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Residual wheat peptides after complete in vitro digestion: type, amount, immunogenicity (and why wheat diversity matters)

by luciano

(in-depth focus 5 of Genetic potential and processing conditions in determining gluten strength, digestibility, and immunogenicity)

Simulated gastrointestinal digestion and gluten residues
The studies reported below show that, after simulated gastrointestinal digestion, there is not a single “gluten residue,” but rather a peptide profile (“fingerprint”) that varies as a function of:

1 – species/genotype (wheat diversity),
2 – food matrix (flour/bread, etc.),
3 – processing (fermentation, leavening, baking),
4 – digestion conditions (protocol and kinetics),
5 – and type/abundance of epitopes (celiac disease / allergy).
This truly allows one to “build a picture” of how wheat diversity influences digestion and immuno-relevant potential.

Key studies (with concrete results)

Practical note
The 2020 Ogilvie study is often used as a “tool” to put numbers (quantities) on peptide markers, whereas Lavoignat 2024 and Boukid 2019 are more peptidomic “atlases” (quality/type + epitopes).
Di Stasio 2020 and Gianfrani 2015 are excellent for the “wheat diversity → different digestibility/immunogenicity” aspect.

Methodological framework (what “complete digestion” means in a standard way)
Many works use or are inspired by the INFOGEST protocol (international standard), which makes results comparable across studies:

1 – A standardised static in vitro digestion method suitable for food — an international consensus (Minekus M. et al., 2014, Food & Function) — DOI: 10.1039/C3FO60702J
2 – INFOGEST static in vitro simulation of gastrointestinal food digestion (Brodkorb A. et al., 2019, Nature Protocols) — DOI: 10.1038/s41596-018-0119-1
Concluding message to include
The response to gluten exposure does not depend on a single factor (e.g., “gluten strength” or “ancient vs modern wheat”), but on the combination of genotype/species, matrix and technological process, and above all on the final profile of residual peptides after digestion: which peptides (type), how many (abundance/markers), and how immuno-relevant they are (epitopes).

Peptidomic studies and targeted quantification studies show that both composition and release patterns of peptides change as a function of wheat type and processing.

Immunogenicity and resistance to digestion in gluten (and why they do not always coincide)

by luciano

(In-depth article 6 of: Genetic potential and processing conditions in determining gluten strength, digestibility, and immunogenicity)

In gluten (especially gliadins and, partly, glutenins) there is a strong overlap between:

  • resistance to gastrointestinal digestion

  • immunogenic potential (especially in celiac disease)

but the two concepts are not equivalent: resistance is often a facilitating condition, whereas immunogenicity also requires specific rules of immunological recognition.

1) Why many immunogenic sequences are also resistant

The most “problematic” regions of gluten are rich in proline (P) and glutamine (Q). This profile:

  • hampers cleavage by the main human proteases (pepsin, trypsin, chymotrypsin), which have low ability to cut near proline;

  • favors the persistence of long oligopeptides (10–30+ aa) in the intestinal lumen.

This point is well described in reviews and experimental studies on gluten digestion and on the persistence of peptides such as the 33-mer. (Cambridge University Press & Assessment)

2) Why resistance increases the probability of “remaining immunogenic” after digestion

A peptide that resists digestion:

  • remains long enough to contain complete epitopes (or multiple overlapping epitopes);

  • can generate, through partial cleavage, sub-fragments that still retain recognizable sequences.

In other words: it is not just “surviving” digestion, but surviving while maintaining sequence motifs compatible with immune presentation.

Peptidomic/in vitro digestion studies on wheat products show that the residual peptide profile often includes regions known for epitope density and resistance. (ScienceDirect)

3) What makes a peptide truly immunogenic (beyond resistance)

To trigger a T-cell response in celiac disease, a peptide must:

  1. be presentable by HLA-DQ2/DQ8 (sequence constraints and “anchor” residues);

  2. often become more affine through deamidation by tissue transglutaminase (TG2) (conversion of Q→E in specific contexts);

  3. be recognized by specific T cells.

Therefore, it is possible to have highly resistant peptides that nevertheless:

  • do not bind HLA-DQ2/DQ8 efficiently,

  • are not good substrates for TG2, and/or

  • do not correspond to known T-cell epitopes.

A classic reference on HLA-DQ2 presentation of gluten peptides is available on PNAS. (pnas.org)

4) Concrete example: resistant but non-immunogenic peptide

A very useful example (although engineered) is described by Bethune et al.: the authors created analogs of the 33-mer in which some key glutamines are substituted (e.g., NNN-33-mer and HHH-33-mer). These analogs:

  • remain resistant to simulated digestion (pepsin and also duodenal digestion with pancreatic/brush border proteases),

  • but are not appreciably recognized by TG2, HLA-DQ2, or celiac-specific T cells.

