This work was aimed at showing the ability of sourdough lactobacilli to hydrolyze wheat prolamins extensively and at finding a novel protocol for manufacturing a sourdough bread that can be tolerated by CS patients.
In a previous paper (18
), we showed that selected lactobacilli have the ability to hydrolyze either albumins, globulins, and gliadins during wheat sourdough fermentation or the 31-43 fragment of A-gliadin in vitro and that, after hydrolysis, they greatly reduced the agglutination of the K 562(S) subclone cells of human myelogenous leukemia origin by a toxic PT digest of gliadins. In this study, we first showed that the pool of L. alimentarius
15M, L. brevis
14G, L. sanfranciscensis
7A, and L. hilgardii
51B has a pattern of specialized peptidases capable of hydrolyzing all of the different peptide bonds that potentially include the imino acid proline. Overall, biologically active peptides contain a large proportion of Pro residues within the sequence, which makes them very resistant to hydrolysis by peptidases, which are not specific for Pro bonds (17
). The hydrolysis by the four lactobacilli also concerned oligopeptides such as fragment 62-75 of A-gliadin and the 33-mer peptide. Peptide 62-75 is part of a longer fragment, 56-75, of A-gliadin that reacts with tissue transglutaminase and is one of the dominant epitopes responsible for the inappropriate T-cell-mediated immune response in CS patients (1
). These hydrolyzing activities are not widespread in dairy lactic acid bacteria. Generally, pure prolyl-endopeptidases have very poor activity on long-chain peptides (30
). In our experiments, we used a pool of selected lactobacilli that was supplemented with a pool of their cytoplasmic enzymes.
Notwithstanding the heterogeneity of T-cell epitopes in gluten, a few epitopes appear to account for most of the α-gliadin recognition by CD4+
T cells from CS patients (1
). The most important is probably the 33-mer peptide, for the following reasons: (i) it remains intact despite prolonged exposure to gastric and pancreatic proteases, (ii) other patient-specific T-cell epitopes are present in its sequence, (iii) hydrolysis of the 33-mer peptide (100 μM) by small intestinal brush border membrane enzymes is less than 20% over 20 h of incubation (40
), and (iv) it remains intact for a long time (ca. 24 h) in the small intestine and even at a low concentration is able to act as a potential antigen for T-cell proliferation and intestinal toxicity in genetically susceptible individuals (40
). Although peptidases capable of hydrolyzing Pro- and Glu-rich peptides are located in the intestinal brush border (2
), these epitopes withstand enzymatic processing (27
). To our knowledge, the only enzyme proposed as a detoxifying agent for the 33-mer peptide is the prolyl-endopeptidase from F. meningosepticum
), which is not related to bread biotechnology. In this study, we first showed that sourdough lactobacilli have the ability to hydrolyze the 33-mer peptide extensively or almost totally during prolonged incubation (12 to 24 h).
Previously, a wheat sourdough was produced with the same Lactobacillus species; considerable, but not total, hydrolysis of gliadins was found (18
). In this study, the amount of wheat flour was decreased to 30% by mixing with nontoxic (26
) oat, millet, and buckwheat flours (ratio, 3:1:4:2). These flours are nutritionally and technologically suitable also (33
). Under these conditions, we achieved almost complete hydrolysis of wheat gliadins while prolamins from oats, millet, and buckwheat were affected less or not at all. A comparison with a chemically acidified dough or with a dough started with baker's yeast alone showed that the hydrolysis was due to the proteolytic activity of sourdough lactobacilli and that prolamin fractions were not affected during dough fermentation with yeast. The great extent of hydrolysis during sourdough fermentation was confirmed on various-size, alcohol-soluble polypeptides that were analyzed by RP-FPLC and by determination of free amino acids. Addition of CE to the sourdough started with selected lactobacilli for 12 h markedly increased the concentration of free amino acids, showing a considerable activity of CE toward low- to medium-molecular-mass peptides.
Prior to initiating the in vivo acute challenge, we wanted to confirm our results based on agglutination tests. Overall, a relatively high correlation was found between the agglutination activity of cereal components against K 562(S) cells and their toxicities in clinical and in vitro trials on the basis of biopsy samples of intestinal mucosa from CS patients (6
). The MAC of the sourdough started with selected lactobacilli was ca. 250 times higher than those of the control and of the dough started with baker's yeast. It was also confirmed that CE plays a role in the further degradation of intermediate polypeptides from gliadins, which probably still have a toxic effect.
For the in vivo challenge, a bread produced with a very long fermentation time was compared to a bread fermented for 2 h with baker's yeast. A very long fermentation time is a common feature of an ancient tradition for the production of typical wheat sourdough breads (23
). After this fermentation, the structure of the dough is obviously collapsed and sourdough is traditionally reused as a starter for a new and very short (2- to 4-h) fermentation process. Under our experimental conditions, the fermented (24 h) wheat sourdough was subsequently mixed with nontoxic flours in the optimal ratio, allowed to ferment for 2 h, and baked at 220°C for 20 min. This type of bread was technologically suitable: the volume was ca. one-half of that started with baker's yeast, and the texture was comparable to that of wheat sourdough breads. To our knowledge, this is the first report of tolerance of CS patients for a bread containing 30% wheat flour on the basis of determination of intestinal permeability during an acute in vivo challenge. Thirteen of the 17 CS patients recruited showed a marked alteration of intestinal permeability after ingestion of baker's yeast bread, while when fed the sourdough bread, the same 13 patients had intestinal permeability values that did not differ significantly from the baseline values. The other four CS patients did not respond to the two types of bread.
The following multidisciplinary research efforts are currently being carried out in several directions to deal with the pathogenesis of CS: (i) engineering of gluten-free grains, (ii) search for the CS genes in humans (20
), (iii) use of some protective substances (e.g., mannan and oligomers of N
), and (iv) use of bacterial prolyl-endopeptidase from F. meningosepticum
as an oral supplementary therapy (40
). None of these efforts considered strategies that are pertinent in bread biotechnology. This study shows that CS patients subjected to an acute challenge tolerated breads produced with sourdough better than those started with baker's yeast. These results showed that a bread biotechnology that uses selected lactobacilli, nontoxic flours, and a long fermentation time is a novel tool for decreasing the level of human intolerance to a certain amount of wheat flour. Work is in progress to confirm these results with a long-term in vivo challenge.