Studio preliminare per l'applicazione dell'analisi Multi-Locus Sequence Typing in Lactobacillus sanfranciscensis

For centuries, sourdough has been the only starter used for cereal fermentation in baked products. It is a mixture of wheat four and water characterized by lactic acid bacteria (LAB) and yeast in mutualistic relationship. Within bacterial population Lactobacillus sanfranciscensis is the dominant species.
The aim of this thesis was the quantitative and qualitative evaluation of sourdough stability during time, though the analysis of Lactobacillus sanfranciscensis strains isolated from different doughs. MLST (Multi-Locus Sequence Typing) was proposed in 1998 as a nucleotide-based approach that provided accurate and portable data appropriate for epidemiological investigations. Bacterial characterization relis on distinguishing genetic variation among isolates sequencing multiple housekeeping loci at nucleotide level. This information feflected population evolution and biology.
In the first part of the work 21 samples coming from 3 different producers were analyzed to evaluate microbiological composotion (dominant and minoritary lactic acid bacteria, yeast count). Quantitative analysis showed that LAB were found at level of 10^8 CFU/g; whilr yeast were present in a concentration of 10^7 CFU/g. Species identification of dominant strains was then carried out through the partial sequencing of 16S rDNA gene for bacteria and 18S-26S rDNA spacer region for yeast isolates. Bacterial species found were: Lactobacillus sanfranciscensis (isolation incidence: 74%), Lactobacillus zymae (21%), Enterococcus faecium (5%), all typical for sourdough. Yeast species were: Candida humilis (68%), Dabaryomyces hansenii (16%), Saccharomycete sp. (16%). In the second part of the work, 28 strains of Lb. sanfranciscensis coming from different places (Italy, Germany, USA, Brazil) and isolated in different times (1971-2006) were collected for MLST analysis. After a preliminary study on 8 strains, the following genes were choosen: gdh (glucose-6-phosphate dehydrogenase), gyrA (subunit A of DNA-gyrase), pta (phosphotransacetylase), nox (NADH-oxidase), mapA (maltose phosphorilase A), pgmA (phosphoglucomutase A). analysis of the loci sequences within these genes made posssible to discern all the 8 strains that were examined. Each strain showed an exclusive allelic profile. The less polymorfic locus was PGM2 with 2 alleles, followed by MAP2 and GDH1 with 3; while PTA1 and GYRA1 generated 4 alleles and NOX1, with 5, was the most variable. To study if nucleotide differences, found in sequenced strains, were present in other 20 isolated coming from the same type of doughs produced in different period of the year, restriction analysis was performed. The results showed that strains from the same producer had the same allelic profile. MLST, as subtyping method, is able to discriminate strains of different geographic origin but did not distinguish those isolated from a sourdough supported in the same way for years. Sourdough for sweet baked products hold the stability of the bacterial association for years, also at strain level.