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

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Food Chemistry 448 (2024) 139148

Available online 26 March 2024

(http://creativecommons.org/licenses/by/4.0/).

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

M´

elanie Lavoignat

, Ang´

ela Juh´

asz

, Utpal Bose

, Thierry Sayd

, Christophe Chambon

Miguel Ribeiro

, Gilberto Igrejas

, S´

ebastien D´

ejean

, Catherine Ravel

Emmanuelle Bancel

Universit´

e Clermont Auvergne, INRAE, UMR1095 GDEC, F-63000 Clermont-Ferrand, France

Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, Edith Cowan University, School of Science, 270 Joondalup Dr,

Joondalup, WA 6027, Australia

CSIRO Agriculture and Food, 306 Carmody Rd, St Lucia, QLD 4067, Australia

INRAE, Plateforme d’Exploration du M´

etabolisme Composante Prot´

eomique (PFEMcp), F-63122 Saint-Gen`

es Champanelle, France

Chemistry Research Centre-Vila Real (CQ-VR), University of Tr´

as-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal

Department of Genetics and Biotechnology, School of Life Sciences and Environment, University of Tr´

as-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal

Functional Genomics and Proteomics Unit, University of Tr´

as-os-Montes and Alto Douro (UTAD), Vila Real, Portugal

Associated Laboratory for Green Chemistry (LAQV-REQUIMTE), University NOVA of Lisboa, 1099-085 Lisboa, Portugal

Institut de Math´

ematiques de Toulouse, UMR5219, Universit´

e de Toulouse, CNRS, UPS, F-31062 Toulouse Cedex 9, France

ARTICLE INFO

Keywords:

Triticum aestivum

Wheat

Immunogenic epitopes

In vitro digestion

Mass spectrometry

ABSTRACT

Wheat proteins can trigger immunogenic reactions due to their resistance to digestion and immunostimulatory

epitopes. Here, we investigated the peptidomic map of partially digested bread samples and the ngerprint of

epitope diversity from 16 wheat genotypes grown in two environmental conditions. Flour protein content and

composition were characterized; gastric and jejunal peptides were quantied using LC-MS/MS, and genotypes

were classied into high or low bread protein digestibility. Differences in our protein content and peptide

composition distinguish high from low digestibility genotypes in both growing environments. No common

peptide signature was found between high- and low-digestible genotypes; however, the celiac or allergen epi-

topes were noted not to be higher in low-digestible genotypes. Overall, this study established a peptidomic and

epitope diversity map of digested wheat bread and provided new insights and correlations between weather

conditions, genotypes, digestibility and wheat sensitivities such as celiac disease and wheat allergy.

1. Introduction

Wheat protein content and composition are critical determinants of

grain techno-functional properties and health quality indicators.

Roughly, wheat grain proteins consist of about 20% of albumins-

globulins and 80% of storage proteins (or gluten proteins) (Shewry,

2019).

Techno-functional properties of the wheat grain primarily rely on the

storage proteins, consisting of about 50–60% of gliadins and 40–50% of

glutenins (Johansson et al., 2013). Gliadins are alcohol-soluble mono-

meric proteins classied into

/β-, γ- and

-gliadins. Glutenins, classi-

ed into low molecular weight (LMW) and high molecular weight

(HMW) subunits are polymeric proteins that form intra- and intermo-

lecular disulphide bonds and hydrophobic interactions to stabilize their

structure. After our hydration and kneading, glutenins and gliadins

form the polymeric gluten network responsible for the dough’s rheo-

logical properties (Johansson et al., 2013).

Wheat grain proteins can trigger IgE-mediated wheat allergy (WA)

and celiac disease (CD), an autoimmune disorder induced by gluten

proteins to genetically susceptible individuals with the human leukocyte

antigens HLA-DQ2 and/or HLA-DQ8 haplotypes (Brouns, van Rooy,

Shewry, Rustgi, & Jonkers, 2019). Gluten proteins are characterized by

unique repetitive domains that account for 30% to >85% of the protein

length. These domains consist of repetitions of peptide sequences that

* Corresponding author.

