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To characterize the lactobacilli community of 20 sourdoughs using a novel polyphasic approach.A polyphasic approach, consisting of a two-step multiplex polymerase chain reaction (PCR) system, 16S rRNA gene sequence analysis and physiological features, was applied to identify 127 isolates, representing about 37% of the presumptive lactobacilli collected from sourdough samples. Multiplex PCR successfully identified 111 isolates, while 16S rRNA gene sequencing was applied for the other 16 isolates, two of which could not be associated with any previously described lactic acid bacteria (LAB) species. Strain diversity was evaluated by phenotypic and random amplified polymorphic DNA-PCR analysis. Molecular detection of Lactobacillus group species was also performed on total DNA extracted from the doughs.Abruzzo region sourdough lactobacilli biodiversity, reflected in both Lactobacillus species composition and strain polymorphism, is similar to that of other Italian regions and is a source of novel LAB species.Within culture-independent methods, multiplex PCR is a rapid tool to study the lactobacilli population of sourdoughs.