Long-Term Fungal Inhibition by Pisum sativum Flour Hydrolysate during Storage of Wheat Flour Bread

In order to identify antifungal compounds from natural sources to be used as ingredients in the bakery industry, water/salt-soluble extracts (WSE) from different legume flour hydrolysates obtained by the use of a fungal protease were assayed against Penicillium roqueforti DPPMAF1. The agar diffusion assays allowed the selection of the pea (Pisum sativum) hydrolysate as the most active. As shown by the hyphal radial growth rate, the WSE had inhibitory activity towards several fungi isolated from bakeries. The MIC of the WSE was 9.0 mg/ml. Fungal inhibition was slightly affected by heating and variations in pH. The antifungal activity was attributed to three native proteins (pea defensins 1 and 2 and a nonspecific lipid transfer protein [nsLTP]) and a mixture of peptides released during hydrolysis. The three proteins have been reported previously as components of the defense system of the plant. Five peptides were purified from WSE and were identified as sequences encrypted in leginsulin A, vicilin, provicilin, and the nsLTP. To confirm antifungal activity, the peptides were chemically synthesized and tested. Freeze-dried WSE were used as ingredients in leavened baked goods. In particular, breads made by the addition of 1.6% (wt/wt) of the extract and fermented by baker''s yeast or sourdough were characterized for their main chemical, structural, and sensory features, packed in polyethylene bags, stored at room temperature, and compared to controls prepared without pea hydrolysate. Artificially inoculated slices of a bread containing the WSE did not show contamination by fungi until at least 21 days of storage and behaved like the bread prepared with calcium propionate (0.3%, wt/wt).     

Application of novel starter cultures for sourdough bread production

Sourdough application has been extensively increased in the last years due to the consumers demand for food consumption without the addition of chemical preservatives. Several starter cultures have been applied in sourdough bread making targeting the increase of bread self-life and the improvement of sensorial character. More specific, Lactobacillus acidophilus and Lactobacillus sakei as single and mixed cultures were used for sourdough bread making. Various sourdough breads were produced with the addition of sourdough perviously prepared with 10% w/w L. acidophilus, 10% w/w L. sakei and 5% w/w L. acidophilus and 5% w/w L. sakei at the same time. Various chemical parameters were determined such as lactic acid, total titratable acidity and pH. The results revealed that the produced sourdough bread made with sourdough containing the mixed culture was preserved for more days (12 days) than all the other breads produced in the frame of this study, since it contained lactic acid in higher concentrations. The respective total titratable acidity varied between 10.5 and 11 ml NaOH N/10. The same sourdough bread had a firmer texture, better aroma, flavor and overall quality compared to other sourdough breads examined in this study, as shown by sensory evaluation tests and results obtained through SPME GC–MS analysis, which revealed significant differences among the different bread types.     

Antifungal activity of Wickerhamomyces anomalus and Lactobacillus plantarum during sourdough fermentation: identification of novel compounds and long-term effect during storage of wheat bread

This study aimed at investigating the antifungal activity of Wickerhamomyces anomalus and sourdough lactic acid bacteria to extend the shelf life of wheat flour bread. The antifungal activity was assayed by agar diffusion, growth rate inhibition, and conidial germination assays, using Penicillium roqueforti DPPMAF1 as the indicator fungus. Sourdough fermented by Lactobacillus plantarum 1A7 (S1A7) and dough fermented by W. anomalus LCF1695 (D1695) were selected and characterized. The water/salt-soluble extract of S1A7 was partially purified, and several novel antifungal peptides, encrypted into sequences of Oryza sativa proteins, were identified. The water/salt-soluble extract of D1695 contained ethanol and, especially, ethyl acetate as inhibitory compounds. As shown by growth inhibition assays, both water/salt-soluble extracts had a large inhibitory spectrum, with some differences, toward the most common fungi isolated from bakeries. Bread making at a pilot plant was carried out with S1A7, D1695, or a sourdough started with a combination of both strains (S1A7-1695). Slices of the bread manufactured with S1A7-1695 did not show contamination by fungi until 28 days of storage in polyethylene bags at room temperature, a level of protection comparable to that afforded by 0.3% (wt/wt) calcium propionate. The effect of sourdough fermentation with W. anomalus LCF1695 was also assessed based on rheology and sensory properties.     

