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Molecular Identification and Phylogenetic Inference of Lactic Acid Bacteria Isolated from Goat’s Raw Milk Cheese
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1  Centro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Campus de Santa Apolónia 5300-253 Bragança, Portugal.
2  Laboratório para a Sustentabilidade e Tecnologia em Regiões de Montanha, Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal.
Academic Editor: Cristina A. Fente

Abstract:

Lactic acid bacteria (LAB) have a wide genetic potential and are applied in areas such as food production, intestinal health, and genetic engineering. Thus, the objectives of this work were: to identify at the species level twenty-eight 16S ribosomal gene sequences of LABs from cheese; and to evaluate the phylogenetic relationship among the sequences. Colony PCR was performed with the primers 15f 5'-GCTCAGGAYGAACGCYGG-3' and 687r 5'-CACCGCTACACATGRADTTC-3'. The identification of the nucleotides was carried out using the Sanger method. The species of the obtained sequences were determined with the BLAST tool. In the R software, a multiple sequence alignment was performed with ClustalW program. Subsequently, an identity matrix containing the squared root of the pairwise distances was calculated, and a phylogenetic tree was built with the Neighbor-Joining method. In the MEGA software, distance between and within species were calculated. LAB sequences were identified as Lacticaseibacillus paracasei (43%), Lactiplantibacillus pentosus (3.6%), Lactiplantibacillus plantarum (10.7%), Lactococcus cremoris (10.7%), Lactococcus lactis (7.1%) and Leuconostoc mesenteroides (25%). The phygenetic tree revealed variability among sequences of the same species from the positions assumed in the cladogram, and allowed the identification of clusters, formed by the species L. mesenteroides, and L. paracasei. The species L. lactis and L. cremoris proved to be the most genetic distant from each other, with 1.6 as the average number of nucleotide substitutions per position. Therefore, L. paracasei has been shown to be the most frequent in the analyzed samples. Further research will yield more understanding of LAB's diverse and phylogenetic characteristics.

Keywords: fermentation; taxonomy; phylogenetic tree; BigDye™ Terminator v3.1
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