• Phytase activity
• Leavening ability
• Lactobacillus sanfranciscensis
• Kazachstania humilis
• Inter-delta regions analysis
• Candida milleri
• biotyping
• Antioxidant activity
• RAPD-PCR
• rep-PCR
• Saccharomyces cerevisiae
• sourdough bread

Sourdough fermentation, through microbial metabolism, positively affects the nutritional, organoleptic and technological traits of leavened baked goods. Each sourdough harbours different lactic acid bacteria (LAB) and yeast communities, and their diversity depends on process technologies, types of flour and other ingredients traditionally associated with local culture and origin. For this reason, the molecular and functional characterization of the sourdough microbiota is pivotal to characterize traditional sourdough breads, including special types selected as worthy of either Protected Geographical Indication (PGI) or Protected Designation of Origin (PDO), such as PDO Tuscan bread. Moreover, the increasing demand for healthy baked goods boosted studies on sourdough microbiota with beneficial metabolic traits, to be used as potential functional starters. Actually, as microbial functional traits are strictly related to strains, the characterization of sourdough microbial isolates is crucial for their biotechnological exploitation to obtain functional baked end-products. The overall objective of this research project was the study of molecular and functional diversity of the microbiota isolated from sourdough breads, in order to characterize the final product PDO Tuscan bread, reveal taxonomic characteristics of isolates and select autochthonous LAB and yeast strains with interesting functional traits, to be used as starter cultures, for the production of fermented baked goods and cereal-based beverages with enhanced nutritional and nutraceutical properties.
Firstly, the species composition of PDO Tuscan bread sourdough was investigated on 96 LAB and 68 yeast isolates, using Amplified Ribosomal DNA Restriction Analysis (ARDRA) and PCR-RFLP analysis of the ITS region, respectively, and sequencing. Results showed that PDO Tuscan bread sourdough is characterized by a peculiar tripartite species association, encompassing a large number of Lactobacillus sanfranciscensis and Candida milleri (now Kazachstania humilis) strains, along with few strains of Saccharomyces cerevisiae. A culture-independent approach (PCR Denaturating Gradient Gel Electrophoresis, DGGE) confirmed such data, revealing that no microorganisms in the Viable But Non-Culturable (VBNC) state occurred. LAB and yeast isolates were further characterized at strain level (typization), after a preliminary identification of the best intraspecific molecular tools. RAPD-PCR with P4, P7 and M13 primers was used to reveal a huge degree of intraspecific variability, discriminating 43 biotypes out of the 96 L. sanfranciscensis isolates characterizing PDO Tuscan bread sourdough. Concerning yeast community of PDO Tuscan bread sourdough, inter-delta regions was able to discriminate two biotypes among the three S. cerevisiae isolates, and rep-PCR with (GTG)5 primer method was used to reveal a high degree of intraspecific polymorphism, discriminating 9 biotypes out of the 65 K. humilis isolates. The relative composition and specific physiological characteristics of such highly complex microbial community is at the basis of the peculiar organoleptic, rheological, nutritional and potentially nutraceutical features of PDO Tuscan bread, as suggested by the qualitative functional characterization of the isolates. Phytase, amylase and protease activities were assessed, showing a significant diversity among strains.
Afterwards, LAB and yeast populations of traditional sourdoughs collected from four Tuscan bakeries were investigated, in order to select the best performing strains, based on their pro- technological, nutritional and functional traits, to be used as starters for making sourdough baked goods or functional cereal-based beverages. Among 200 yeasts and 135 LAB isolated, 78 and 45, respectively, were randomly selected and molecularly characterized, at genera, species and strain levels. Restriction analyses of ITS region showed that S. cerevisiae was dominant, representing the only species detected in three out of the four sourdoughs. The fourth one harboured also K. humilis. Inter-delta regions analysis revealed a high intraspecific polymorphism discriminating 16 biotypes of S. cerevisiae isolates, which clustered based on their origin. Concerning bacteria, L. sanfranciscensis species-specific PCR showed that all the LAB isolates belonged to L. sanfranciscensis species and RAPD-PCR analysis with P4, P7 and M13 primers showed the occurrence of 33 L. sanfranciscensis biotypes, revealing a wide degree of variability (73%).
Based on genetic differences 20 yeast and 12 LAB strains were selected and individually used to ferment wheat flour, aiming at evaluating their pro-technological, nutritional and functional features. During fermentation under standardized conditions, all yeast strains were able to grow of ca. 2 log cycles, reaching the highest volumes of the dough after 16 h of fermentation. S. cerevisiae IMA D2Y produced the highest dough volume increase. K. humilis IMA G23Y was the only strain able to increase the total free amino acids concentration of the doughs. Moreover, K. humilis IMA G23Y, S. cerevisiae IMA D20Y and S. cerevisiae IMA D24Y showed a threefold higher phytase activity than spontaneously fermented control, and the highest concentration of total phenols. Almost all the strains led to increases of antioxidant activity, without significant differences among them. Investigations on the resistance of the strains to simulated gastric and intestinal conditions, that is considered a pre-requisite for the selection of probiotics, revealed the ability to survive in vitro by many of the strains considered.
Regarding LAB, no strain was able to produce exopolysaccharides (EPS). During fermentation under standardized conditions, all strains were able to acidify wheat doughs, showing median values of ΔpH of 2.57 ± 0.05. L. sanfranciscensis IMA D30LAB, D27LAB, D8LAB and D22LAB showed significantly higher values of phytase activity than spontaneously fermented control. All the strains led to increases of antioxidant activity, without significant differences among them, but significantly greater than control.
Results obtained allowed us to select S. cerevisiae IMA D24Y, K. humilis IMA G23Y, L. sanfranciscensis IMA D34LAB and IMA D8LAB isolates, to be used in association to produce potential functional starters. During fermentation under standardized conditions, all strains were able to grow and acidify the doughs. Lower pH values and correspondingly higher TTA values were reached using both L. sanfranciscensis isolates in combination with K. humilis IMA G23Y than with S. cerevisiae IMA D24Y. The former combinations were further characterized for their phytase and antioxidant activities. Although the co-presence of two strains didn’t show a synergistic effect, values resulted significantly higher than not inoculated doughs for both activities. The textural properties of the experimental breads, started with the consortia L. sanfranciscensis IMA D34LAB – K. humilis IMA G23Y (Bread C) and L. sanfranciscensis IMA D8LAB – K. humilis IMA G23Y (Bread D), were determined after baking; a sensory analysis showed that the use of such starters can improve the overall quality of bread, compared with baker’s yeast bread.