Antifungal activity of phenyllactic acid against molds isolated from bakery products

Phenyllactic acid (PLA) has recently been found in cultures of Lactobacillus plantarum that show antifungal activity in sourdough breads. The fungicidal activity of PLA and growth inhibition by PLA were evaluated by using a microdilution test and 23 fungal strains belonging to 14 species of Aspergillus, Penicillium, and Fusarium that were isolated from bakery products, flours, or cereals. Less than 7.5 mg of PLA ml(-1) was required to obtain 90% growth inhibition for all strains, while fungicidal activity against 19 strains was shown by PLA at levels of < or = 10 mg ml(-1). Levels of growth inhibition of 50 to 92.4% were observed for all fungal strains after incubation for 3 days in the presence of 7.5 mg of PLA ml(-1) in buffered medium at pH 4, which is a condition more similar to those in real food systems. Under these experimental conditions PLA caused an unpredictable delaying effect that was more than 2 days long for 12 strains, including some mycotoxigenic strains of Penicillium verrucosum and Penicillium citrinum and a strain of Penicillium roqueforti (the most widespread contaminant of bakery products); a growth delay of about 2 days was observed for seven other strains. The effect of pH on the inhibitory activity of PLA and the combined effects of the major organic acids produced by lactic acid bacteria isolated from sourdough bread (PLA, lactic acid, and acetic acid) were also investigated. The ability of PLA to act as a fungicide and delay the growth of a variety of fungal contaminants provides new perspectives for possibly using this natural antimicrobial compound to control fungal contaminants and extend the shelf lives of foods and/or feedstuffs.     

Detection of rope spoilage in bread caused by Bacillus species

Rope spoilage of bread by eight Bacillus isolates obtained from a bakery environment was examined via direct inoculation of slices of bread with bacterial culture. The three types of loaf examined were two soft grain varieties, one containing vinegar and the other containing calcium propionate as the preservative agent, and a white variety containing calcium propionate. Differences in rope production caused by batch variation were studied by comparing seven loaves of each type of bread. Not all isolates were capable of causing extensive rope, but isolates of Bacillus subtilis , B. licheniformis , B. megaterium and B. pumilus were able to produce such spoilage. Limited rope was also caused by pre-existing Bacillus species within the loaves. The amount of rope production by an isolate was not constant on all loaf types or even between different batches of the same variety, indicating that approaches that rely on direct inoculation of loaves with culture are not applicable for assessing the rope-inducing potential of Bacillus isolates. However, it was clear from this study that vinegar in soft grain loaves was more effective than calcium propionate at inhibiting rope.     

Medical nutrition therapy: use of sourdough lactic acid bacteria as a cell factory for delivering functional biomolecules and food ingredients in gluten free bread

Celiac disease (CD) is an immune-mediated disease, triggered in genetically susceptible individuals by ingesting gluten from wheat, rye, barley, and other closely related cereal grains. Currently, the estimated prevalence of CD is around 1 % of the population in the western world and medical nutritional therapy (MNT) is the only accepted treatment for celiac disease. To date, the replacement of gluten in bread presents a significant technological challenge for the cereal scientist due to the low baking performance of gluten free products (GF). The increasing demand by the consumer for high quality gluten-free (GF) bread, clean labels and natural products is rising. Sourdough has been used since ancient times for the production of rye and wheat bread, its universal usage can be attributed to the improved quality, nutritional properties and shelf life of sourdough based breads. Consequently, the exploitation of sourdough for the production of GF breads appears tempting. This review will highlight how sourdough LAB can be an efficient cell factory for delivering functional biomolecules and food ingredients to enhance the quality of gluten free bread.     

Antifungal Activity of Phenyllactic Acid against Molds Isolated from Bakery Products

Phenyllactic acid (PLA) has recently been found in cultures of Lactobacillus plantarum that show antifungal activity in sourdough breads. The fungicidal activity of PLA and growth inhibition by PLA were evaluated by using a microdilution test and 23 fungal strains belonging to 14 species of Aspergillus, Penicillium, and Fusarium that were isolated from bakery products, flours, or cereals. Less than 7.5 mg of PLA ml⁻¹ was required to obtain 90% growth inhibition for all strains, while fungicidal activity against 19 strains was shown by PLA at levels of ≤10 mg ml⁻¹. Levels of growth inhibition of 50 to 92.4% were observed for all fungal strains after incubation for 3 days in the presence of 7.5 mg of PLA ml⁻¹ in buffered medium at pH 4, which is a condition more similar to those in real food systems. Under these experimental conditions PLA caused an unpredictable delaying effect that was more than 2 days long for 12 strains, including some mycotoxigenic strains of Penicillium verrucosum and Penicillium citrinum and a strain of Penicillium roqueforti (the most widespread contaminant of bakery products); a growth delay of about 2 days was observed for seven other strains. The effect of pH on the inhibitory activity of PLA and the combined effects of the major organic acids produced by lactic acid bacteria isolated from sourdough bread (PLA, lactic acid, and acetic acid) were also investigated. The ability of PLA to act as a fungicide and delay the growth of a variety of fungal contaminants provides new perspectives for possibly using this natural antimicrobial compound to control fungal contaminants and extend the shelf lives of foods and/or feedstuffs.     

