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Applied and Environmental Microbiology, September 2008, p. 5662-5673, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.00418-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Polyphasic Approach to Bacterial Dynamics during the Ripening of Spanish Farmhouse Cheese, Using Culture-Dependent and -Independent Methods{triangledown}

Antonio M. Martín-Platero, Eva Valdivia, Mercedes Maqueda, Inés Martín-Sánchez, and Manuel Martínez-Bueno*

Departamento de Microbiología, Facultad de Ciencias, Fuentenueva s/n, 18071 Granada, Spain

Received 19 February 2008/ Accepted 15 July 2008

We studied the dynamics of the microbial population during ripening of Cueva de la Magahá cheese using a combination of classical and molecular techniques. Samples taken during ripening of this Spanish goat's milk cheese in which Lactococcus lactis and Streptococcus thermophilus were used as starter cultures were analyzed. All bacterial isolates were clustered by using randomly amplified polymorphic DNA (RAPD) and identified by 16S rRNA gene sequencing, species-specific PCR, and multiplex PCR. Our results indicate that the majority of the 225 strains isolated and enumerated on solid media during the ripening period were nonstarter lactic acid bacteria, and Lactobacillus paracasei was the most abundant species. Other Lactobacillus species, such as Lactobacillus plantarum and Lactobacillus parabuchneri, were also detected at the beginning and end of ripening, respectively. Non-lactic-acid bacteria, mainly Kocuria and Staphylococcus strains, were also detected at the end of the ripening period. Microbial community dynamics determined by temporal temperature gradient gel electrophoresis provided a more precise estimate of the distribution of bacteria and enabled us to detect Lactobacillus curvatus and the starter bacteria S. thermophilus and L. lactis, which were not isolated. Surprisingly, the bacterium most frequently found using culture-dependent analysis, L. paracasei, was scarcely detected by this molecular approach. Finally, we studied the composition of the lactobacilli and their evolution by using length heterogeneity PCR.

* Corresponding author. Mailing address: Departamento de Microbiología, Facultad de Ciencias, Fuentenueva s/n, 18071 Granada, Spain. Phone: 34 958 243184. Fax: 34 958 249486. E-mail: mmartine{at}ugr.es

{triangledown} Published ahead of print on 25 July 2008.

Applied and Environmental Microbiology, September 2008, p. 5662-5673, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.00418-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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