Metabolic and proteomic adaptation of Lactobacillus rhamnosus strains during growth under cheese-like environmental conditions compared to de Man, Rogosa, and Sharpe medium.

The aim of this study was to demonstrate the metabolic and proteomic adaptation of Lactobacillus rhamnosus strains, which were isolated at different stages of Parmigiano Reggiano cheese ripening. Compared to de Man, Rogosa, and Sharpe (MRS) broth, cultivation under cheese-like conditions (cheese broth, CB) increased the number of free amino acids used as carbon sources. Compared with growth on MRS or pasteurized and microfiltrated milk, all strains cultivated in CB showed a low synthesis of d,l-lactic acid and elevated levels of acetic acid. The proteomic maps of the five representative strains, showing different metabolic traits, were comparatively determined after growth on MRS and CB media. The amount of intracellular and cell-associated proteins was affected by culture conditions and diversity between strains, depending on their time of isolation. Protein spots showing decreased (62 spots) or increased (59 spot) amounts during growth on CB were identified using MALDI-TOF-MS/MS or LC-nano-ESI-MS/MS. Compared with cultivation on MRS broth, the L. rhamnosus strains cultivated under cheese-like conditions had modified amounts of some proteins responsible for protein biosynthesis, nucleotide, and carbohydrate metabolisms, the glycolysis pathway, proteolytic activity, cell wall, and exopolysaccharide biosynthesis, cell regulation, amino acid, and citrate metabolism, oxidation/reduction processes, and stress responses.

Evaluation of genetic polymorphism among Lactobacillus rhamnosus non-starter Parmigiano Reggiano cheese strains.

Parmigiano Reggiano (PR) is an Italian cooked, long-ripened cheese made with unheated cow's milk and natural whey starter. The microflora is involved in the manufacturing of this cheese, arising from the natural whey starter, the raw milk and the environment. Molecular studies have shown that mesophilic non-starter lactic acid bacteria (NSLAB) are the dominant microflora present during the ripening of PR. In this study, a characterisation of Lactobacillus rhamnosus isolated from a single PR manufacturing and ripening process is reported, using a combination of genotypic fingerprinting techniques (RAPD-PCR and REP-PCR). The intraspecies heterogeneity evidenced for 66 strains is correlated to their abilities to adapt to specific environmental and technological conditions. The detection of biotypes that correlate with specific moments in cheese ripening or differential development throughout this process suggests that these strains may have specific roles closely linked to their peculiar technological properties.

A polyphasic approach to detect enterotoxigenic Staphylococcus aureus and diarrheagenic Escherichia coli in raw milk Italian cheeses by multiplex PCR.

A polyphasic approach was evaluated for the detection of eight staphylococcal enterotoxin (SE)-encoding genes (sea, sec, sed, seg, seh, sei, sej, sel) and the Escherichia coli genes most commonly associated with virulence factors (eae, elt, ipaH, stx) in traditional soft cheeses, manufactured artisanally from whole raw milk in the Lombardy region (northern Italy). To determine the presence of the target genes, two multiplex PCRs were performed on DNA extracted both directly from cheese samples (culture-independent approach) and from whole cultivable cells, formed by harvesting colonies from the first serial dilution agar plates of selective media, as representative of cultivable community ("bulk"). Genes associated with enteroinvasive E. coli, ipaH, and Shiga toxin-producing E. coli, stx, were detected in two of the bulk samples analyzed; no virulence genes were found by amplifying DNA directly extracted from cheeses. SE-encoding genes were found in three cheeses (sea in all three samples, associated with sed and sej in two of these). More SE-encoding genes were detected by amplifying DNA obtained from bulk samples: sea, sed, sej, sec, seg, sel, and sei. No samples harbored the gene encoding for SE type H. The polyphasic approach followed has been useful in enhancing detection of target genes. Our results indicate that some of the artisanal cheeses examined may constitute a potential hazard for consumer health.

Application of AFLP fingerprint analysis for studying the biodiversity of Streptococcus thermophilus.

Streptococcus thermophilus is a lactic acid bacteria (LAB) widely used in milk fermentation processes as a starter culture. In this work the genetic diversity of S. thermophilus isolates from different sources was analyzed using Amplified Fragment Length Polymorphism fingerprinting (AFLP). Since this is the first report that indicates the application of AFLP in order to study genotypic polymorphism in S. thermophilus species, an optimization of experimental conditions was carried out to decide the optimal AFLP analysis protocol. Furthermore the fingerprinting resolutions of AFLP and RAPD (Random Amplified Polymorphic DNA) were evaluated and compared. The overall data suggest that genotypic characterization performed by AFLP provide a better view of microbial diversity of S. thermophilus, indicating that RAPD is less discriminating than AFLP. The successful use of AFLP analysis in the characterization of S. thermophilus strains reported in this study suggests the potential uses for this technique to define the whole-genome diversity of each specific strain, as an alternative to the fingerprinting methods used till now.