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Grains, Immunogenicity, and Gluten Strength: Genetic Bases and Applied Markers

by luciano

(In-depth article 4 of: Genetic potential and processing conditions in determining gluten strength, digestibility, and immunogenicity)

When creating a new wheat cultivar, breeders aim to obtain strong wheats.

In breeding, this becomes central because:

  • if you want to increase the probability of obtaining “strong” lines, you select parents with favorable alleles/subunits;

  • then, in the progeny, you use rapid tests (and increasingly molecular markers / rapid proteomics) to choose the best lines.

Clear examples:

  • Near-isogenic lines (NILs) or lines with targeted deletions: these are specifically used to isolate the effect of a single HMW-GS on dough strength/elasticity. A recent study shows that the absence of individual HMW-GS reduces elasticity/strength and alters alveographic parameters. (ScienceDirect)

  • Studies on populations (DH lines) comparing combinations of HMW-GS and their effect on quality traits: they show that the effect is not only “presence/absence,” but also depends on interactions among subunits. (PLOS)

  • Accelerated screening of breeding lines with rapid gluten-strength tests: useful because breeding programs must evaluate thousands of samples. (MDPI)

Wheats with lower genetic potential but greater ability to create new bonds.

The “genetic potential” (subunits, cysteines, fraction ratios) sets an upper limit: if certain structural components are missing, you cannot build a large network from nothing.
However, the “capacity to express” that potential also depends on factors that vary among varieties and lots: accessibility of reactive groups, thiol–disulfide exchange kinetics, initial distribution of polymeric fractions, etc.
This is why, in practice, proxies such as GMP and polymeric fraction analyses are also used to understand how much the network actually develops. (ResearchGate)

These biological differences translate into the possibility of using specific genetic and proteomic markers as predictive tools.

Comparative Table – Markers and Tests for Bread vs. Pasta

Practical markers (also usable in professional contexts)

1) HMW-GS profile at Glu-1 loci (Glu-A1 / Glu-B1 / Glu-D1)

  • What it is: which HMW-GS subunits are present (e.g., at Glu-D1: 5+10 vs 2+12).

  • Why it matters: some combinations are repeatedly associated with better rheological and baking properties; in particular, allele 5+10 (Glu-D1d) and 17+18 (Glu-B1i) are often reported among the most “effective.” (PMC)

  • How it is measured (practical): SDS-PAGE; in screening contexts also MALDI-TOF. (PMC)

2) “Polymeric vs monomeric” ratio (P/M) or proportion of high-MW polymers (SE-HPLC / extractability)

  • What it is: how much protein is in polymeric form (glutenins, especially high MW) relative to smaller/monomeric fractions.

  • Why it matters: higher polymeric fraction (and especially large polymers) → greater potential elastic “framework.”

  • How it is measured: SE-HPLC (size distribution) or extractability proxies (SDS-soluble vs SDS-insoluble).

3) GMP / UPP (glutenin macropolymer; unextractable polymeric protein)

  • What it is: fraction of very large polymers (often SDS-insoluble) considered tightly linked to network strength.

  • Why it matters: one of the most widely used proxies for “how much polymeric network” can be built and expressed.

4) Free thiol (–SH) content and redox state

  • What it is: how many –SH groups are free (and therefore potentially involved in thiol–disulfide exchange).

  • Why it matters: it does not indicate “how high W will be,” but helps explain disulfide reorganization dynamics (expressibility of potential), i.e., how easily the network can remodel.

Documented examples

A) Example (bread): cultivars named in a study with moderate-to-strong gluten

A study on Indian varieties combining markers and rheological evaluations reports that only four varieties among those analyzed combined high protein content and moderately strong gluten: K307, DBW39, NI5439, DBW17. (PMC)
Note: this is a “named” example within a specific study (useful as proof that literature lists cultivars), but obviously relative to the germplasm and context of that work.

B) Example (Italy, durum): named varieties and differences in composition (glutenins/gliadins)

In a study on durum wheat genotypes, varieties such as Svevo and Saragolla are reported (higher in glutenins and lower in gliadins in the considered set) and Cappelli shows opposite behavior in the reported comparison. (doi.org).

This type of evidence links starting composition (fraction ratios) to a potentially more or less favorable profile for “strong gluten.”

C) Example (Italy, durum): technological quality and W in “old cultivars”

A study evaluates “historical” durum cultivars with technological measurements including W (alveograph) to discuss whether the quality of old cultivars is comparable to modern ones. (PMC)
(The study is useful because it shows that the question “which cultivars have high W” is addressed experimentally on real varietal sets.)

Existing “strong” cultivars and new ones

  • Screening of existing cultivars: HMW-GS genotyping/profiling and measurement of rheology or polymeric proxies. (PMC)

  • Breeding (hybridization/new lines): the same scheme is used as a selection criterion, but it does not originate “only” there. (PMC)

SOFT wheats (bread) — cultivars with HMW-GS profiles associated with high quality

Examples from studies on glutenin alleles and profiles associated with quality traits (mainly for bread) (ResearchGate)

Cultivar/Genotipo

Combinazione HMW-GS Glu-1

Nota sul potenziale di qualità

Fonte

Genotipo con “1, 7+9, 5+10”

1 (Glu-A1), 7+9 (Glu-B1), 5+10 (Glu-D1)

Combinazione associata a migliori qualità di grano (contenuto proteico, WGC ecc.)

