of surface continuity in Series I (Photo 9; Photo 10, Series I), whereas in Series II they produce only a partial reorganization (Photo 3, Series II). The term “reaggregation of GMP” must be used as a structural inference consistent with the literature on resting, not as a direct measurement. The images of Series I and the work of Feng et al. make this interpretation highly plausible [2].

8.6. State F: stabilization of the functional network

The final result bifurcates into two outcomes:

1)relatively stable functional network, with orderly growth and a sufficiently homogeneous crumb (Photo 17; Photo 18; Photo 19; Photo 20; Photo 21, Series I);

2)still functional but less uniform network, with less controlled growth and a heterogeneous crumb (Photo 8; Photo 9, Series II).

This second outcome is precisely that of Series II. The crucial point is that the model does not distinguish only between “success” and “failure,” but between optimal network, functional network, and collapse. In the present experiment the first two levels were observed, not the third.
In synthetic form, the behavior of the dough follows the cycle: dispersion → rupture →

When the temperature is correct: network → dispersion → reaggregation → stable structure.
When the temperature is too high: network → dispersion → degradation → rupture.

9. Qualitative rheological interpretation.

9.1. Methodological premise

Here the term “rheology” is used in a qualitative sense. We do not have instrumental curves of G’, G’’, tan delta, farinograms, or alveograms relating to the doughs. However, the rheology of a dough can also be inferred from its response to manipulation, shape retention, surface quality, the dynamics of oven expansion, and crumb morphology. The literature on gluten, GMP, and dough properties justifies this kind of interpretive reading, provided that it is declared qualitative and not as a direct measurement [1][2].

9.2. Four key rheological properties

In the protocol under examination, four properties matter above all:

1)Elasticity: the ability to recover at least part of the shape after deformation.
2)Extensibility: the ability to stretch without breaking.

3)Resistance to deformation: the ability to oppose gas pressure and manipulation without yielding too early.

4)Gas retention capacity: the most important integrated property, requiring continuity of the matrix and coordination of the surface skin.

This distinction is consistent with Wieser’s classical framework on the complementary function of gliadins and glutenins [1].

9.3. Rheological reading of Series I

Advanced Method for Producing Bread Doughs with Flours Having Limited Gluten Development Capacity

by luciano

sangiogio.l@libero.it

Related artiche to: 

Experimental application of the advanced method for the production of bread doughs with flours with limited gluten development capacity: analysis of the results. (Analysis carried out by ChatGPT)

Foreword:
The method described here represents an evolution of the procedure covered by the patent:
NEW METHOD FOR MAKING (FERMENTED) BAKERY PRODUCTS
Publication date: 11.04.2018 – Bulletin 2018/15
Application number: 17194677.5
EP 3 305 078 A1

Based on this conceptual framework, several experimental tests were carried out using stone-milled whole einkorn wheat flour. The method is specifically designed for flours characterized by:
1 – weak gluten,
2 – high gliadin/glutenin ratio,
3 – redominantly viscous-cohesive rheological behavior.

The presentation of this method derives from one of the tests conducted, selected because it is representative of the full expression of the process.

1 – Technological context of the approach

Whole flours obtained from stone-milled ancient grains have technological characteristics that differ profoundly from those of modern strong flours commonly used in industrial breadmaking. The “strength” of a flour, in breadmaking terms, is linked to the ability of the protein fraction to build an elastic, continuous gluten network resistant to the pressure of fermentation gases. Comparative studies on wheat species show that, even in the presence of a high total protein content, ancient species such as einkorn may present a higher gliadin/glutenin ratio than modern soft wheat. The result is a less elastic and less structurally “load-bearing” network [1] (cf. comparative studies published in the MDPI field).

In practical terms: in other words, a high protein content does not necessarily imply the presence of a functional gluten structure for breadmaking purposes.

The reduced capacity of such flours to develop a continuous and elastic gluten network entails:
1 – difficulty in retaining the gases produced during fermentation;
2 – limited tolerance to mechanical processing;
3 – structural instability of the dough during the proofing stages;
4 – greater sensitivity to fermentation peaks.

In current practice, these critical issues are often compensated for by:
1 – blending with high-strength flours;
2 – use of improver additives;
3 – use of technological aids.

