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...

The growth of pure cultures of Bacteroides thetaiotaomicron LMG 11262 and Bacteroides fragilis LMG 10263 on fructose and oligofructose was examined and compared to that of Bifidobacterium longum BB536 through in vitro laboratory fermentations. Gas chromatography (GC) analysis was used to determine the different fractions of oligofructose and their degradation during the fermentation process. Both B. thetaiotaomicron LMG 11262 and B. fragilis LMG 10263 were able to grow on oligofructose as fast as on fructose, succinic acid being the major metabolite produced by both strains. B. longum BB536 grew slower on oligofructose than on fructose. Acetic acid and lactic acid were the main metabolites produced when fructose was used as the sole energy source. Increased amounts of formic acid and ethanol were produced when oligofructose was used as an energy source at the cost of lactic acid. Detailed kinetic analysis revealed a preferential metabolism of the short oligofructose fractions (e.g., F 2 and F 3 ) for B. longum BB536. After depletion of the short fractions, the larger oligofructose fractions (e.g., F 4 , GF 4 , F 5 , GF 5 , and F 6 ) were metabolized, too. Both Bacteroides strains did not display such a preferential metabolism and degraded all oligofructose fractions simultaneously, transiently increasing the fructose concentration in the medium. This suggests a different mechanism for oligofructose breakdown between the strain of Bifidobacterium and both strains of Bacteroides, which helps to explain the bifidogenic nature of inulin-type fructans.

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...

Thirty-one lactic acid bacterial strains from different species were evaluated for exopolysaccharide (EPS) production in milk. Thermophilic strains produced more EPS than mesophilic ones, but EPS yields were generally low. Ropiness or capsular polysaccharide formation was strain dependent. Six strains produced high-molecular-mass EPS. Polymers were classified into nine groups on the basis of their monomer composition. EPS from Enterococcus strains were isolated and characterized.Certain lactic acid bacteria (LAB) produce exopolysaccharides (EPS), either capsular polysaccharides (CPS) that are tightly associated with the cell surface or slime EPS that are secreted into the extracellular environment. EPS from LAB can be divided into homopolysaccharides, which are polymers composed of one type of monosaccharide, and heteropolysaccharides (HePS), which are polymers of repeating units that are composed of two or more types of monosaccharides (5,6,7,11). A large biodiversity of HePS from LAB exists regarding their composition and structure, molecular mass (MM), yield, and functionalities (5,7,35). Further, polymer formation is strongly influenced by culture conditions (1,7,22,32). Recently, the molecular genetics of HePS biosynthesis have been studied for different LAB species (6, 25). Several glycosyltransferases involved in the assemblage of the HePS repeating units have been discovered (17,18,35). EPS can act as viscosifying, stabilizing, gel-forming, and/or water-binding agents in various foods (6, 10). Additionally, they have been claimed to display properties beneficial to health (6, 29). Little attention has been paid to CPS formation by food grade LAB. Exploration of the biodiversity of wild LAB strains is the most suitable approach to search for a desired EPS phenotype (28,33,34). The aims of this study were to seek new EPS-producing LAB strains, to characterize their biopolymers, and to explore their diversity to find novel or interesting HePS or HePS-producing strains.EPS screening, isolation, and characterization. Two hundred one thermophilic (11 strains of Lactobacillus acidophilus, 79 of Lactobacillus delbrueckii subsp. bulgaricus, 1 of L. delbrueckii subsp. lactis, 1 of Streptococcus macedonicus, and 42 of Streptococcus thermophilus) and mesophilic (23 strains of Enterococcus faecalis, 1 of Enterococcus faecium, 5 of Lactobacillus casei, 29 of Lactobacillus paracasei, and 9 of Lactobacillus rhamnosus) LAB strains (CERELA Culture Collection, Tucumán, Argentina) were used throughout this study. All bacteria were stored as previously described (22). Before experimental use, cultures were propagated twice in MRS (Britania, Buenos Aires, Argentina) for lactobacilli or in LAPTg (27) for streptococci and enterococci.Screening for EPS-producing LAB was performed with reconstituted skim milk (10%, wt/vol) at 37°C for 16 h using cultures propagated in milk as the inoculum (1%, vol/vol). Noninoculated medium was used as a control. Cell counts, expressed as numbers of CFU per milliliter, were determined by pour pla...

The growth of Bifidobacterium animalis DN-173 010 on different energy sources was studied through smalland large-scale fermentations. Growth on both more common energy sources (glucose, fructose, galactose, lactose, and sucrose) and inulin-type fructans was examined. High-performance liquid chromatography analysis was used to investigate the kinetics. Gas chromatography was used to determine the fructan degradation during the fermentation process. B. animalis DN-173 010 was unable to grow on a medium containing glucose as the sole energy source. In general, monosaccharides were poor growth substrates for the B. animalis strain. The fermentations with the inulin-type fructans resulted in changes in both growth and metabolite production due to the preferential metabolism of certain fructans, especially the short-chain oligomers. Only after depletion of the shorter chains were the larger fractions also metabolized, although to a lesser extent. Acetic acid was the major metabolite produced during all fermentation experiments. At the beginning of the fermentation, high levels of lactic acid were produced, which were partially replaced by formic acid at later stages. This suggests a shift in sugar metabolism to gain additional ATP that is necessary for growth on oligofructose, which is metabolized more slowly.

Several strains belonging to the genus Bifidobacterium were tested to determine their abilities to produce succinic acid. Bifidobacterium longum strain BB536 and Bifidobacterium animalis subsp. lactis strain Bb 12 were kinetically analyzed in detail using in vitro fermentations to obtain more insight into the metabolism and production of succinic acid by bifidobacteria. Changes in end product formation in strains of Bifidobacterium could be related to the specific rate of sugar consumption. When the specific sugar consumption rate increased, relatively more lactic acid and less acetic acid, formic acid, and ethanol were produced, and vice versa. All Bifidobacterium strains tested produced small amounts of succinic acid; the concentrations were not more than a few millimolar. Succinic acid production was found to be associated with growth and stopped when the energy source was depleted. The production of succinic acid contributed to regeneration of a small part of the NAD ؉ , in addition to the regeneration through the production of lactic acid and ethanol.

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...

Sourdough fermentations are characterized by the combined activity of lactic acid bacteria and yeasts. An investigation of the microbial composition of 21 artisan sourdoughs from 11 different Belgian bakeries yielded 127 yeast isolates. Also, 12 spontaneous 10-day laboratory sourdough fermentations with daily backslopping were performed with rye, wheat, and spelt flour, resulting in the isolation of 217 yeast colonies. The isolates were grouped according to PCR-fingerprints obtained with the primer M13. Representative isolates of each M13 fingerprint group were identified using the D1/D2 region of the large subunit rRNA gene, internal transcribed spacer sequences, and partial actin gene sequences, leading to the detection of six species. The dominant species in the bakery sourdoughs were Saccharomyces cerevisiae and Wickerhamomyces anomalus (formerly Pichia anomala), while the dominant species in the laboratory sourdough fermentations were W. anomalus and Candida glabrata. The presence of S. cerevisiae in the bakery sourdoughs might be due to contamination of the bakery environment with commercial bakers yeast, while the yeasts in the laboratory sourdoughs, which were carried out under aseptic conditions with flour as the only nonsterile component, could only have come from the flour used.