Wang et al. (2024) (MDPI)

Genotipo con “1, 7, 5+10”

1, 7, 5+10

Effetti positivi su parametri qualitativi del grano

Wang et al. (2024) (MDPI)

Genotipo con “1, 14+15, 2+12”

1, 14+15, 2+12

Buone correlate a qualità (proteine, WGC)

Wang et al. (2024) (MDPI)

Genotipo con “1, 6+8, 5+10”

1, 6+8, 5+10

Correlazione positiva con qualità

Wang et al. (2024) (MDPI)

GW-273

profilo HMW-GS non esplicitato

Glu-1 score alto (10/10), indicativo di superiori caratteristiche di impasto per pane

Patil (2015) (Tandfonline)

GW-322

profilo HMW-GS non esplicitato

Glu-1 score elevato (10/10)

Patil (2015) (Tandfonline)

What do these data indicate?

  • HMW-GS allele combinations with 5+10 and certain Glu-B1 variants (such as 7+9, 14+15) are frequently associated with better qualitative parameters (e.g., WGC, dough performance) in studies on many soft wheat genotypes.

  • Some genotypes have very high “Glu-1 scores” (≈ 9–10), a phenomenon correlated with higher genetic potential for strong gluten quality. (ResearchGate)

DURUM wheats (pasta / durum bread) — examples and considerations (protein profile and quality)

For durum wheat (Triticum durum), the literature is more variable and often focuses on local collections or genetic variability rather than on specific cultivar names “classified by gluten quality.” However, useful documentation exists on glutenin allelic profiles in durum lines and their relationship with quality traits (including uses other than pasta). (Springer Nature Link)

Creso (historical Italian durum wheat cultivar)
Cultivar obtained in the 1970s through mutagenesis and selection, widely used as a parent in breeding programs to combine yield, adaptation, and technological quality.

Scientific references:

  • De Vita, P., et al. (2007). Genetic improvement effects on yield stability in durum wheat cultivars grown in Italy. Euphytica.

  • De Vita, P., et al. (2010). Effects of genetic improvement on protein content and gluten quality in durum wheat grown in Italy. European Journal of Agronomy.

  • Laidò, G., et al. (2013). Genetic diversity and population structure of durum wheat (Triticum durum) landraces and cultivars using SSR markers. Genetic Resources and Crop Evolution.

Why relevant here:

Creso often appears as a parent or reference in studies analyzing progressive improvement of parameters such as protein content, gluten index, and dough characteristics, showing how breeding has increased technological quality in durum wheat.

Simeto (modern Italian durum wheat cultivar)

Variety selected in Italy and widely cultivated, used as a reference for good semolina quality and balanced technological performance.

Scientific references:

  • De Vita, P., et al. (2007). Euphytica.

  • Troccoli, A., et al. (2000). Variation in grain quality traits among durum wheat cultivars grown in southern Italy. Cereal Chemistry.

  • Ficco, D. B. M., et al. (2014). Genetic variability in quality traits of durum wheat for pasta making. Journal of Cereal Science.

Why relevant here:

Simeto is frequently included in comparisons among modern cultivars, showing good levels of protein, gluten index, and semolina quality, parameters that can also be linked to glutenin composition.

Varietà / linea (durum)

Nota qualitativa / genotipica

Fonte

Varietà marocchine (Henkrar et al.)

Diverse combinazioni alleliche HMW-GS correlate a qualità di trasformazione

Henkrar et al., 2017

Isly, Massa, Anouar, Sboula, Chaoui

Profili variabili di glutenine HMW

Henkrar et al., 2017

Creso

Cultivar storica, genitore chiave nel breeding italiano; miglioramento progressivo di proteine e qualità glutine

De Vita et al., 2007; De Vita et al., 2010

Simeto

Cultivar moderna, buona qualità semola/pasta; riferimento in studi su qualità tecnologica

Troccoli et al., 2000; Ficco et al., 2014

In durum wheats, the association between glutenin allelic profile, protein composition, and technological quality is documented mainly through comparative studies on varietal collections and reference cultivars such as Creso and Simeto. This allows these cultivars to be used as scientific benchmarks, not merely as commercial names.

Important note on gluten quality in durum wheat:

For durum wheat, superior technological quality is not always defined by the same “strength” parameters used for bread (W, alveograph); often the focus is on viscoelasticity, tenacity, extensibility, and ability to form semolina/pasta. Nevertheless, the presence of certain HMW-GS combinations (also in tetraploids) has been documented and correlated with grain quality (total proteins, glutenin content, etc.). (Springer Nature Link)

What these examples tell us

✅ In soft wheats, certain HMW-GS profiles combined with specific subunits (e.g., 5+10 and Glu-B1 variants) are scientifically associated with better gluten quality parameters (and thus higher genetic potential). (MDPI)

✅ Some genotypes (such as GW-273 and GW-322) show very high quality scores, used as reference examples in technical publications. (Tandfonline)
✅ In durum wheats, the literature often includes lists of cultivars/lines with glutenin allelic profiles, useful for breeding and for correlating genetic profiles with quality (even if data are not always reported with standardized “commercial” names). (Springer Nature Link)

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.”