Long-Term Fungal Inhibitory Activity of Water-Soluble Extracts of Phaseolus vulgaris cv. Pinto and Sourdough Lactic Acid Bacteria during Bread Storage

The antifungal activity of proteinaceous compounds from different food matrices was investigated. In initial experiments, water-soluble extracts of wheat sourdoughs, cheeses, and vegetables were screened by agar diffusion assays with Penicillium roqueforti DPPMAF1 as the indicator fungus. Water-soluble extracts of sourdough fermented with Lactobacillus brevis AM7 and Phaseolus vulgaris cv. Pinto were selected for further study. The crude water-soluble extracts of L. brevis AM7 sourdough and P. vulgaris cv. Pinto had a MIC of 40 mg of peptide/ml and 30.9 mg of protein/ml, respectively. MICs were markedly lower when chemically synthesized peptides or partially purified protein fractions were used. The water-soluble extract of P. vulgaris cv. Pinto showed inhibition toward a large number of fungal species isolated from bakeries. Phaseolin alpha-type precursor, phaseolin, and erythroagglutinating phytohemagglutinin precursor were identified in the water-soluble extract of P. vulgaris cv. Pinto by nano liquid chromatography-electrospray ionization-tandem mass spectrometry. When the antifungal activity was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all three proteins were inhibitory. A mixture of eight peptides was identified from the water-soluble extract of sourdough L. brevis AM7, and five of these exhibited inhibitory activity. Bread was made at the pilot plant scale by sourdough fermentation with L. brevis AM7 and addition of the water-soluble extract (27%, vol/wt; 5 mg of protein/ml) of P. vulgaris cv. Pinto. Slices of bread packed in polyethylene bags did not show contamination by fungi until at least 21 days of storage at room temperature, a level of protection comparable to that afforded by 0.3% (wt/wt) calcium propionate.     

Antifungal activity of Leuconostoc citreum and Weissella confusa in rice cakes

The antifungal activity of organic acids greatly improves the shelf life of bread and bakery products. However, little is known about the effect of lactic acid fermentation on fungal contamination in rice cakes. Here, we show that lactic acid fermentation in rice dough can greatly retard the growth of three fungal species when present in rice cakes, namely Cladosporium sp. YS1, Neurospora sp. YS3, and Penicillium crustosum YS2. The antifungal activity of the lactic acid bacteria against these fungi was much better than that of 0.3% calcium propionate. We found that organic acids including lactic and acetic acid, which are byproducts of lactic fermentation or can be artificially added, were the main antifungal substances. We also found that some Leuconostoc citreum and Weissella confusa strains could be good starter species for rice dough fermentation. These results imply that these lactic acid bacteria can be applicable to improve the preservation of rice cakes.     

Effect of selected natural antimicrobials on Baker''s yeast activity

AIMS: To evaluate the responses of Baker's yeast (Saccharomyces cerevisiae) activity to the natural antimicrobials acetic acid, calcium lactate, a lactate-containing cocktail and lactic acid compared to calcium propionate. METHODS AND RESULTS: A dough fermentometer test was used to measure Baker's yeast activity in the presence of these natural antimicrobials and calcium propionate. Yeast activity generally decreased as a function of increasing antimicrobial concentrations, but the lactate-containing cocktail showed no relationship between concentration and yeast activity reduction. At in situ concentrations, calcium propionate resulted in the highest yeast activity reduction, followed by calcium lactate, acetic acid, the lactate-containing cocktail and lactic acid in decreasing order. CONCLUSION: Based on yeast activity reduction, all natural antimicrobials tested showed potential as possible replacements for calcium propionate. SIGNIFICANCE AND IMPACT OF THE STUDY: This has practical implications since calcium propionate inhibits Baker's yeast activity and attracts negative consumer perceptions as a chemical bread preservative.     