E-mail address: catherine.ravel@inrae.fr (C. Ravel).

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier.com/locate/foodchem

https://doi.org/10.1016/j.foodchem.2024.139148

Received 13 November 2023; Received in revised form 26 February 2024; Accepted 24 March 2024

Food Chemistry 448 (2024) 139148

are three to nine amino acids long and rich in proline and glutamine

(Shewry, 2019). The high repetition of glutamine and proline content in

gluten proteins makes them partially resistant to proteolytic cleavage by

human digestive enzymes. The resulting undigested gluten epitopes can

cross the small intestinal barrier and trigger immune reactions. The

deamidation of these peptides by transglutaminase 2 (TG2) in the gut

can enhance the binding afnity of these peptides to the antigen-

presenting heterodimers HLA-DQ2 or HLA-DQ8 and activate the

CD4+T cells (Brouns et al., 2019). This activation leads to the pro-

duction of anti-gluten and anti-TG2 antibodies and cytokines such as IL-

15 to damage the intestinal epithelial cells, leading to celiac enteropa-

thy. In addition, plant defence-related proteins such as alpha-amylase

trypsin inhibitors (ATIs), wheat germ-agglutinins, and serpins present

in wheat can also trigger immune responses in some people (Aziz,

Hadjivassiliou, & Sanders, 2015; Zevallos et al., 2017). Non-celiac wheat

sensitivity (NCWS) is increasingly reported by patients with intestinal

and extra-intestinal responses. Patients diagnosed with NCWS test

negative for CD and WA, but the key cause for this disease symptoms is

currently unknown (Brouns et al., 2019), and the role of gluten proteins

in NCWS pathology remains elusive. Although wheat consumption can

trigger adverse immune reactions in a small part of the population, most

of the human population still depend on wheat as their primary diet. As

such, it is important to understand the immunostimulatory peptide

contents precisely so that the majority of people can meet their regular

dietary requirements.

Studies have reported quantitative and qualitative differences in

epitope content and composition between the genotypes of different

Triticum species (Ribeiro et al., 2016) and T. aestivum varieties (Denery-

Papini et al., 2007; Prandi, Tedeschi, Folloni, Galaverna, & Sforza, 2017;

Ronga et al., 2020; Schalk, Lang, Wieser, Koehler, & Scherf, 2017). The

environmental variability of epitope-containing proteins has been

investigated (Juh´

asz et al., 2018; Juh´

asz, Haraszi, & B´

ek´

es, 2020;

Landol et al., 2021; Ronga et al., 2020) and more broadly, the inuence

of environment on protein composition and content (and thus impacting

epitopes) has been extensively studied (Johansson et al., 2020). In

addition, several studies have shown the immunostimulatory potential

of wheat products due to the presence of IgE-binding WA epitopes and

immunogenic celiac epitopes (Denery-Papini et al., 2007; Schalk et al.,

2017). In addition to the availability of >16 high-resolution bread wheat

genome sequence resources, advancements in mass-spectrometry (MS)-

based proteomics and bioinformatics workows have allowed us to

understand better the diversity of immunostimulatory peptides involved

in wheat-related disorders. The MS-based proteomics approach can

precisely identify and quantify immunogenic epitopes from complex

our samples and processed food products (Ribeiro et al., 2021). The

identication and quantitation of epitopes in the experimental samples

depend on the precise characterization of sub-classes of proteins upon

digestion (Landol et al., 2021; Ogilvie et al., 2020). For instance,

-gliadins generate the most diverse CD-related epitopes (Landol et al.,

2021), and these peptides were reported among the most immunodo-

minant (Juh´

asz et al., 2018; Tye-Din et al., 2010). However, the γ-gli-

adins have been shown to generate the largest number of CD epitopes

during digestion (Ogilvie et al., 2020). Two studies monitored the

release of immunogenic peptides during bread (Ogilvie et al., 2020) or

pasta (Mamone et al., 2015) digestion and showed that a few immu-

nogenic peptides survived the in vitro digestion, i.e., they resisted to

proteolysis.

