Dana Urminská

Immunochemical evaluation of proteolysis of cereal proteins causing celiac disease by microbial proteases

ABSTRACT Celiac disease is one of the most prevalent food intolerances worldwide, with a gluten-free diet as the only effective therapy. Therefore, searching for new alternative approaches is a priority, and one of them is the idea of using proteinases for degradation of gluten proteins. The aim of the study was a trial of enzymatic cleavage of prolamins extracted from wheat, barley, rye, triticale and oat. The proteolysis was performed by microbial proteases from Bacillus stearothermophilus, Bacillus licheniformis, Bacillus thermoproteolyticus and Streptomyces griseus. The reaction was performed up to 60 min, stopped by addition of appropriate synthetic inhibitor and products of limited proteolysis were analyzed by SDS-PAGE. Most of the prolamins were susceptible to proteolysis by the examined microbial enzymes and as the result of hydrolysis the molecules of lower molecular weight and peptides occurred (<30,000 Da). The immunoreactivity of proteolysis products was evaluated using the reaction with polyclonal and monoclonal antibodies by Western blot and ELISA analysis, respectively. Immunochemical analyses confirmed that celiac-active sequences of amino acids in prolamins were at least partially destroyed and have lost their “toxicity”, so application of microbial proteases seems to be an alternative method for decreasing of celiac activity of prolamins.

APPLICATION OF WESTERN BLOT ANALYSIS FOR DETECTION OF PROLAMIN PROTEINS IN CEREAL GRAINS AND BREAD

Celiac disease is an inflammatory condition of the small intestine in genetically susceptible individuals caused by ingestion of wheat gluten and corresponding proteins from barley and rye. Cereal storage proteins (prolamins) are responsible for immunological response of patients with celiac disease. Prolamins are alcohol soluble fractions, namely gliadins (wheat), hordeins (barley) and secalins (rye). The main triggering factor is wheat fraction with low molecular weight (20-30 kDa) called α-gliadins. Immunochemical detection of celiac active proteins is based on reactivity of gluten-detecting antibodies with prolamins extracted from cereals. In our study, we used Western blot analysis for detection of prolamin complex in cereal grains and processed foods (breads). Western blot was carried out by polyclonal antibody raised against wheat gluten. Reaction was positive for all kind of cereal grains. The samples of wheat and spelt wheat show much more positive affinity to antibody than rye and oat. As well as for cereal grains, all samples of bread showed positive immunological reaction with used antibody. Western blot analysis with gluten polyclonal antibody is suitable method for qualitative detection of prolamin complex in cereal grains and processed foods.