A Rapid, Reliable RP-UPLC Method for Large-Scale Analysis of Wheat HMW-GS Alleles

High-molecular-weight glutenin subunits (HMW-GS) account for only 10% of total wheat storage proteins, but play an important role in the processing quality of wheat flour. Therefore, identifying HMW-GS alleles associated with good end-use quality provides important information for wheat breeders. To rapidly, accurately and reproducibly identify HMW-GS, we established an optimized reversed-phase ultra-performance liquid chromatography (RP-UPLC) method. Separation parameters were optimized using an ACQUITY UPLC Protein BEH C4 column and stepwise ACN gradient, and the separation patterns and retention times (RTs) of 22 subunits were comparatively analyzed in 16 standard wheat cultivars. All HMW-GS proteins were well separated within about 5.5 min, and all analyses were complete within 12 min. We distinguished the 16 subunits based on RT, although three subunits in 1Bx (1Bx7/1Bx7OE and 1Bx17) and three subunits in 1By (1By8*, 1By9 and 1By15) had overlapping RTs; these were differentiated by SDS-PAGE. To distinguish 1Bx7 and 1Bx7OE, which differ in protein abundance, RP-UPLC was combined with PCR analysis of DNA junction markers. The optimized method was successfully applied to determine HMW-GS alleles in a large collection of bread wheat germplasm (1787 lines). This protocol is an appropriate option for selecting lines harboring favorable HMW-GS alleles in wheat breeding.

Reducing Immunoreactivity of Gliadins and Coeliac-Toxic Peptides Using Peptidases from L. acidophilus 5e2 and A. niger

Wheat storage proteins and products of their hydrolysis may cause coeliac sprue in genetically predisposed individuals with high expression of main histocompatibility complex HLA-DQ2 or DQ8, since by consuming wheat, they become exposed to proline- (P) and glutamine (Q)-rich gluten. In bread-making, the hydrolysis of gliadins and coeliac-toxic peptides occurs with varied efficiency depending on the fermentation pH and temperature. Degradation of gliadins catalysed by Lactobacillus acidophilus 5e2 peptidases and a commercial prolyl endopeptidase synthesised by A. niger, carried out at pH 4.0 and 37 °C, reduces the gliadin concentration over 110-fold and decreases the relative immunoreactivity of the hydrolysate to 0.9% of its initial value. Hydrolysis of coeliac-toxic peptides: LGQQQPFPPQQPY (P1) and PQPQLPYPQPQLP (P2) under the same conditions occurs with the highest efficiency, reaching 99.8 ± 0.0% and 97.5 ± 0.1%, respectively. The relative immunoreactivity of peptides P1 and P2 was 0.8 ± 0.0% and 3.2 ± 0.0%, respectively. A mixture of peptidases from L. acidophilus 5e2 and A. niger may be used in wheat sourdough fermentation to reduce the time needed for degradation of proteins and products of their hydrolysis.

Effects of Cold Jet Atmospheric Pressure Plasma on the Structural Characteristics and Immunoreactivity of Celiac-Toxic Peptides and Wheat Storage Proteins

The present research reported the effects of structural properties and immunoreactivity of celiac-toxic peptides and wheat storage proteins modified by cold jet atmospheric pressure (CJAP) plasma. It could generate numerous high-energy excited atoms, photons, electrons, and reactive oxygen and nitrogen species, including O3, H2O2, •OH, NO2− and NO3− etc., to modify two model peptides and wheat storage proteins. The Orbitrap HR-LC-MS/MS was utilized to identify and quantify CJAP plasma-modified model peptide products. Backbone cleavage of QQPFP and PQPQLPY at specific proline and glutamine residues, accompanied by hydroxylation at the aromatic ring of phenylalanine and tyrosine residues, contributed to the reduction and modification of celiac-toxic peptides. Apart from fragmentation, oxidation, and agglomeration states were evaluated, including carbonyl formation and the decline of γ-gliadin. The immunoreactivity of gliadin extract declined over time, demonstrating a significant decrease by 51.95% after 60 min of CJAP plasma treatment in vitro. The CJAP plasma could initiate depolymerization of gluten polymer, thereby reducing the amounts of large-sized polymers. In conclusion, CJAP plasma could be employed as a potential technique in the modification and reduction of celiac-toxic peptides and wheat storage proteins.