The duration and intensity of protein hydrolysis within the gastro-

intestinal tract can increase the generation of shorter peptides, resulting

in a lower immunostimulatory effect in the gut. Protein digestibility can

be inuenced by several factors, including human gastro-intestinal

conditions (Torcello-G´

omez et al., 2020), the complexity of the food

matrix (Freitas, G´

omez-Mascaraque, & Brodkorb, 2022), composition

(Wu, Taylor, Nebl, Ng, & Bennett, 2017) and the food processing steps

such as baking. Heat treatments applied during baking or cooking can

alter the protein structure by unfolding the proteins through subsequent

re-arrangements of disulde bonds (Ogilvie et al., 2021; Pasini, Simo-

nato, Giannattasio, Peruffo, & Curioni, 2001). Contradictory results

have been published on the effect of baking on the protein digestibility

of wheat. According to Bredariol, Carvalho, and Vanin (2020), there are

optimum time and temperature baking parameters that would improve

proteolysis during digestion. In addition, the impact of protein di-

gestibility varied considerably for the bread crumb and crust; the crumb

portion showed similar digestibility to the uncooked dough, while the

bread crust had lower digestibility (Pasini et al., 2001). The thermal

treatment also inuences the recognition of WA epitopes by the IgE

receptors (Lupi et al., 2019). This study reported that the thermal

treatment initially reduces gliadin recognition by IgE due to the pro-

duction of large aggregates. However, upon hydrolysis under acidic

stomach conditions, the epitopes can be unmasked and recognized by

the T cells in the intestine. From our to processed and baked products,

epitopes can undergo modications that would alter their immunosti-

mulatory potential (Liu et al., 2023). Thus, to study the diversity of

peptides released from wheat-based food during human digestion, the

choices of the food matrix as well as the digestion model are essential. In

addition to wheat our’s baking or cooking process, the protein di-

gestibility varies across wheat genotypes. Notably, the protein compo-

sitional variability in the grain our and, thus, breads between

genotypes have shown a different spectrum of digestibility (Lavoignat

et al., 2022). Although many studies have reported the impact of the

baking process and genotype-dependent variations that inuence the

digestibility and release of immunogenic epitopes, the combination of

genotype and environmental variations and their food processing on the

human food digestibility assessment remains unknown.

The present study aims to investigate how wheat genotype-

dependent variability can impact the bread protein digestibility and

epitope release during the human digestion process. We hypothesized

that peptides with a specic signature could discriminate between high-

and low-digestible genotypes. First, we performed the in vitro digestion

assay on the bread samples and collected and measured the peptides

generated during the gastric and intestinal digestion process. The

measured peptide abundances were associated with the our composi-

tion and protein content to separate the genotype with high digestibility

(HD) and low digestibility (LD). Moreover, we detected and compre-

hensively mapped the epitopes identied during the digestion process to

the different sub-types of gliadins, glutenins and albumins-globulins

using an in-silico approach. Together, this study establishes the pepti-

domic map of partially digested bread samples and the ngerprint of

epitope diversity from 16 wheat genotypes grown at two different

environmental conditions.

2. Materials and methods

The plant material and wholemeal our phenotyping were previ-

ously described in (Lavoignat et al., 2023).

2.1. Meteorological data

In total, 16 bread wheat genotypes were grown in the eld in two

different locations: Clermont-Ferrand (CF) and Estr´

ees-Mons (EM) in

duplicates. To assess the weather pattern for CF and EM, the maximal

(Tmax), minimal (Tmin) and mean (Tmean) temperatures (◦C) and cu-

mulative rainfall (CumR, mm) of the 2016–2017 growing season and the

last 30 years (Tmax30, Tmin30, Tmean30, CumR30) were retrieved

(https://agroclim.inrae.fr/). The cumulative growing degree days

(CumDD_MJJ) and the cumulative rainfall (CumR_MJJ) over the grain

lling period, i.e., May to July, were calculated for two locations based

on the heading date of genotype.

2.2. R5 competitive ELISA assay on wholemeal our

For the 64 grains samples (16 genotypes ×2 locations ×2

M. Lavoignat et al.

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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

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