RESUMO
A genus-specific PCR analysis method was developed for a rapid and reliable differentiation between the two heterofermentative lactic acid bacteria genera Leuconostoc and Weissella. Primer sets specific for target regions of the 16S rRNA genes were designed and the specificity of the PCR was evaluated using the type strains of 13 species of Leuconostoc and 11 species of Weissella. In addition, the newly developed genus-specific PCR analysis was applied to characterize 72 lactic acid bacteria (LAB) strains isolated from coffee fermentation and which were presumptively classified as Leuconostoc or Weissella species. Additionally, a total of 34 LAB isolates from various other fermented foods were included. The investigations of these strains were conducted to test the effectiveness of correct characterization of field isolates using the genus-specific PCR approach. The correct assignment to one of these two genera by the application of the genus-specific primers was confirmed by further identifying the strains using repetitive extragenic palindromic-PCR and 16S rRNA gene sequencing.
Assuntos
Microbiologia de Alimentos , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/genética , Reação em Cadeia da Polimerase/métodos , Café/metabolismo , Primers do DNA/genética , DNA Bacteriano/genética , DNA Ribossômico/genética , Fermentação , Genes de RNAr/genética , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
A Gram-positive, ovoid lactic acid bacterium, strain LMG 23990(T), was isolated from Ethiopian coffee fermentation. 16S rRNA gene sequence analysis indicated that the novel strain belongs to the genus Leuconostoc, with Leuconostoc citreum and Leuconostoc lactis as the closest neighbours (99.6 and 99.0 % 16S rRNA gene sequence similarity, respectively). Genotypic fingerprinting by fluorescent amplified fragment length polymorphism, whole-cell protein electrophoresis, DNA-DNA hybridizations, comparative sequence analysis of pheS, rpoA, atpA, and physiological and biochemical tests allowed us to differentiate strain LMG 23990(T) from all established Leuconostoc species. Strain LMG 23990(T) (=CCUG 54536(T)) therefore represents a novel species, for which the name Leuconostoc holzapfelii sp. nov. is proposed.