RESUMO
The diversity and dynamics of Lactobacillus populations in traditional raw milk Camembert cheese were monitored throughout the manufacturing process in 3 dairies. Culture-dependent analysis was carried out on isolates grown on acidified de Man - Rogosa - Sharpe agar and Lactobacillus anaerobic de Man Rogosa Sharpe agar supplemented with vancomycin and bromocresol green media. The isolates were identified by polymerase chain reaction - temperature gradient gel electrophoresis (PCR-TGGE) and (or) species-specific PCR and (or) sequencing, and Lactobacillus paracasei and Lactobacillus plantarum isolates were characterized by pulsed field gel electrophoresis (PFGE). Milk and cheese were subjected to culture-independent analysis by PCR-TGGE. Presumed lactobacilli were detected by plate counts throughout the ripening process. However, molecular analysis of total DNA and DNA of isolates failed to detect Lactobacillus spp. in certain cases. The dominant species in the 3 dairies was L. paracasei. PFGE analysis revealed 21 different profiles among 39 L. paracasei isolates. Lactobacillus plantarum was the second most isolated species, but it occurred nearly exclusively in one dairy. The other species isolated were Lactobacillus parabuchneri, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus helveticus, a Lactobacillus psittaci/delbrueckii subsp. bulgaricus/gallinarum/crispatus group, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, Lactobacillus brevis, Lactobacillus kefiri, and Lactobacillus perolens. Lactobacilli diversity at the strain level was high. Dynamics varied among dairies, and each cheese exhibited a specific picture of species and strains.
Assuntos
Biodiversidade , Queijo/microbiologia , Lactobacillus/classificação , Lactobacillus/crescimento & desenvolvimento , Animais , Contagem de Colônia Microbiana , Meios de Cultura/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Eletroforese em Gel de Campo Pulsado , Eletroforese em Gel de Poliacrilamida/métodos , Genes de RNAr , Lactobacillus/isolamento & purificação , Leite/microbiologia , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Temperatura de TransiçãoRESUMO
Lactobacilli are Gram positive rods belonging to the Lactic Acid Bacteria (LAB) group. Their phenotypic traits, such as each species' obligate/facultative, homo/heterofermentation abilities play a crucial role in souring raw milk and in the production of fermented dairy products such as cheese, yoghurt and fermented milk (including probiotics). An up to date safety analysis of these lactobacilli is needed to ensure consumer safety. Lactobacillus genus is a heterogeneous microbial group containing some 135 species and 27 subspecies, whose classification is constantly being reshuffled. With the recent use of advanced molecular methods it has been suggested that the extreme diversity of the Lactobacillus genomes would justify recognition of new subgeneric divisions. A combination of genotypic and phenotypic tests, for example DNA-based techniques and conventional carbohydrate tests, is required to determine species. Pulsed-Field gel Electrophoresis (PFGE) has been successfully applied to strains of dairy origin and is the most discriminatory and reproducible method for differentiating Lactobacillus strains. The bibliographical data support the hypothesis that the ingestion of Lactobacillus is not at all hazardous since lactobacillemia induced by food, particularly fermented dairy products, is extremely rare and only occurs in predisposed patients. Some metabolic features such as the possible production of biogenic amines in fermented products could generate undesirable adverse effects. A minority of starter and adjunct cultures and probiotic Lactobacillus strains may exceptionally show transferable antibiotic resistance. However, this may be underestimated as transferability studies are not systematic. We consider that transferable antibiotic resistance is the only relevant cause for caution and justifies performing antibiotic-susceptibility assays as these strains have the potential to serve as hosts of antibiotic-resistance genes, with the risk of transferring these genes to other bacteria. However, as a general rule, lactobacilli have a high natural resistance to many antibiotics, especially vancomycin, that is not transferable. Safety assessment requirements for Lactobacillus strains of technological interest should be limited to an antibiotic profile and a study to determine whether any antibiotic resistance(s) of medical interest detected is (or are) transferable. This agrees with the recent EFSA proposal suggesting attribution of a QPS status for 32 selected species of lactobacilli.