Such interventions, although effective from a volumetric standpoint, frequently entail a reduction in the nutritional, sensory, and identity-related peculiarities of the original flours, in addition to modifying aspects of digestibility and tolerability.

1.1 The specific case of einkorn

Triticum monococcum is one of the earliest cereals domesticated by humans and is considered one of the oldest wheat species still cultivated. Compared with modern wheats widely used in breadmaking, einkorn has peculiar nutritional and technological characteristics that significantly influence its behavior in doughs.

Several studies have shown that this species often has a relatively high total protein content; however, breadmaking functionality does not depend exclusively on the amount of protein, but rather on the structure and organization of the gluten protein system.

According to the structural models proposed by Peter R. Shewry and Peter R. Halford (2002) and subsequently further developed by Herbert Wieser (2007), the rheological properties of wheat doughs mainly depend on the interaction between three main protein classes:
1 – high molecular weight glutenins (HMW-GS);

2 – low molecular weight glutenins (LMW-GS);

3 -gliadins.

In einkorn, the relative distribution of these fractions differs significantly from that of modern soft wheat. In particular, the following have been observed:

1 – lower formation of high molecular weight protein polymers;
2 – greater proportion of monomeric proteins;
3 – limited ability to generate a continuous and structurally stable gluten network.

From a technological standpoint, this generally translates into doughs characterized by:
1 – lower stability during processing;
2 – greater extensibility;
3 – lower ability to retain fermentation gases.

Despite these structural limitations, recent studies have shown that the breadmaking performance of ancient grains can be significantly improved through proper management of the technological process. In particular, techniques based on controlled fermentations, prolonged maturation, and careful management of the thermal conditions of the dough can promote a progressive reorganization of the protein system and improve structural stability during proofing and baking (Luca Cappelli et al., 2019; Gabriele Brandolini and Adriano Hidalgo, 2014).

In light of these considerations, the present work experimentally analyzes the behavior of the einkorn protein network during a breadmaking process characterized by a prolonged low-temperature maturation phase followed by controlled thermal reactivation of the dough.

2. Process objective

Whole flours — particularly those derived from ancient grains such as einkorn — pose specific technological challenges:

1 – greater water absorption and competition for water;

2 – interference of the bran fraction with the formation and continuity of the protein network;
3 – fermentative variability;

4 – presence of phytates and other compounds with potential nutritional interference.

Indirect fermentation and sourdough strategies are commonly employed to mitigate these critical issues through:

  1. controlled acidification
  2. activation of endogenous enzymes (e.g. phytase)
  3. modification of the fibrous fraction (e.g. arabinoxylans)
  4. aroma development and improved shelf life

The objective of the methodology described here is to:
1 – concentrate and “condition” the most critical fraction of the dough (rich in bran and fiber) within a biga-type pre-dough;
2 – maintain rigorous thermal control during the different stages of the process, to preserve fermentative predictability and avoid undesired accelerations;
3 – introduce an innovative step of mechanical dispersion of the pre-dough in cold water, with air incorporation, aimed at improving the homogeneity of the dough system and the volumetric contribution during the baking stage.

2. Materials and Methods: complete description of the method

2.1 Raw material and functional fractionation

Raw material

1 – Whole einkorn wheat flour (Triticum monococcum), stone-milled
2 – Total quantity: 1800 g

Stone milling is known to fully preserve the grain fractions (endosperm, germ, bran), with a higher content of bioactive compounds and fibers than refined flours, but with a significant impact on the rheological properties of the dough (Shewry & Hey, 2015; Hidalgo & Brandolini, 2014).

Sieving at 600 µm
The flour was entirely sieved through a 600 µm mesh, obtaining:
1 – 85 g residual bran fraction
2 – 1715 g passing flour

The fractionation is not aimed at refining, but at functional reorganization of the components, in order to modulate the fiber load in the pre-dough..

Distribution in the process stages
Final dough: 1000 g flour passing 600 µm
Pre-dough (Biga):
715 g passing flour
85 g residual bran
= 800 g total

Methodological consideration
The pre-dough does not contain only bran, but is a classic pre-dough that ferments a whole flour made richer in the fibrous fraction. The fiber load is increased in the pre-dough, concentrating the most critical phase (arabinoxylans, phytates, insoluble fraction) in the indirect fermentation stage.

