Sourdough fermentation is one of the oldest biotechnological process of bread-making. Sourdough is the mixture of flour and water, spontaneous fermented by presented metabolically active microflora composed of lactic acid bacteria and yeasts. During fermentation, lactic acid bacteria produce organic acids, which improve flavor of bread, increase gas retention, help the swelling of gluten and shorten mixing time [1]. Microbial acidification is crucial for proteolysis in cereal substrates. After fermentation, sourdough have a final pH between 3.5 – 4.0, which is ideal for activation of endogenous cereal proteinases and initiation of a primary proteolysis of cereal proteins. Secondary proteolysis progress into the LAB cells by intracellular peptidases, because LAB do not possess extracellular proteolytic activity [2]. LAB are cell factories for a broad scale of enzymes, including specific peptidases capable of hydrolyzing of proline-rich celiac active peptides. Sourdoughs produced by addition of selected LAB according their substrate specifity may lead to degradation of gluten and production of gluten-low bakery products [3].

The aim of this study was to evaluate metabolic activity and survivability of lactobacilli starter culture in cereal-based substrate and preparation of sourdough samples for further analysis with an emphasis on cleavage at proline-rich proteins.

Substrates were prepared from commercial available rye bread flour (70 %) and wheat flour (30 %). Water was added into the flour mix in ratio 2.5 : 1 (w/v). Control sample was prepared as a mixture of flours and water and experimental sourdough was enriched by the addition of selected lactobacilli (~10⁸ CFU.ml^-1 water of each strain). The lactobacilli starter culture composed of lactobacilli (LAB) in the late exponential phase of growth and LAB were selected according to their prolyl-aminopeptidase activity (Lactobacillus plantarum CCM 3626, L. plantarum CCM 3627, L. zymae CCM 7241 and L. fermentum CCM 7192). Prepared sourdoughs were fermented for 24 hours at 37 °C, which is optimum growth temperature of LAB.

LAB and yeast counts were determined before and after fermentation (CFU.g^-1 of dough). Samples of sourdoughs were analyzed every 4 hours of fermentation process, while pH values and total titrable acidity of sourdoughs were observed.

Low colony counts of LAB and yeast populations (10⁵ CFU.g^-1 and 10⁴ CFU.g^-1 of dough, respectively) were found at the begining of fermentation. After 24 hours fermentation of control sample, LAB and yeasts numerous increased to 2,9 x 10⁹ CFU.g^-1 and 1,5 x 10⁵ CFU.g^-1, respectively. Cell colony numbers of LAB grew to 7,6 x 10¹⁰ CFU.g^-1 and yeasts increased to 4,2 x 10⁸ CFU.g^-1 in sample leavening by starter culture. Measuring the pH values of sourdough samples showed that LAB had active carbohydrate metabolism. The first change of pH value was observed after 8 hours of control sample fermentation. The pH value of experimental sample dropped bellow 4.5 after 4 hours of total 24 hours fermentation process. Presented microflora (with or without starter culture) produced such amount of organic acids, which decrease pH from initial 6,5 to less than 4.5 in both cases, but starter culture significantly accelerate an acidification of substrate. Total titrable acidity depends on the presence of yeasts in the initial and intermadiate phase of fermentation, and after that depends on the metabolic activity of LAB [1]. For neutralization of control and experimental samples were used 0,3 ml and 0,6 ml of NaOH before fermentation and 3,6 ml and 6,6 ml after fermentation, respectively.

The resulting sourdough samples will be used for further immunochemical tests (ELISA assay, Western blotting) for detection of harmful peptides for celiac patients.

The incorporation of selected lactobacilli as a starter culture for sourdough preparation may lead to production of cereal products with defined characteristics and possibly health-promoting properties [4]. Lactobacilli which produce prolyl-specific peptidases can be an alternative tool for degradation of prolin-rich celiac active peptides during food-making process.