This experimentally demonstrates that resistance to digestion ≠ immunogenicity, even when length and “proline-richness” remain similar. (PMC)

Note: this is a “clean” example because it preserves the resistance feature while breaking (through targeted modifications) the immunological recognition requirements.

5) Summary

Immunogenic gluten sequences tend to be overrepresented among digestion-resistant fragments because resistance allows the persistence of sufficiently long, epitope-rich peptides; however, immunogenicity also requires compatibility with HLA-DQ2/DQ8 presentation and often TG2-mediated modification (deamidation).

Further discussion

So far, the genetic and technological variability of the entire pool of digestion-resistant fragments has not been explored in a systematic and in-depth manner, because most studies focus on known immunogenic peptides rather than on the complete repertoire of proteolysis-resistant fragments in relation to genotype/process. (Frontiers)

Read more

Key evidence-supported points:

1. Peptidomic studies show richness of resistant peptides, but rarely investigate non-immunogenic ones

Analyses based on simulated digestion and mass spectrometry (LC-MS/MS) reveal hundreds or thousands of peptides after gluten digestion. Only a minority of these coincide with known immunogenic epitopes; most resistant peptides identified in digests are not directly associated with immunogenicity in published studies. (Frontiers)

2. The prevailing interpretation is still “epitope-focused”

Recent literature summarizes the state of the art of methodologies to assess potential immunogenicity (digestion + peptide profiling); however, these reviews also underline that analytical techniques tend to isolate and quantify immunogenic epitopes rather than delineate a complete catalog of persistent, non-immunogenic peptides. (Frontiers)

3. Genotypic variability has been analyzed, but with focus on immunogenic epitopes

Studies on different wheat genotypes show that:

  • digestion and peptide-release profiles vary with genotype,

  • some genotypes show differences in the amount of immunogenic epitopes released,

  • but the pool of resistant non-immunogenic peptides is rarely systematically characterized. (ScienceDirect)

This means that, even though very large peptidomic datasets exist, studies have so far not exploited the “non-immunogenic” component—i.e., digestion-resistant residues lacking immune-presentation motifs—as an object of genotypic and technological comparison aimed at reducing overall biological impact.

4. Research concentrates on clinically relevant immunogenicity

Much of the literature (and analytical strategies) focuses on identification or quantification of so-called Gluten Immunogenic Peptides (GIP), which are fragments detectable in digests and biological matrices that correlate with immune responses in celiac patients and also serve as diagnostic/monitoring markers. (ResearchGate)

This directs attention toward what activates the immune system rather than toward the full profile of non-activating fragments.

Summary

✔ Digestion-resistant but non-immunogenic peptides exist in in vitro digests
✔ There are studies that observe them indirectly (as part of the total peptidome)
❌ There is not yet a systematic body of research that:

  • exhaustively maps resistant non-immunogenic peptides,

  • compares this variability among genotypes,

  • explores how different processes (fermentation, enzymes, baking) quantitatively influence the overall pool of resistant peptides.

In other words: research has the tools (in vitro digestion + LC-MS/MS) to do this, and some preliminary data indicate genotypic variability in digestion profiles, but a comprehensive evaluation of the biological weight of resistant non-immunogenic peptides in relation to genotype/technology has not yet been completed. (ScienceDirect)

Useful references

Boukid, F. et al. (2019) – A Complete Mass Spectrometry (MS)-Based Peptidomic Description of Gluten Peptides Generated During In Vitro Gastrointestinal Digestion of Durum Wheat. J. Am. Soc. Mass Spectrom. DOI:10.1007/s13361-019-02212-8 — describes the complete peptidome after digestion of durum wheat, highlighting many resistant sequences without focusing only on immunogenic epitopes. (Springer Nature)

Lavoignat, M. et al. (2024) – Peptidomics analysis of in vitro digested wheat breads: Effect of genotype and environment on protein digestibility and release of celiac disease and wheat allergy related epitopes — lays the groundwork for studying genotypic variability in production of resistant peptides and epitopes, but does not yet provide an exhaustive classification of non-immunogenic ones. (ScienceDirect)

Mamone, G. et al. (2023) – Analytical and functional approaches to assess the immunogenicity potential of gluten proteins. Front. Nutr. — methodological review reflecting the current epitope-oriented approach. (Frontiers)

Concise conclusion

Robust peptidomic data show the abundance of proteolysis-resistant fragments in digested gluten; however, the literature has so far prioritized identification and quantification of immunogenic peptides only, leaving largely unexplored the genetic and technological variability in the overall production of resistant non-immunogenic residues and their possible biological role. (Frontiers)

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)