Ilse Scheirlinck

Culture-Independent Analysis of Probiotic Products by Denaturing Gradient Gel Electrophoresis

ABSTRACT 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 culture-dependent 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.

Population Dynamics and Metabolite Target Analysis of Lactic Acid Bacteria during Laboratory Fermentations of Wheat and Spelt Sourdoughs

ABSTRACT 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.

Influence of Geographical Origin and Flour Type on Diversity of Lactic Acid Bacteria in Traditional Belgian Sourdoughs

ABSTRACT 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.

Taxonomic Structure and Stability of the Bacterial Community in Belgian Sourdough Ecosystems as Assessed by Culture and Population Fingerprinting

ABSTRACT 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 culture-independent 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.

Molecular source tracking of predominant lactic acid bacteria in traditional Belgian sourdoughs and their production environments

Aims:  To investigate the circulation of predominant sourdough lactic acid bacteria (LAB) species in the production environment of two Belgian artisan sourdough bakeries.

Lactic acid bacteria community dynamics and metabolite production of rye sourdough fermentations share characteristics of wheat and spelt sourdough fermentations.

Four spontaneous rye sourdough fermentations were performed over a period of ten days with daily back-slopping. Samples taken at all refreshment steps were used for culture-dependent and culture-independent characterization of the microbiota present. Furthermore, an extensive metabolite target analysis was performed through a combination of various chromatographic methods, including liquid chromatography coupled to mass spectrometry (LC/MS) and gas chromatography coupled to mass spectrometry (GC/MS). Spearmans rank correlation coefficients were calculated and a principal component analysis (PCA) was performed on the data obtained in this study combined with data obtained previously for wheat and spelt sourdoughs. In general, the establishment of a stable microbial ecosystem occurred through a three-phase evolution, with mainly Lactobacillus plantarum and Lactobacillus fermentum dominating the rye sourdough ecosystems. PCA revealed that ornithine and mannitol were positively correlated with rye sourdoughs, contributing to bacterial competitiveness at the onset of sourdough production. Wheat and spelt sourdoughs showed a high degree of similarity, although certain compounds (e.g. indolelactic acid) appeared to be specific for spelt sourdoughs. The production of amino acid metabolites, mainly hydroxy acids (e.g. phenyllactic acid) and alcohols (e.g. 3-methyl-1-butanol), contributed to the equilibration of the redox balance and further enhanced the competitiveness of dominant species in stable sourdoughs.

Diversity and dynamics of bacterial populations during spontaneous sorghum fermentations used to produce ting, a South African food

Ting is a spontaneously fermented sorghum food that is popular for its sour taste and unique flavour. Insight of the microbial diversity and population dynamics during sorghum fermentations is an essential component of the development of starter cultures for commercial production of ting. In this study, bacterial populations associated with spontaneous sorghum fermentations were examined using a culture-independent strategy based on denaturing gradient gel electrophoresis and sequence analysis of V3-16S rRNA gene amplicons, and a culture-dependent strategy using conventional isolation based on culturing followed by 16S rRNA and/or pheS gene sequence analysis. The entire fermentation process was monitored over a 54 h period and two phases were observed with respect to pH evolution and microbial succession. The first phase of the process (0-6h) was characterized by relatively high pH conditions and the presence of Enterococcus mundtii, albeit that this species was only detected with the culture-dependent approach. The second phase of the fermentation process (12-54 h) was characterized by increased acidity and the predominance of a broader range of lactic acid bacteria, including Lactococcus lactis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, Weissella cibaria, Enterococcus faecalis, and a close relative of Lactobacillus curvatus, as well as some members of the Enterobacteriaceae family. The Lb. curvatus-like species was only detected with PCR-DGGE, while the majority of the other species was only detected using the culture-dependent approach. These findings highlighted the fact that a combination of both approaches was essential in revealing the microbial diversity and dynamics during spontaneous sorghum fermentations.

Polyphasic taxonomic characterization of Lactobacillus rossiae isolates from Belgian and Italian sourdoughs reveals intraspecific heterogeneity

(GTG)5-PCR fingerprinting and pheS sequence analysis of 18 Lactobacillus rossiae isolates, mainly originating from Belgian and Italian artisan sourdoughs, revealed intraspecies grouping as evidenced by the delineation of three and two subgroups, respectively. On the other hand, 16S rRNA and rpoA gene sequence analysis and DNA-DNA hybridizations supported the accommodation of all isolates in a single species. No correlation between genetic and phenotypic heterogeneity was observed. Collectively, these data do not warrant taxonomic division of L. rossiae. On the other hand, the considerable differences in intraspecies sequence variation of L. rossiae isolates displayed by the pheS (9.8%) and rpoA (1.1%) genes highlight that the discriminatory power of housekeeping genes as alternative genomic markers for the 16S rRNA gene in the identification of Lactobacillus species may significantly differ from gene to gene. In conclusion, this study has demonstrated that a polyphasic approach remains highly useful for identification of isolates belonging to genotypically heterogeneous species such as L. rossiae.