Note:
A – Biga is a dry/coarse pre-dough. “Pre-dough” is a more generic term that includes both solid methods (biga) and liquid ones (such as poolish or Giorilli biga).

B – Preferment vs pre-dough: biga or pre-dough are themselves “ferments.” It is therefore NOT correct to define them as pre-ferments, since they are themselves ferments intended for fermentation: fermentation and/or metabolic activity occurs immediately in time and NOT afterwards.

Scientific reference
The concentration of the bran fraction in the pre-dough stage is consistent with studies showing that fermentation of bran-rich fractions can:
1 – increase arabinoxylan solubilization
2 – reduce the antagonistic effect of fiber on the protein network
3 – promote activation of endogenous phytase through acidification
(Katul et al., 2019; Rizzello et al., 2010; Lopez et al., 2001).

2.2 Pre-dough (12 hours, controlled temperature)
Pre-dough ingredients
1 – Flour: 800 g.
2 – Water: 340 g.
3 – LiCoLi: 180 g (assumed 100% hydration: 90 g water + 90 g flour)
3 – Fresh compressed baker’s yeast: 3 g.

Actual” pre-dough composition (accounting for the LiCoLi)
1 – Total flour in biga = 800 + 90 = 890 g
2 – Total water in biga = 340 + 90 = 430 g
3 – Actual biga hydration = 430 / 890 = 48.3%
4 – PH: 4.75
5 – actual weight: 1321 g.

Fermentation
2 – Temperature: 18 °C
3 – Initial biga temperature: 16 °C
4 – Final temperature: 18.2 °C

Thermal control
The initial temperature of the pre-dough must not exceed 18 °C in order to avoid early entry into the exponential phase of yeast growth.

In the test (02/03/2026; ambient T 21 °C):
1 – Water temperature: 5 °C
2 – Flour temperature: 10 °C
3 – Initial pre-dough temperature: 16 °C

In summer, the water may go down to 2 °C and the flour even to 5C° in order to maintain the thermal constraint.

Technological rationale
A 48% pre-dough on high-bran flour generates a matrix:
1 – compact
2 – with reduced water mobility
3 – with limited metabolite diffusion

A condition consistent with slower and more structured fermentation (Gobbetti et al., 1994).

Acidification (pH about 4.75) falls within the range favorable to activation of endogenous wheat phytase, responsible for phytate reduction (Lopez et al., 2001; Leenhardt et al., 2005).

Structural note: with 48% hydration on high-bran flour, the pre-dough is consistently “pasty/compact”: this favors slower and more structured fermentation and reduces the risk of rapid and unmanageable rising, but makes direct incorporation into the final dough more difficult.

2.3 Li.Co.Li. (liquid culture starter)
The Li.Co.Li is made with the same whole einkorn flour.

Refreshment
Ratio 1:1:1
(e.g. 50 g starter + 50 g water + 50 g flour)

Fermentation:
3–4 h
25 °C
until volume doubles

Choice of the same flour
In current practice, strong flours are often used for managing sourdough starter. In this protocol, the same whole einkorn flour is used in order to:
1 – avoid the introduction of external strong gluten
1 – maintain protein consistency of the system
1 – not alter the rheological profile of the final product

Function of Li.Co.Li in the system
In the present protocol, LiCoLi:
does not have the primary function of a leavening agent (because limited with einkorn) has the prevailing function of biochemical maturation

The literature shows that sourdough fermentation:
1 – promotes partial protein hydrolysis
2 – reduces phytates
3 – increases mineral solubility
4 – modifies the structure of arabinoxylans
(Rizzello et al., 2010; Gänzle, 2014).

The use of whole flour in Li.Co.Li increases the availability of fermentable substrates and bioactive compounds, supporting a more diversified microbiota (Gobbetti et al., 2016).

2.4 Final dough: nominal ingredients (without pre-dough)
Final dough ingredients:
1 – Flour (Fraction A): 1000 g
2 – Water: 660 g
3 – EVO oil: 72 g
4 – Salt: 30 g
5 – Malt: 22 g
6 – Fresh compressed baker’s yeast: 4 g

Final dough data:

a – Water temperature: 4 °C
b – Flour temperature: 7 °C
c – Final dough pH: 5.25
d – Dough temperature: 16.2 °C
e – Total preparation time: about 30 min.
f – Biga dispersion time: about 5 min.
g – Total dough weight: 3117 g

2.5 Overall dough balance (pre-dough + final dough)
Total flour in the system
890 g. (biga) + 1000 g. (final dough) = 1890 g.

Total water in the system
430 g. (pre-dough) + 660 g. (final dough) = 1090 g.

Actual total hydration
1090 / 1890 = 57.7% (≈ 58%)

Baker’s % (based on total flour = 100%)
Water: 57.7%
EVO oil: 72/1890 = 3.8%
Salt: 30/1890 = 1.6% (must be combined with the flour)
Malt: 22/1890 = 1.2%
Total compressed yeast: (3+4)/1890 = 0.37%
Li.Co.Li (total weight): 180/1890 = 9.5% (note: already included in the flour+water balance)

In this test: water temperature 4C°; flour temperature 7C°; pH at end of dough mixing: 5.25; dough temperature before resting: 16.2C°; total dough preparation time 30 minutes; time to dissolve dough 5 minutes; dissolved biga temperature: 16.2C°. Dough weight: 3117 (divided in two to perform two separate baking tests; moreover the first dough will come out of the chamber after 24 hours at about 5 C°; the second dough 4 hours later).

Important note:
After preparation in the mixer, the dough is placed on a work surface lightly greased with oil (NOT with dusting flour) in order to be divided into two parts.

2.6 The role of oil (extra virgin). Oil in bread dough greatly improves consistency and shelf life, making the crumb softer, more elastic, and more fragrant. It acts as a natural lubricant, promotes regular alveolation, and increases bread shelf life (preservation) by retaining moisture.
Oil in weak-gluten doughs plays an important structural as well as sensory function.

Documented effects:
1 – reduction of friction between proteins
2 – increase in extensibility
3 – improvement in crumb softness
4 – slowing of staling through interaction with starch
(Cauvain & Young, 2007; Primo-Martín et al., 2006).

In weak flours, the presence of lipids can contribute to stabilizing the alveolar structure by acting as a plasticizer of the protein matrix.

2.7 The role of malt Malt acts in bread dough as a natural improver. Rich in enzymes, it breaks down starches into simple sugars, constantly feeding the yeast, improving fermentation, increasing volume, and giving a more golden and crisp crust.
Malt provides amylase enzymes that:

hydrolyze starch → maltose and fermentable sugars

support yeast nutrition

increase CO₂ production

promote crust coloration (Maillard reaction). (Lynch et al., 2009).

In weak flours, the balance is delicate: excess amylase activity can further weaken the structure, while controlled dosages improve fermentation without compromising stability.
Diastatic malt is a malt that has a high concentration of these enzymes and is therefore capable of breaking down starches and producing simple sugars (maltose and maltodextrins). It is very active …. with einkorn a reflection is necessary and …control tests.

References
Geisslitz et al., 2019 – einkorn protein composition
Lopez et al., 2001 – phytate reduction sourdough
Leenhardt et al., 2005 – endogenous phytase
Rizzello et al., 2010 – fermentation and bioactivity
Gänzle, 2014 – sourdough microbiology
Cauvain & Young, 2007 – bakery technology
Primo-Martín et al., 2006 – lipid–starch interactions
Lynch et al., 2009 – malt enzymes and fermentation

3. Process innovation: water dispersion and “controlled aeration” of the pre-dough
3.1 Practical and technological rationale

At the end of 12 h, the biga is “pasty” and difficult to incorporate homogeneously with water/flour without leaving lumps and microbial/hydration inhomogeneities. To overcome this limitation, an intermediate step is adopted that also has another important function:

3.2 Procedure

  1. 550 g of water (part of the final dough water), at about 5 °C, is added to the mature biga.

  2. An immersion blender (with blades) is used for 5–7 minutes, at medium-low speed, with the main objectives of breaking/dispersing the biga (not whipping, not emulsifying) and, above all, incorporating air.

