Biodiversity, dynamics and ecology of bacterial community during grape marc storage for the production of grappa Petros A. Maragkoudakis a, 1, 2 , Tiziana Nardi b, 1, 3 , Barbara Bovo a , Maura D'Andrea b , Kate S. Howell c , Alessio Giacomini a, b, ⁎, Viviana Corich a, b a DAFNAE — Department of Agronomy, Food, Natural Resources, Animals and Environment, Viale dell'Università 16, Legnaro (PD), Italy b Centro di Ricerca Interdipartimentale per la Viticoltura e l'Enologia, Università di Padova, via XXVIII Aprile 14, Conegliano (TV), Italy c Melbourne School of Land and Environment, University of Melbourne, 3010 Parkville, Australia abstract article info Article history: Received 27 April 2012 Received in revised form 21 December 2012 Accepted 2 January 2013 Available online 11 January 2013 Keywords: Grape marc Lactobacillus plantarum Antimicrobial activity Biofilm Genotyping The Italian spirit obtained from grape marc, grappa, is produced by an extended storage of the marc which allows alcoholic fermentation. Bacterial populations can develop and are associated with off-flavour production. Grape marc acidification before storage is a common practice in distilleries to control bacterial proliferation. Few studies have been published on the microbial biodiversity in grape marc and no information exists about microbiology of acidified marcs and physiological properties needed for colonizing such an environment. The aim of this study was to investigate the composition and dynamics of grape marc bacterial populations during the long-period storage by microbiological analyses of acidified and untreated marcs. Eight bacterial species were identified by ARDRA — 16s rRNA sequencing at the beginning of the fermentation. Among them the bacterial species of Tatumella terrea, Acetobacter ghanensis and Tatumella ptyseos were identified for the first time in a wine environ- ment. In later stages Oenococcus oeni and members of the Lactobacillus plantarum group became dominant in acidified and non-acidified grape marc, respectively. Further molecular typing of L. plantarum isolates yielded 39 strains. To explain the prevalence of L. plantarum in untreated samples, all strains were tested for potential antimicrobial activity and for biofilm formation ability. Although no antimicrobial activity was found, many strains exhibited the ability to form a biofilm, which may confer an ecological advantage to these strains and their dominance during marc storage. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Grape marc consists of the solid parts of grapes, containing grape skins, seeds and sometimes stalks which remain after juice extraction, and represents the main solid waste of the wine-making process. This type of by-product has been traditionally utilised in southern Europe, and spirits obtained from grape marc distillation are produced in almost all the Mediterranean countries (Gerogiannaki-Christopoulou et al., 2006). In addition, this material can be used for animal feed or recycled as soil conditioner due to its organic and nutrient contents. Moreover, grape marcs are also burnt after distillation for heat pro- duction (Lo Curto and Tripodo, 2001). Grappa is a traditional alcoholic beverage produced in Italy from steam distillation of grape marc (EEC, 1989). During the manufactur- ing process of white grape varieties, marc is separated from grape juice before must fermentation (De Rosa and Castagner, 1994). The marc is sent to the distillery where they are stored, generally in sealed plastic tunnels, for a period ranging from a few days to several weeks. During this period sugars present at 5 to 15% (w/w) in grape marc, are converted into ethanol to reach a final concentration of 4–10% (w/w). Generally, microbial starter cultures are not added and thus many undefined biochemical reactions take place due to the activity of the natural microflora. Although yeasts are essential to the process of al- coholic fermentation, they can also be responsible for the production of spoilage compounds. In particular yeasts often produce excess of higher alcohols that are then concentrated into the distillate (Bovo et al., 2010; Cortés et al., 2010; De Pina and Hogg, 1999; Nykanen, 1986; Weinberg et al., 1988). Bacteria are responsible for the most frequent off-flavours present in the distillate via the production of 2-butanol and ethyl lactate (Bae et al., 2006; Davis et al., 1985; Manitto et al., 1994; Pozo-Bayon et al., 2005; Versini and Margheri, 1979). For these reasons a well-controlled storage process that prevents the growth of spoilage microflora is considered essential. To control International Journal of Food Microbiology 162 (2013) 143–151 ⁎ Corresponding author at: DAFNAE — Department of Agronomy, Food, Natural Resources, Animals and Environment, Viale dell'Università 16, Legnaro (PD), Italy. Tel.: +39 049 8272924; fax: +39 049 8272929. E-mail address: alessio.giacomini@unipd.it (A. Giacomini). 1 These authors contributed equally to this work. 2 Present address: European Commission, Joint Research Institute, via E. Fermi 2749, 21027 Ispra (VA), Italy. 3 Present address: Lallemand Italia, via Rossini 14/B, 37060 Castel d'Azzano (VR), Italy. 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.01.005 Contents lists available at SciVerse ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro