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Why intermolecular bonds “make” gluten strong

by luciano

(In-depth note 1 of Genetic potential and processing conditions in determining gluten strength, digestibility, and immunogenicity)

Gluten is a protein network that emerges when gliadins and glutenins are hydrated and subjected to mechanical energy (mixing/kneading) or thermal energy (heating). Its “strength” (tenacity/elasticity and ability to withstand stress) depends on two families of interactions:

1 – Covalent disulfide bonds (S–S).
These are the most “structural” cross-links. In glutenins (especially HMW-GS and LMW-GS), intermolecular disulfide bonds build polymers (often referred to as GMP / glutenin macropolymer) that provide the elastic backbone of the network.
2 – Non-covalent interactions (hydrophobic, hydrogen bonds, ionic interactions).
They are weaker but extremely numerous and “modulate” the network: they contribute to cohesion, viscosity, reorganization, and the response to hydration/temperature/solvents. Many studies show that changes in secondary structure and non-covalent interactions accompany (and sometimes amplify) the effects of disulfide bonds.
A key point:

the network is not static. During processing, thiol–disulfide exchange reactions (–SH/–S–S–) occur that remodel network connectivity: more opportunities to form/reorganize S–S bonds → generally a “stronger” and/or more resilient network.

Practical implications for dough

From an operational standpoint, gluten strength does not depend only on the genetic potential of the flour, but also on how the system is “set up” to express and organize its intermolecular bonds.

Adequate hydration:
Water acts as a plasticizer and allows proteins to move, interact, and realign. Hydration levels that are too low limit network formation; higher hydration promotes molecular mobility and bond reorganization, making gluten more extensible.

Mixing energy:
Mechanical action facilitates contact between protein chains and accelerates thiol–disulfide exchange reactions. Insufficient mixing leads to an incomplete network; excessive energy, on the other hand, can cause bond breakage and excessive reorganization, with a loss of structure.

Resting time:
Rest phases (autolyse, bulk fermentation) allow non-covalent interactions and disulfide bonds to redistribute toward more stable configurations, improving the balance between elasticity and extensibility.

Chemical conditions:
pH, salts, and the presence of oxidizing or reducing agents directly influence the equilibrium between –SH groups and –S–S– bridges, thereby modulating cross-link density within the network.

Genetic Potential and Processing Conditions in Determining Gluten Strength, Digestibility, and Immunogenicity

by luciano

Introduction
Gluten is the protein complex that forms when wheat storage proteins—mainly gliadins and glutenins—are hydrated and subjected to mechanical work. During this process, they organize into a continuous three-dimensional network responsible for the viscoelastic properties of dough.
Gluten strength is not an intrinsic and immutable property of individual wheat proteins, but rather an emergent characteristic of the supramolecular organization that develops when storage proteins are hydrated and exposed to mechanical energy during mixing (Shewry & Tatham, 1997; Wieser, 2023). Gluten quality therefore results from the interaction between the initial molecular composition and process-induced structural transformations.
In the grain, gliadins consist mainly of monomeric proteins stabilized by intramolecular disulfide bonds, whereas glutenins are also present as polymers stabilized by intermolecular disulfide bonds, which constitute the structural basis of gluten elasticity (Shewry & Tatham, 1997; Wieser, 2023). Disulfide bridges therefore represent the main covalent cross-links responsible for the formation of a continuous protein network.
It is essential to distinguish between the strength of an individual bond and the ability to form an extended network of bonds. From a chemical perspective, the bond energy of a disulfide bridge is essentially constant; differences among varieties do not arise from “stronger” bonds, but from variations in the number, position, and accessibility of cysteine residues, as well as from the composition of high- and low-molecular-weight glutenin subunits (Wieser, 2023). These features define the genetic cross-linking potential, namely the intrinsic predisposition of proteins to participate in the formation of intermolecular bonds.
The existence and structural importance of disulfide bonds in gluten have been confirmed through direct identification of S–S connections by mass spectrometry, which enabled mapping of specific intra- and intermolecular bonds in gluten proteins (Lutz et al., 2012). This evidence supports the concept that the gluten network is stabilized by a dense web of covalent connections.
During mixing, genetic potential is converted into real structure through dynamic processes of disulfide bond breakage and reformation, mainly via thiol–disulfide exchange reactions (Lagrain et al., 2010). Consequently, the gluten network does not simply correspond to the polymers already present in the grain, but rather represents a reorganized structure that develops as a function of hydration, mechanical energy, temperature, and redox conditions.
Protein composition also influences the architecture of the polymers that form. It has been shown that certain gliadins containing an odd number of cysteine residues can be incorporated into polymeric fractions and act as elements that limit or modulate chain extension (Vensel et al., 2014). This highlights that network quality depends not only on the amount of polymeric proteins, but also on their molecular nature.
In parallel, classic studies have shown that glutenin polymers undergo depolymerization and repolymerization during dough processing, and that the content of glutenin macropolymer (GMP) is closely correlated with dough and gluten strength (Weegels et al., 1996). This dynamic behavior underscores the decisive role of processing conditions in modulating the expression of genetic potential.

Structural Implications for Digestibility
Gluten strength and protein network structure influence not only dough rheology, but also the accessibility of proteins and starch to digestive enzymes. Recent studies show that glutens characterized by a more compact and extensive network are associated with a lower rate of starch digestion and different kinetics of protein degradation, suggesting that the gluten matrix functions as a physical barrier to enzymatic action (Zou et al., 2022).
At the molecular level, gluten proteins are rich in proline and glutamine, a composition that confers intrinsic resistance to major gastrointestinal proteases. As a result, gluten digestion frequently leads to the formation of relatively long and poorly degradable peptides (Di Stasio et al., 2025).
Among these, fragments derived from α-gliadins—such as the well-known 33-mer peptide—exhibit high resistance to proteolysis and contain epitopes recognized by the immune system in individuals with celiac disease (Hernández-Figueroa et al., 2025). The likelihood of formation and persistence of such peptides is influenced by both wheat genotype and gluten structural organization.

Role of Processing in Modulating Peptides
Processing conditions, particularly fermentation, can significantly modify gluten structure and the peptide profile generated during digestion. Sourdough fermentation, through the combined activity of endogenous flour enzymes and microbial proteases, can partially hydrolyze gluten proteins and alter the distribution of released immunogenic peptides (Ogilvie et al., 2021).
Peptidomic analyses of breads subjected to in vitro digestion reveal considerable peptide diversity, correlated with wheat genotype, agronomic conditions, and processing technologies (Lavoignat et al., 2024). This confirms that the final peptide profile is not determined solely by protein sequence, but also by network architecture and its processing history.
The use of standardized semi-dynamic digestion protocols (such as INFOGEST) allows realistic simulation of oral, gastric, and intestinal phases, enabling quantification of resistant and potentially toxic peptide formation (Freitas et al., 2022). Advanced liquid chromatography–mass spectrometry techniques allow absolute quantification of these fragments and comparative evaluation of varieties and processes.
In parallel, the use of supplemental enzymes or selected microorganisms has been explored as a strategy to enhance degradation of particularly resistant gluten peptides, demonstrating that targeted interventions can significantly reduce the concentration of problematic fragments (Dunaevsky et al., 2021).

Integrated Perspective
Taken together, these findings lead to an integrated view:
The initial molecular composition defines the upper limit of possible gluten connectivity.
Mixing and fermentation determine how much of this potential is actually expressed.
The resulting network structure influences not only technological strength, but also digestibility and the profile of released peptides.

In summary:
✔ What matters most is the network that forms in gluten
✔ But this network is constrained by what exists at the origin
✔ And the resulting network also determines the digestive fate of proteins

Einkor wheat bread 100%: the strength of gluten makes the difference

by luciano

The aim of the study
The aim of the study is to evaluate the role of gluten strength of the same genotype (equal genetic imprinting) but with different cultivation on the final volume of bread.

Test
Two loaves were made with two einkorn wheat flours which are completely identical in quantity of ingredients and methods of execution. Both flours used belong to the einkorn genotype type ID331; one (A) grown without any fertilizer or other chemical compounds, the other (B) grown with the supply of nitrogen.
Both loaves were prepared with the same method:
“New Method for making fermented bakery products n. EP 3305078B1: at the bottom of https://glutenlight.eu/en/2019/09/27/einkorn-bread100/”.
The result clearly shows how the strength of gluten (1) played a decisive role in giving bread (B) a higher volume, a more open and regular crumb (Photo NN. 3, 4, 5, 6, 7, 8).
It is known that the supply of nitrogen contributes to increasing both the quantity and strength of gluten (2). This was a decisive factor for the development of agrotechnics which allowed flour to be produced with better workability from an industrial point of view; the increase in the strength of gluten, however, led in parallel to a less digestible (3) and less tolerable (4) gluten.

Gluten: digestibility

by luciano

Gluten which is a compound formed by gliadin and glutenin which is the basis of baked products (bread and other) is not, as such, assimilable by the intestine but must be reduced to the amino acids components or small series (peptides) of them. The reduction occurs by different enzymes such as trypsin in the stomach, pepsin in the small intestine and other enzymes [1]. In normal health the intestine expels the parts of gluten that are not digested because they are too large to be assimilated. The digestibility of gluten is not only, however, dependent on the “strength of the gluten”, that is on the strength of the different types of bonds that “connect” the proteins of gluten but also on the type of enzymes that hydrolyse “break” the gluten and from the environment in which these processes take place. For example, trypsin in the stomach is activated (ie works), only in an acid environment. Furthermore, all digestive enzymes have the possibility of working better if directly in contact with gluten: something that can only occur in laboratory experiments, since these enzymes will have to “work” on in the stomach and intestines a “complex” of foods and not on gluten [2]. Knowledge of the digestibility of gluten is therefore extremely complex being affected by multiple factors, not least the variability of the conditions of the environment where it occurs (stomach and intestine).

The method of preparation of the finished product should not be overlooked. Indeed the digestibility of gluten, and more specifically, of the finished product is greatly influenced by the preparation method and the ingredients used [3]. Among these a primary role is played by the type of flour and the use of sour dough and / or yeasts. Certainly the use of flours that have little and weak * gluten favor the digestive process but a fundamental role is played by the sourdough (better if associated with very limited quantities of brewer’s yeast). The sourdough with its lactobacilli carries out a strong action of hydrolysis (chopping) of the gluten proteins both directly and by activating the proteases of the flour. Many studies and researches have been devoted to this subject, one in particular:

Protein Digestibility of Cereal Products Iris Joye
Department of Food Science, University of Guelph, Guelph, ON N1G 2W1, Canada; ijoye@uoguelph.ca; Tel.: +1-519-824-4120 (ext. 52470). Published: 8 June 2019
“Abstract: Protein digestibility is currently a hot research topic and is of big interest to the food industry. Different scoring methods have been developed to describe protein quality. Cereal protein scores are typically low due to a suboptimal amino acid profile and low protein digestibility. Protein digestibility is a result of both external and internal factors. Examples of external factors are physical inaccessibility due to entrapment in e.g., intact cell structures and the presence of antinutritional factors. The main internal factors are the amino acid sequence of the proteins and protein folding and crosslinking. Processing of food is generally designed to increase the overall digestibility through affecting these external and internal factors. However, with proteins, processing may eventually also lead to a decrease in digestibility. In this review, protein digestion and digestibility are discussed with emphasis on the proteins of (pseudo)cereals.”