The genus Lactobacillus comprises 261 species (at March 2020) that are extremely diverse at phenotypic, ecological and genotypic levels. This study evaluated the taxonomy of Lactobacillaceae and Leuconostocaceae on the basis of whole genome sequences. Parameters that were evaluated included core genome phylogeny, (conserved) pairwise average amino acid identity, clade-specific signature genes, physiological criteria and the ecology of the organisms. Based on this polyphasic approach, we propose reclassification of the genus Lactobacillus into 25 genera including the emended genus Lactobacillus, which includes host-adapted organisms that have been referred to as the Lactobacillus delbrueckii group, Paralactobacillus and 23 novel genera for which the names Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus are proposed. We also propose to emend the description of the family Lactobacillaceae to include all genera that were previously included in families Lactobacillaceae and Leuconostocaceae. The generic term 'lactobacilli' will remain useful to designate all organisms that were classified as Lactobacillaceae until 2020. This reclassification reflects the phylogenetic position of the micro-organisms, and groups lactobacilli into robust clades with shared ecological and metabolic properties, as exemplified for the emended genus Lactobacillus encompassing species adapted to vertebrates (such as Lactobacillus delbrueckii, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensensii, Lactobacillus johnsonii and Lactobacillus acidophilus) or invertebrates (such as Lactobacillus apis and Lactobacillus bombicola).

A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae.

PCR-DGGE Fingerprinting: Novel Strategies for Detection of Microbes in Food

http://sgpwe.izt.uam.mx/files/users/uami/acym/articulo_1.pdf

Food fermentations: Microorganisms with technological beneficial use

The production of sourdough bread can be traced back to ancient times. Sourdough is a mixture of flour and water that is fermented with lactic acid bacteria (LAB). Sourdough is an intermediate product and contains metabolically active yeast and LAB strains. The LAB that develop in the dough may originate from selected natural contaminants in the flour or from a starter culture containing one or more known species of LAB. Sourdough can be produced in bakeries or obtained from commercial suppliers. The microbial ecology of the sourdough fermentation is determined by ecological factors. Microbiological studies have revealed that more than 50 species of LAB, mostly species of the genus Lactobacillus, and more than 20 species of yeasts, especially species of the genera Saccharomyces and Candida, occur in this ecological niche. The sourdough microflora is composed of stable associations of lactobacilli and yeasts, in particular due to metabolic interactions. As shown for certain industrial sourdough processes, such microbial associations may endure for years, although the fermentation process runs under non-aseptic conditions. A reproducible and controlled composition and activity of the sourdough microflora is indispensable to achieve a constant quality of sourdough bread.

The sourdough microflora: biodiversity and metabolic interactions

Lactobacilli in sourdough fermentation

Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L. mindensis, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L. crispatus and L. pontis became the predominant species in sourdough B and L. crispatus, L. panis, and L. frumenti became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L. johnsonii and L. reuteri were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L. fermentum was also detected. Isolates of the species L. sanfranciscensis and L. fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively.

Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis

Four sourdoughs (A to D) were produced under practical conditions, using a starter obtained from a mixture of three commercially available sourdough starters and baker's yeast. The doughs were continuously propagated until the composition of the microbiota remained stable. A fungi-specific PCR-denaturing gradient gel electrophoresis (DGGE) system was established to monitor the development of the yeast biota. The analysis of the starter mixture revealed the presence of Candida humilis, Debaryomyces hansenii, Saccharomyces cerevisiae, and Saccharomyces uvarum. In sourdough A (traditional process with rye flour), C. humilis dominated under the prevailing fermentation conditions. In rye flour sourdoughs B and C, fermented at 30 and 40°C, respectively, S. cerevisiae became predominant in sourdough B, whereas in sourdough C the yeast counts decreased within a few propagation steps below the detection limit. In sourdough D, which corresponded to sourdough C in temperature but was produced with rye bran, Candida krusei became dominant. Isolates identified as C. humilis and S. cerevisiae were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. The yeast species isolated from the sourdoughs were also detected by PCR-DGGE. However, in the gel, additional bands were visible. Because sequencing of these PCR fragments from the gel failed, cloning experiments with 28S rRNA amplicons obtained from rye flour were performed, which revealed Cladosporium sp., Saccharomyces servazii, S. uvarum, an unculturable ascomycete, Dekkera bruxellensis, Epicoccum nigrum, and S. cerevisiae. The last four species were also detected in sourdoughs A, B, and C.

Identification and population dynamics of yeasts in sourdough fermentation processes by PCR-denaturing gradient gel electrophoresis.

Characterisation of the Microbiota of Rice Sourdoughs and Description of Lactobacillus spicheri sp. nov.

The identification of lactic acid bacteria in a complex microbiota using bacteriological culture in combination with phenotypic and genotypic identification techniques is laborious and time-consuming. New molecular methods permit a fast and culture-independent characterisation of such microbiota. Denaturing gradient gel electrophoresis (DGGE) of PCR fragments of the 16S rRNA gene has been proven to be a suitable tool. Here the use of PCR-DGGE with group specific primers is described to investigate the dynamic of sourdough microbiota from addition of the starter until the microbiota remained stable. Species were identified by applying an identification ladder obtained from reference strains or by sequence analysis of the PCR fragments. Furthermore, a method for detection of strains in complex microbiota is described. A strain specific chromosomal DNA fragment of Lactobacillus paracasei LTH 2579 was isolated applying the subtraction hybridisation. Based on the acquired target sequence a specific PCR system was established and combined with a PCR system specific for the species L. paracasei. Use of this detection system permitted to identify and quantitatively detect L. paracasei LTH 2579 in fermented sausages and upon consumption in faecal samples.

Christiane B. Meroth

Papers

Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis

Identification and population dynamics of yeasts in sourdough fermentation processes by PCR-denaturing gradient gel electrophoresis.

Characterisation of the Microbiota of Rice Sourdoughs and Description of Lactobacillus spicheri sp. nov.

Species and strain specific identification of lactic acid bacteria in complex microflora