Diversity, Dynamics, and Activity of Bacterial Communities during Production of an Artisanal Sicilian Cheese as Evaluated by 16S rRNA Analysis

Abstract

The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and cultureindependent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of Leuconostoc, Lactococcus lactis, and Macrococcus caseolyticus were dominant in the raw milk, while Streptococcus thermophilus prevailed during lactic fermentation. Other thermophilic LAB, especially Lactobacillus delbrueckii and Lactobacillus fermentum, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the Lactobacillus delbrueckii species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology.

Safety and quality of farm fresh goat's cheese in the Czech Republic

“…In contrast to our study that focused on a single farm, the aforementioned authors analysed cheese samples of different origins. The average count of lactic acid bacteria was of the order of magnitude of 10 8 CFU/g in both monitored types of cheese (Table 4) and was in agreement with the data generally reported for raw cheese (Randazzo et al 2002). The presence of B. cereus was not detected in any of the samples.…”

Microbiota of Karakačanski skakutanac, an artisanal fresh sheep cheese studied by culture-independent PCR-ARDRA and PCR-DGGE

“…Taking into account this drawback of culture-independent PCR approach, identical ARDRA profiles could be explained although initial diverse profiles were expected due to the use of raw milk in the cheese production. It has been reported by other authors [3,4,11,14,15] that population structure of LAB community present in the traditional raw milk cheeses is diverse and complex. The dominance of only one species was also confirmed by PCR-DGGE.…”

“…Molecular analysis of the Stilton cheese demonstrated that the use of different targets of the same DNA template revealed remarkably different results [6]. Moreover, partial 16S rDNA sequences do not always allow discrimination between closely related species and one band may represent two species with identical partial sequence [12,[14][15][16].…”

Multifactorial diversity sustains microbial community stability

“…Similarly, milk can accommodate numerous microbial species (Delbes et al, 2007). However, strong selective pressures like heat treatment (Van Hoorde et al, 2008), dairy processing conditions (Randazzo et al, 2002), intrinsic properties of lactic acid fermentation and antagonistic interactions (Lindgren and Dobrogosz 1990), enrich the microbiota toward a few lactic acid bacteria species.…”