METHODS Bacterial biodiversity and dynamics during malolactic fermentation of Tempranillo wines as determined by a culture-independent method (PCR-DGGE) Patricia Ruiz & Susana Seseña & Pedro Miguel Izquierdo & María Llanos Palop Received: 2 December 2009 / Revised: 1 February 2010 / Accepted: 1 February 2010 / Published online: 9 March 2010 # Springer-Verlag 2010 Abstract The bacterial population during malolactic fer- mentation of Tempranillo wine was studied using the polymerase chain reaction-denaturing gradient gel electro- phoresis, a culture-independent method successfully used for identification and monitoring of bacterial population in different habitats included food fermentations. The results showed that Oenococcus oeni was the predominant species in the malolactic fermentation of Tempranillo wines, although the presence of Gluconobacter oxydans, Asaia siamensis, Serratia sp., and Enterobacter sp. was also observed. These results were partly coincidental with those obtained from a culture-dependent method, using a selective medium. Therefore, it may be concluded that for a more complete knowledge of the bacterial community present during malolactic fermentation of Tempranillo wine, an approach that combines a culture-independent method and a culture-dependent method would be advisable. Keywords Malolactic fermentation . Tempranillo wine . RAPD-PCR . PCR-DGGE Introduction Malolactic fermentation (MLF), a process in which L-malate is converted into L-lactate and carbon dioxide, has been described as having a significant influence on wine quality (Henick-Kling 1993; Lonvaud-Funel 1999). Lactic acid bacteria (LAB) are responsible for this process, although other species of bacteria may also be present (Bae et al. 2006; Renouf et al. 2007). Information regarding the composition and dynamics of microbial communities throughout the vinification process is always useful to control the process, which will contribute to improving wine quality. Both traditional and molecular methods have been used to study the microbial population dynamics during wine fermentation, mainly those of yeasts and LAB, which has allowed for a better understanding of the relations and interactions between the different species involved (Andorrà et al. 2008; Reguant and Bordons 2003; Renouf et al. 2007). In recent years, randomly amplified polymorphic DNA- polymerase chain reaction (RAPD-PCR) has been frequent- ly used in the genetic characterization of strains of bacteria participating in different food fermentations, including MLF (Coppola et al. 2006; Lechiancole et al. 2006; Rodas et al. 2005; Ruiz et al. 2010; Sánchez et al. 2004; Zapparoli et al. 2000). This culture-dependent method provides significant insight into specific isolates and microbial populations, but it is well known that only a small proportion of microorganisms are cultivable and, therefore, culture-dependent techniques often result in an incomplete representation of the true bacterial diversity present (Amann et al. 1995; Hugenholtz et al. 1998). Thus, recent microbial ecology studies of foods have employed novel culture- independent molecular approaches, such as those that use polymerase chain reaction amplification with different P. Ruiz : S. Seseña : M. L. Palop (*) Departamento de Química Analítica y Tecnología de Alimentos, Facultad de Ciencias del Medio Ambiente, Universidad de Castilla—La Mancha, Avda. Carlos III s/n, 45071 Toledo, Spain e-mail: MariaLlanos.Palop@uclm.es P. M. Izquierdo Instituto de la Vid y del Vino de Castilla—La Mancha, Crta. Toledo-Albacete s/n, Tomelloso, 13700 Ciudad Real, Spain Appl Microbiol Biotechnol (2010) 86:1555–1562 DOI 10.1007/s00253-010-2492-8