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Sourdough is renowned for improving bakery products’ nutritional and quality characteristics through the enzymes produced by its microbiota. Among the enzymatic framework present in sourdough fermentation, amylase, cellulase, and peptidase are responsible for many of the properties valued in sourdough products. Furthermore, there is an increasing concern regarding the allergenic potential of gluten, which motivates the investigation of enzymatic gluten hydrolysis. This study aimed to select probiotics, isolate and identify microorganisms from sourdough, and assess their amylase, cellulase, and peptidase profiles. Additionally, a rapid screening method was developed for gluten and wheat flour hydrolysis, and gluten zymography and enzymography were performed. As a result, 18 microorganisms were isolated from sourdough and identified. The probiotic Bacillus licheniformis LMG-S 28935, and three microorganisms isolated from sourdough, the Limosilactobacillus fermentum, Pediococcus pentosaceus, and Saccharomyces cerevisiae, completed the profile of analyzed hydrolases and presented the capacity to hydrolyze gluten. These findings contribute to a better understanding of sourdough microorganisms’ hydrolase activities in the bakery science and technology field. In addition, an efficient, fast, and economical method for screening extracellular glutenase, produced by microorganisms, was applied. To our knowledge, it was the first time that amylase, cellulase, and peptidase activities were assessed from sourdough microorganisms.

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