Rope-producing strains of Bacillus spp. from wheat bread and strategy for their control by lactic acid bacteria

Two types of white wheat bread (high- and low-type loaves) were investigated for rope spoilage. Thirty of the 56 breads tested developed rope spoilage within 5 days; the high-type loaves were affected by rope spoilage more than the low-type loaves. Sixty-one Bacillus strains were isolated from ropy breads and were characterized on the basis of their phenotypic and genotypic traits. All of the isolates were identified as Bacillus subtilis by biochemical tests, but molecular assays (randomly amplified polymorphic DNA PCR assay, denaturing gradient gel electrophoresis analysis, and sequencing of the V3 region of 16S ribosomal DNA) revealed greater Bacillus species variety in ropy breads. In fact, besides strains of B. subtilis, Bacillus licheniformis, Bacillus cereus, and isolates of Bacillus clausii and Bacillus firmus were also identified. All of the ropy Bacillus isolates exhibited amylase activity, whereas only 32.4% of these isolates were able to produce ropiness in bread slices after treatment at 96 degrees C for 10 min. Strains of lactic acid bacteria previously isolated from sourdough were first selected for antirope activity on bread slices and then used as starters for bread-making experiments. Prevention of growth of approximately 10(4) rope-producing B. subtilis G1 spores per cm(2) on bread slices for more than 15 days was observed when heat-treated cultures of Lactobacillus plantarum E5 and Leuconostoc mesenteroides A27 were added. Growth of B. subtilis G1 occurred after 7 days in breads started with Saccharomyces cerevisiae T22, L. plantarum E5, and L. mesenteroides A27.     

Antifungal activity of strains of lactic acid bacteria isolated from a semolina ecosystem against Penicillium roqueforti, Aspergillus niger and Endomyces fibuliger contaminating bakery products

Thirty samples of Italian durum wheat semolina and whole durum wheat semolina, generally used for the production of Southern Italy's traditional breads, were subjected to microbiological analysis in order to explore their lactic acid bacteria (LAB) diversity and to find strains with antifungal activity. A total of 125 presumptive LAB isolates (Gram-positive and catalase-negative) were characterized by repetitive extragenic palindromic-PCR (REP-PCR) and sequence analysis of the 16S rRNA gene, leading to the identification of the following species: Weissella confusa, Weissella cibaria, Leuconostoc citreum, Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus rossiae and Lactobacillus plantarum. The REP-PCR results delineated 17 different patterns whose cluster analysis clearly differentiated W. cibaria from W. confusa isolates. Seventeen strains, each characterized by a different REP-PCR pattern, were screened for their antifungal properties. They were grown in a flour-based medium, comparable to a real food system, and the resulting fermentation products (FPs) were tested against fungal species generally contaminating bakery products, Aspergillus niger, Penicillium roqueforti and Endomyces fibuliger. The results of the study indicated a strong inhibitory activity – comparable to that obtained with the common preservative calcium propionate (0.3% w/v) – of ten LAB strains against the most widespread contaminant of bakery products, P. roqueforti. The screening also highlighted the unexplored antifungal activity of L. citreum, L. rossiae and W. cibaria (1 strain), which inhibited all fungal strains to the same or a higher extent compared with calcium propionate. The fermentation products of these three strains were characterized by low pH values, and a high content of lactic and acetic acids.     

Effect of Breadmaking Process on In Vitro Gut Microbiota Parameters in Irritable Bowel Syndrome

A variety of foods have been implicated in symptoms of patients with Irritable Bowel Syndrome (IBS) but wheat products are most frequently cited by patients as a trigger. Our aim was to investigate the effects of breads, which were fermented for different lengths of time, on the colonic microbiota using in vitro batch culture experiments. A set of in vitro anaerobic culture systems were run over a period of 24 h using faeces from 3 different IBS donors (Rome Criteria–mainly constipated) and 3 healthy donors. Changes in gut microbiota during a time course were identified by fluorescence in situ hybridisation (FISH), whilst the small -molecular weight metabolomic profile was determined by NMR analysis. Gas production was separately investigated in non pH-controlled, 36 h batch culture experiments. Numbers of bifidobacteria were higher in healthy subjects compared to IBS donors. In addition, the healthy donors showed a significant increase in bifidobacteria (P<0.005) after 8 h of fermentation of a bread produced using a sourdough process (type C) compared to breads produced with commercial yeasted dough (type B) and no time fermentation (Chorleywood Breadmaking process) (type A). A significant decrease of δ-Proteobacteria and most Gemmatimonadetes species was observed after 24 h fermentation of type C bread in both IBS and healthy donors. In general, IBS donors showed higher rates of gas production compared to healthy donors. Rates of gas production for type A and conventional long fermentation (type B) breads were almost identical in IBS and healthy donors. Sourdough bread produced significantly lower cumulative gas after 15 h fermentation as compared to type A and B breads in IBS donors but not in the healthy controls. In conclusion, breads fermented by the traditional long fermentation and sourdough are less likely to lead to IBS symptoms compared to bread made using the Chorleywood Breadmaking Process.     

