Microbial Ecology of Greek Wheat Sourdoughs, Identified by a Culture-Dependent and a Culture-Independent Approach

Abstract

The aim of the present study was to assess the microecosystem of 13 homemade spontaneously fermented wheat sourdoughs from different regions of Greece, through the combined use of culture-dependent (classical approach; clustering by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) and identification by PCR species-specific for Lactiplantibacillus plantarum, and sequencing of the 16S-rRNA and 26S-rRNA gene, for Lactic Acid Bacteria (LAB) and yeasts, respectively) and independent approaches [DNA- and RNA-based PCR-Denaturing Gradient Gel Electrophoresis (DGGE)]. The pH and Total Titratable Acidity (TTA) values ranged from 3.64–5.05 and from 0.50–1.59% lactic acid, respectively. Yeast and lactic acid bacteria populations ranged within 4.60–6.32 and 6.28–9.20 log CFU/g, respectively. The yeast: LAB ratio varied from 1:23–1:10,000. A total of 207 bacterial and 195 yeast isolates were obtained and a culture-dependent assessment of their taxonomic affiliation revealed dominance of Lb. plantarum in three sourdoughs, Levilactobacillus brevis in four sourdoughs and co-dominance of these species in two sourdoughs. In addition, Companilactobacillusparalimentarius dominated in two sourdoughs and Fructilactobacillussanfranciscensis and Latilactobacillus sakei in one sourdough each. Lactococcus lactis, Lb. curvatus, Leuconostoc citreum, Ln. mesenteroides and Lb. zymae were also recovered from some samples. Regarding the yeast microbiota, it was dominated by Saccharomyces cerevisiae in 11 sourdoughs and Pichia membranifaciens and P. fermentans in one sourdough each. Wickerhamomyces anomalus and Kazachstania humilis were also recovered from one sample. RNA-based PCR-DGGE provided with nearly identical results with DNA-based one; in only one sample the latter provided an additional band. In general, the limitations of this approach, namely co-migration of amplicons from different species to the same electrophoretic position and multiband profile of specific isolates, greatly reduced resolution capacity, which resulted in only partial verification of the microbial ecology detected by culture-dependent approach in the majority of sourdough samples. Our knowledge regarding the microecosystem of spontaneously fermented Greek wheat-based sourdoughs was expanded, through the study of sourdoughs originating from regions of Greece that were not previously assessed.