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Applied and Environmental Microbiology, April 2002, p. 1882-1892, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1882-1892.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Cinzia L. Randazzo,1,2 Sandra Torriani,2,{ddagger} Antoon D. L. Akkermans,1 Willem M. de Vos,1 and Elaine E. Vaughan1*

Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 CT Wageningen, The Netherlands,1 Laboratory of Microbiology, Dipartimento di Orto-Floro-Arboricoltura e Tecnologie Agroalimentari, University of Catania, Catania, Italy2

Received 10 August 2001/ Accepted 14 January 2002

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 culture-independent 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.

* Corresponding author. Mailing address: Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT, Wageningen, The Netherlands. Phone: 31 317 483486. Fax: 31 317 483829. E-mail: elaine.vaughan{at}algemeen.micr.wau.nl.

{dagger} Paper no. 1 from the Ministry of Agricultural, Food and Forest Resources, Italy (MIRAAF), within the program "Valorizzazione e salvaguardia della microflora autoctona caratteristica delle produzioni casearie italiane."

{ddagger} Present address: Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Verona, Italy.

Applied and Environmental Microbiology, April 2002, p. 1882-1892, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1882-1892.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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