Antifungal Hydroxy Fatty Acids Produced during Sourdough Fermentation: Microbial and Enzymatic Pathways,and Antifungal Activity in Bread

Lactobacilli convert linoleic acid to hydroxy fatty acids; however, this conversion has not been demonstrated in food fermentations and it remains unknown whether hydroxy fatty acids produced by lactobacilli have antifungal activity. This study aimed to determine whether lactobacilli convert linoleic acid to metabolites with antifungal activity and to assess whether this conversion can be employed to delay fungal growth on bread. Aqueous and organic extracts from seven strains of lactobacilli grown in modified De Man Rogosa Sharpe medium or sourdough were assayed for antifungal activity. Lactobacillus hammesii exhibited increased antifungal activity upon the addition of linoleic acid as a substrate. Bioassay-guided fractionation attributed the antifungal activity of L. hammesii to a monohydroxy C_18:1 fatty acid. Comparison of its antifungal activity to those of other hydroxy fatty acids revealed that the monohydroxy fraction from L. hammesii and coriolic (13-hydroxy-9,11-octadecadienoic) acid were the most active, with MICs of 0.1 to 0.7 g liter⁻¹. Ricinoleic (12-hydroxy-9-octadecenoic) acid was active at a MIC of 2.4 g liter⁻¹. L. hammesii accumulated the monohydroxy C_18:1 fatty acid in sourdough to a concentration of 0.73 ± 0.03 g liter⁻¹ (mean ± standard deviation). Generation of hydroxy fatty acids in sourdough also occurred through enzymatic oxidation of linoleic acid to coriolic acid. The use of 20% sourdough fermented with L. hammesii or the use of 0.15% coriolic acid in bread making increased the mold-free shelf life by 2 to 3 days or from 2 to more than 6 days, respectively. In conclusion, L. hammesii converts linoleic acid in sourdough and the resulting monohydroxy octadecenoic acid exerts antifungal activity in bread.     

Rope-Producing Strains of Bacillus spp. from Wheat Bread and Strategy for Their Control by Lactic Acid Bacteria

Two types of white wheat bread (high- and low-type loaves) were investigated for rope spoilage. Thirty of the 56 breads tested developed rope spoilage within 5 days; the high-type loaves were affected by rope spoilage more than the low-type loaves. Sixty-one Bacillus strains were isolated from ropy breads and were characterized on the basis of their phenotypic and genotypic traits. All of the isolates were identified as Bacillus subtilis by biochemical tests, but molecular assays (randomly amplified polymorphic DNA PCR assay, denaturing gradient gel electrophoresis analysis, and sequencing of the V3 region of 16S ribosomal DNA) revealed greater Bacillus species variety in ropy breads. In fact, besides strains of B. subtilis, Bacillus licheniformis, Bacillus cereus, and isolates of Bacillus clausii and Bacillus firmus were also identified. All of the ropy Bacillus isolates exhibited amylase activity, whereas only 32.4% of these isolates were able to produce ropiness in bread slices after treatment at 96°C for 10 min. Strains of lactic acid bacteria previously isolated from sourdough were first selected for antirope activity on bread slices and then used as starters for bread-making experiments. Prevention of growth of approximately 10⁴ rope-producing B. subtilis G1 spores per cm² on bread slices for more than 15 days was observed when heat-treated cultures of Lactobacillus plantarum E5 and Leuconostoc mesenteroides A27 were added. Growth of B. subtilis G1 occurred after 7 days in breads started with Saccharomyces cerevisiae T22, L. plantarum E5, and L. mesenteroides A27.     

