Four laboratory sourdough fermentations, initiated with wheat or spelt flour and without the addition of a starter culture, were prepared over a period of 10 days with daily back-slopping. Samples taken at all refreshment steps were used for determination of the present microbiota. Furthermore, an extensive metabolite target analysis of more than 100 different compounds was performed through a combination of various chromatographic methods including liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. The establishment of a stable microbial ecosystem occurred through a three-phase evolution within a week, as revealed by both microbiological and metabolite analyses. Strains of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus rossiae, Lactobacillus brevis, and Lactobacillus paraplantarum were dominating some of the sourdough ecosystems. Although the heterofermentative L. fermentum was dominating one of the wheat sourdoughs, all other sourdoughs were dominated by a combination of obligate and facultative heterofermentative taxa. Strains of homofermentative species were not retrieved in the stable sourdough ecosystems. Concentrations of sugar and amino acid metabolites hardly changed during the last days of fermentation. Besides lactic acid, ethanol, and mannitol, the production of succinic acid, erythritol, and various amino acid metabolites, such as phenyllactic acid, hydroxyphenyllactic acid, and indolelactic acid, was shown during fermentation. Physiologically, they contributed to the equilibration of the redox balance. The biphasic approach of the present study allowed us to map some of the interactions taking place during sourdough fermentation and helped us to understand the fine-tuned metabolism of lactic acid bacteria, which allows them to dominate a food ecosystem.Sourdough is a mixture of ground cereals (e.g., wheat or rye) and water that is spontaneously fermented. Sourdough fermentations improve dough properties, enhance both bread texture and bread flavor, and delay bread spoilage (28). Lactic acid bacteria (LAB) and yeasts play a key role in sourdough fermentation processes (9,21,26,28,29,55). Sourdough LAB have been intensively studied with respect to their carbohydrate metabolism (16,24,60), proteolysis and amino acid metabolism (16,23,58,69), lipid metabolism (16), and production of volatile compounds (7,31,32). Besides these general metabolic traits, specific metabolic properties have been recognized in sourdough LAB, such as the use of alternative electron acceptors (59, 61), the production of antifungal compounds (38,45,57), the biosynthesis of exopolysaccharides (36, 63, 64), and arginine catabolism (8, 53). These metabolic traits of sourdough LAB highlight their adaptation to the sourdough environment. For instance, fructose-to-mannitol and arginineto-ornithine conversion favor ATP generation and/or acid stress (16). Also, interactions between sourdough LAB and yeasts have been studied in detail (9, 21).The microbial growth and activity of LAB in so...

A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) in Belgian traditional sourdoughs and to assess the influence of flour type, bakery environment, geographical origin, and technological characteristics on the taxonomic composition of these LAB communities. For this purpose, a total of 714 LAB from 21 sourdoughs sampled at 11 artisan bakeries throughout Belgium were subjected to a polyphasic identification approach. The microbial composition of the traditional sourdoughs was characterized by bacteriological culture in combination with genotypic identification methods, including repetitive element sequence-based PCR fingerprinting and phenylalanyl-tRNA synthase (pheS) gene sequence analysis. LAB from Belgian sourdoughs belonged to the genera Lactobacillus, Pediococcus, Leuconostoc, Weissella, and Enterococcus, with the heterofermentative species Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus plantarum, and Lactobacillus pontis as the most frequently isolated taxa. Statistical analysis of the identification data indicated that the microbial composition of the sourdoughs is mainly affected by the bakery environment rather than the flour type (wheat, rye, spelt, or a mixture of these) used. In conclusion, the polyphasic approach, based on rapid genotypic screening and high-resolution, sequence-dependent identification, proved to be a powerful tool for studying the LAB diversity in traditional fermented foods such as sourdough.Sourdough represents a natural food ecosystem in which the fermentation activities of lactic acid bacteria (LAB) and yeasts largely determine the typical characteristics of the resulting baked goods (26). Based on the production technology, sourdough fermentations can be divided into three types (2, 38): type I, or traditional, sourdoughs are characterized by continuous propagation of the dough at ambient temperatures (20 to 30°C); type II, or industrial, sourdoughs are incubated at high temperatures (Ͼ30°C), with longer fermentation times and a higher water content; and type III sourdoughs are dried preparations of industrial doughs. Traditional Belgian sourdoughs belong to type I sourdoughs. The use of sourdough improves the overall characteristics of bakery products, such as the dough properties, texture, and flavor (16). LAB contribute significantly to these properties, e.g., by acidification of the dough (17), proteolysis of the gluten (10), hydrolysis of the starch (5), and the production of taste and aromatic compounds (15). Moreover, several sourdough LAB inhibit the development of pathogens due to the production of acids and bacteriocins (22). During spontaneous sourdough maturation, LAB occur as the predominant microorganisms, at numbers of Ͼ10 8 CFU/g sourdough. The genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella predominate in this ecosystem, whereas lactococci, enterococci, and streptococci are rarely found. Sourdough LAB may originate from natural contamination of the flour or may be introduced i...

In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culturedependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential.

A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and cultureindependent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.Traditional sourdoughs comprise a complex microbial association of lactic acid bacteria (LAB) and yeasts and are thought to improve sensory, texture, and health-promoting properties of many bakery products (15). During sourdough fermentation, the prevailing LAB produce acids (mainly lactic acid and acetic acid) that lower the pH of the sourdough medium. In addition, these organisms are responsible for the production of ethanol, aroma compounds, bacteriocins, exopolysaccharides, and several enzymes (17). Sourdough LAB may originate with natural contaminants in the flour or with a starter culture which contains one or more LAB strains (6). Sourdough can be cultivated in bakeries or obtained from commercial suppliers. In Belgium, many artisan bakeries still use spontaneously fermented sourdoughs, which are kept metabolically active through the addition of flour and water at regular intervals (backslopping). During this process of continuous propaga...

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