  3. Cold oil and malt are added to the dispersed biga (dense slurry).

  4. Transfer to fork mixer: the remaining water is added (110 g, because 660–550 = 110 g) and mixed for about 10 minutes at low speed. Water temp.: 4; flour temp. 7C°

  5. Dough exit temperature: about 16.2 °C. In this test: time 12 minutes.

  6. Dough PH: 5.25

Considerations (highlighted):

Thermal control: water at 5 °C acts as a “buffer” against shear heating; with output at ~17 °C one remains below the threshold at which leavening activity accelerates rapidly.
Microbiological and metabolic homogenization: dispersion increases the uniform distribution of yeasts/LAB and of the metabolites produced in the biga (acids, aromatic compounds), reducing the risk of “inert” or hyperactive zones.

Air incorporation: even without “emulsifying,” dispersion introduces microbubbles that act as nuclei; in the oven they can contribute to growth because the gases (air+CO₂+steam) expand as temperature rises. The effect does not replace fermentation, but can improve nucleation and alveolar uniformity.

3.3 Role of γ-gliadins in the adhesive behavior of dough
Einkorn wheat is characterized by a peculiar protein profile, in which the gliadin fractions — including the γ-gliadin component — are relatively more represented than in modern soft wheat.

γ-gliadins belong to the class of monomeric gluten proteins and contribute mainly to the viscous and adhesive properties of the dough, rather than to the formation of elastic three-dimensional networks (Shewry et al., 2002; Geisslitz et al., 2019).

A high proportion of gliadins relative to glutenins results in:
1 – greater viscosity;
2 – lower elasticity;
3 – more plastic-adhesive behavior;
4 – greater tendency of the dough to adhere to metal surfaces during mechanical processing.

In einkorn, this characteristic may be particularly evident, with practical effects such as:
adhesion to mixer walls;
1 – difficulties in industrial sheeting;
2 – greater friction in automated processes (e.g. pizza lines, laminated products).

Technological interpretation
Adhesiveness is not a defect in an absolute sense: it is the expression of a protein matrix dominated by less structuring monomeric components. However, in an industrial setting:
1 – high adhesiveness requires lubrication systems (oiling of surfaces);
2 – it limits compatibility with high-speed lines;
3 – it reduces stability in laminated processing.

The addition of a small amount of oil in the initial stage helps to:
1- reduce dough-surface friction;
2 – modulate plasticity;
3 – improve workability without significantly altering the structure.

Additional note

γ-gliadins, although they do not contribute to the formation of high molecular weight gluten polymers, significantly influence dough rheology through modulation of viscosity and adhesiveness. In einkorn, the high gliadin/glutenin ratio amplifies this behavior, making the dough less suited to industrial paradigms based on high elasticity and mechanical tolerance, but potentially more suitable for controlled low-mechanical-energy systems, such as the one described here.

Useful references for this part
Shewry, P. R., Halford, N. G., Belton, P. S., & Tatham, A. S. (2002). The structure and properties of gluten. Philosophical Transactions of the Royal Society B.
Geisslitz et al., 2019 – einkorn protein composition.
Scanlon & Zghal, 2001 – bread structure and dough physics.
Gänzle, 2014 – sourdough microbiology.

4. Cold maturation and kinetic control
4.1 Cell maturation
Finished dough (inside covered container) placed in chamber at ≈ 5 °C for 24 h.
Dough temperature on exiting the chamber: ≈ 6–8 °C.

Consideration (highlighted): starting with dough at ~17 °C, then bringing it to 5 °C, reduces “thermal shocks” and above all avoids entering the chamber already in full fermentative acceleration. It is a fine control of the fermentation curve.

5. Phenomenon of water on the lid in the chamber (“internal” condensation)
It may be observed that, upon opening the lid of the dough container, it is already wet even in the chamber,.

Correct physical interpretation

In a closed container, a small fraction of the dough water passes into the vapor phase (even at 5 °C the vapor pressure is not zero). The internal air tends to become saturated. The coldest point/condensation surface (often the lid) collects the vapor that re-condenses.