DESENVOLVIMENTO E CARACTERIZAÇÃO DE PÃO SEM GLÚTEN À BASE DE FARINHA DE VEGETAIS

<p><em>A Doença Celíaca (DC) é caracterizada por um processo inflamatório no intestino delgado ocasionado pela presença de um composto heterogêneo formado por gliadinas e gluteninas que, basicamente, são proteínas de armazenamento do trigo designadas como glúten. A deficiência em relação à disponibilidade e acesso a produtos isentos de glúten caracteriza uma situação de insegurança alimentar e nutricional do indivíduo celíaco e, devido à dificuldade encontrada por esse público em adquirir produtos básicos que atendam às suas necessidades, o presente trabalho teve como objetivos elaborar farinhas a partir da secagem dos vegetais: beterraba, cenoura e espinafre, desenvolver três formulações de pães sem glúten à base do produto farináceo desses vegetais e realizar a caracterização sensorial - utilizando métodos instrumentais - físico-química e microbiológica. Três formulações foram desenvolvidas e realizaram-se análises físico-químicas de umidade, atividade de água, cinzas, proteínas, fibra alimentar, lipídeos, acidez, pH e carboidratos. Dentre elas, observou-se que o pão de beterraba obteve o maior percentual de umidade e proteína. O pão de espinafre destacou-se pelo alto teor de cinzas, evidenciando seu alto valor nutricional em relação aos minerais. Já o pão de cenoura obteve o menor percentual de lipídeos. As análises instrumentais de cor e textura estão de acordo com o obtido por outros autores que estudaram pães sem glúten. Como critério de segurança, realizou-se análises microbiológicas de coliformes a 45 °C e Salmonella sp. O pão sem glúten à base de farinha de vegetais pode ser uma opção para agregar valor nutricional à alimentação dos pacientes celíacos.</em></p><p><em> </em></p><p><em>ABSTRACT</em></p><p><em>Celiac Disease (DC) is characterized by an inflammatory process in the small intestine caused through the presence of a heterogeneous compound formed by gliadins and glutenins, which are basically wheat storage proteins called gluten. The deficiency in the availability and access to gluten-free products characterizes a situation of food and nutritional insecurity of the celiac individual and, due to the difficulty encountered by this public in acquiring basic products that meet their needs, the present work had as objectives to elaborate flours from the drying of the vegetables: beet, carrot and spinach flour, to develop three gluten-free bread formulations based on the farinaceous product of these vegetables and to perform the sensorial characterization - using instrumental methods - physical-chemical and microbiological. Three bread formulations were developed, and physical-chemical analyzes of moisture, water activity, ashes, proteins, dietary fiber, lipids, acidity, pH and carbohydrates were carried out. Among them, it was observed that the beet bread obtained the highest percentage of moisture and protein. The spinach bread was distinguished due the fact of the high ash content, evidencing its high nutritional value in relation to the minerals. Carrot bread, on the other hand, obtained the lowest percentage of lipids, that is, with less fat content. The instrumental analyzes of color and texture are in agreement with the one obtained by other authors who studied gluten-free breads. As a safety criterion, microbiological analyzes of coliforms at 45 °C and Salmonella sp. Gluten-free bread based on vegetable meal may be an option to add nutritional value to the diet of celiac patients.</em></p>

Molecular characterization and phylogenetic analysis of one Omega-gliadin gene from Aegilops speltoidesl

Gliadins, as the major components of wheat storage proteins, determine the extensibility properties of dough and have important effects on flour processing quality. Wheat related species carries potential storage protein gene resources for quality improvement. In this study, we isolated and characterized the first complete ?-gliadin gene Omega-AS from Aegilops speltoides L. (2n = 2x = 14, SS) by allelic-specific PCR and investigated its phylogenetic relationships among Triticum and Aegilopsspecies. Molecular structure showed that Omega-AS gene consisted of 1122 bp encoding 373 amino acid residues with deduced molecular mass 41379.21 Da. Omega-AS gene was exceptionally rich in prolines and glutamines with fewer methionine and no cysteine. Sequence characterization and epitope analysis showed that three epitopes QQPIPVQPQQ, TQPQQPTPIQ and IQPQQPFPQQ were absent in Omega-AS gene encoded protein, indicating its potential value for wheat quality improvement with less toxic, or no toxic peptides. Phylogenetic analysis revealed that Omega-AS was closely related to gliadin genes of wheat and related species and its divergence from bread wheat was more recently (less than 1.243 MYA). Heterologous expression showed that Omega-AS gene could successfully express with a high level in E. coli under the control of T7promoter. The transcription expression pattern of Omega-AS gene during grain development detected by qRT-PCR revealed that the highest expression level occurred at 17 days post an thesis.