Directed evolution of a maltogenic alpha-amylase from Bacillus sp. TS-25

Directed evolution coupled with a high-throughput robotic screen was employed to broaden the industrial use of the maltogenic alpha-amylase Novamyl from Bacillus sp. TS-25. Wild-type Novamyl is currently used in the baking industry as an anti-staling agent in breads baked at neutral or near neutral pH. However, the enzyme is rapidly inactivated during the baking process of bread made with low pH recipes and Novamyl thus has very limited beneficial effect for this particular application. In an effort to improve the performance of Novamyl for low pH bread applications such as sourdough and rye, two error-prone PCR libraries were generated, expressed in Bacillus subtilis and screened for variants with improved thermal stability and activity under low pH conditions. Variants exhibiting improved performance were iteratively recombined using DNA shuffling to create two generations of libraries. Relative to wild-type Novamyl, a number of the resulting variants exhibited more than 10 degrees C increase in thermal stability at pH 4.5, one of which demonstrated substantial anti-staling properties in low pH breads.     

基于转录组学分析的丙酸钙对酿酒酵母的抑菌机制

[背景]丙酸钙作为在面包等食品中添加的防腐剂,具有一定的抑菌作用,但目前对其的研究大多聚焦于生化、群体层次.[目的]在分子水平上探究丙酸钙对酵母起抑菌作用的机制.[方法]取实验组和对照组中对数生长期的耐高糖酵母BH1进行转录组测序及分析,并进行实时荧光定量PCR验证.[结果]与6h对照组(无丙酸钙处理;Control ...     

Sourdough Bread Made from Wheat and Nontoxic Flours and Started with Selected Lactobacilli Is Tolerated in Celiac Sprue Patients

This work was aimed at producing a sourdough bread that is tolerated by celiac sprue (CS) patients. Selected sourdough lactobacilli had specialized peptidases capable of hydrolyzing Pro-rich peptides, including the 33-mer peptide, the most potent inducer of gut-derived human T-cell lines in CS patients. This epitope, the most important in CS, was hydrolyzed completely after treatment with cells and their cytoplasmic extracts (CE). A sourdough made from a mixture of wheat (30%) and nontoxic oat, millet, and buckwheat flours was started with lactobacilli. After 24 h of fermentation, wheat gliadins and low-molecular-mass, alcohol-soluble polypeptides were hydrolyzed almost totally. Proteins were extracted from sourdough and used to produce a peptic-tryptic digest for in vitro agglutination tests on K 562(S) subclone cells of human origin. The minimal agglutinating activity was ca. 250 times higher than that of doughs chemically acidified or started with baker's yeast. Two types of bread, containing ca. 2 g of gluten, were produced with baker's yeast or lactobacilli and CE and used for an in vivo double-blind acute challenge of CS patients. Thirteen of the 17 patients showed a marked alteration of intestinal permeability after ingestion of baker's yeast bread. When fed the sourdough bread, the same 13 patients had values for excreted rhamnose and lactulose that did not differ significantly from the baseline values. The other 4 of the 17 CS patients did not respond to gluten after ingesting the baker's yeast or sourdough bread. 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 gluten intolerance in humans.     

Sourdough bread made from wheat and nontoxic flours and started with selected lactobacilli is tolerated in celiac sprue patients

This work was aimed at producing a sourdough bread that is tolerated by celiac sprue (CS) patients. Selected sourdough lactobacilli had specialized peptidases capable of hydrolyzing Pro-rich peptides, including the 33-mer peptide, the most potent inducer of gut-derived human T-cell lines in CS patients. This epitope, the most important in CS, was hydrolyzed completely after treatment with cells and their cytoplasmic extracts (CE). A sourdough made from a mixture of wheat (30%) and nontoxic oat, millet, and buckwheat flours was started with lactobacilli. After 24 h of fermentation, wheat gliadins and low-molecular-mass, alcohol-soluble polypeptides were hydrolyzed almost totally. Proteins were extracted from sourdough and used to produce a peptic-tryptic digest for in vitro agglutination tests on K 562(S) subclone cells of human origin. The minimal agglutinating activity was ca. 250 times higher than that of doughs chemically acidified or started with baker's yeast. Two types of bread, containing ca. 2 g of gluten, were produced with baker's yeast or lactobacilli and CE and used for an in vivo double-blind acute challenge of CS patients. Thirteen of the 17 patients showed a marked alteration of intestinal permeability after ingestion of baker's yeast bread. When fed the sourdough bread, the same 13 patients had values for excreted rhamnose and lactulose that did not differ significantly from the baseline values. The other 4 of the 17 CS patients did not respond to gluten after ingesting the baker's yeast or sourdough bread. 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 gluten intolerance in humans.