Conclusion: it is not “starch releasing water” and it is not retrogradation (that occurs after gelatinization during baking). It is migration of the water phase → vapor → water and attainment of an internal hygrometric equilibrium.

Possible secondary contributions

Redistribution of water in the matrix (fibers/proteins hydrate slowly, releasing or making more mobile a fraction of water initially weakly retained). Metabolic production of water (minimal at 5 °C but theoretically present) as a by-product of alcoholic fermentation; in a closed system it may contribute marginally to saturation.

6. Microbial cooperation: LAB + yeasts (and why they “really do collaborate”)
6.1 General principle: sourdough/mixed fermentation ecosystem

In systems with sourdough starter (LiCoLi), the following typically coexist:
lactic acid bacteria (LAB): acidification (lactic/acetic), indirect enzymatic activity, substrate modulation;
yeasts (including added baker’s yeast): production of CO₂, ethanol, metabolism of specific sugars, and aromatic contribution.

Classical literature clearly shows that this is not simple co-presence: there are trophic (cross-feeding) and non-trophic (competition/selection, antimicrobials, pH) interactions. (ScienceDirect)

6.2 Examples “in simple words” but technically correct

Some LAB can hydrolyze maltose and leave glucose available; yeasts unable to use maltose can thus grow thanks to the released sugar (cross-feeding). (Springer Nature Link). Yeasts can release essential amino acids (e.g. valine/leucine), favoring LAB growth under conditions in which they would otherwise grow poorly. (PubMed)

The practical result is often: greater LAB yield/activity (more acidity and metabolites) without necessarily increasing yeast yield in parallel, i.e. true functional synergy. (PubMed)

6.3 Is cooperation different between pre-dough and final dough?
Yes: the ecological environment changes.

In the pre-dough (48%, ≤18 °C, high bran):
1 – more compact and structured environment;
2 – acidification and transformations of the matrix (fiber/phytate) are relatively more “central”;
3 – LAB-yeast cooperation is above all one of biochemical preparation (acidification, metabolites, substrates made available). (Springer Nature Link)

In the final dough (maturation at 5 °C):
microbial growth slowed down; slow processes prevail: water redistribution, enzymatic rebalancing, maturation; cooperation becomes mainly one of maintenance/maturation rather than expansion.

In the 4 hours before the oven (gradual heating and then 30 °C in the basket):
Baker’s yeast becomes the protagonist of the volumetric push; LAB continue to modulate pH and aroma, but volume derives mainly from yeast CO₂.

7. Role of baker’s yeast in the final dough: “push” yes, but with a real thermal curve
7.1 In the system adopted in this test, the commonplace “baker’s yeast requires a long time” does not apply
In a direct dough, the yeast must adapt and multiply from zero. In this system:
1 – a portion of yeast has already gone through 12 h of pre-dough (so the biomass is not “only 3 g” initially);
2 – then 24 h in the cold keeps the system active but slowed;
3 – thermal recovery after the chamber progressively restarts CO₂ production.

7.2 The operational thermal sequence
1 – Chamber exit: 7–8 °C
2 – Rest on plate ~22 °C (TA ≈20 °C) – total pre-oven duration ≈ 4 h
3 – The dough reaches ~20 °C only in the last ~2 h (field observation).
4 – After ~3 h: transfer to proofing basket with plate at 30 °C (push phase).

Interpretation:
1 – first part: reactivation (little expansion);
2 – last 2 hours at ~20 °C: perceptible CO₂ increase;
3 – phase at 30 °C: main push, window to be controlled.

7.3 Why increasing yeast in the final dough can “break everything”
1 – with einkorn + high bran + warm final phase, increasing yeast risks:
2 – accelerating the 30 °C phase too much;
3 – reducing the control window and increasing overproofing/collapse;
4 – altering the LAB/yeast balance and the aromatic profile.

The setup works because the curve is progressive and controlled.

8. Technological and biochemical rationales linked to “enhanced” bran in the pre-dough


8.1 Arabinoxylans and water management (functional fiber)

Studies on pre-fermented bran and on sourdough/wholemeal show that fermentation can increase the solubility of fibrous components (particularly arabinoxylans) with consequences for rheology, water retention, and shelf life. (ScienceDirect)