A methodological approach to screen diverse cheese-related bacteria for their ability to produce aroma compounds Tomislav Poga  ci  c a, b, ** , Marie-Bernadette Maillard a, b , Aur  elie Leclerc c , Christophe Herv  e c , Victoria Chuat a, b , Alyson L. Yee a, b , Florence Valence a, b , Anne Thierry a, b, * a INRA, UMR1253 Science et Technologie du Lait et de l'Œuf, 65 rue de Saint Brieuc, 35000 Rennes, France b AGROCAMPUS OUEST, UMR1253 Science et Technologie du Lait et de l'Œuf, 65 rue de Saint Brieuc, 35000 Rennes, France c Laboratoires Standa, F-14000 Caen, France article info Article history: Received 6 May 2014 Received in revised form 8 July 2014 Accepted 26 July 2014 Available online 8 August 2014 Keywords: Cheese Bacteria Aroma compounds Screening Volatile metabolite profiling Volatilome abstract Microorganisms play an important role in the development of cheese flavor. The aim of this study was to develop an approach to facilitate screening of various cheese-related bacteria for their ability to produce aroma compounds. We combined i) curd-based slurry medium incubated under conditions mimicking cheese manufacturing and ripening, ii) powerful method of extraction of volatiles, headspace trap, coupled to gas chromatography-mass spectrometry (HS-trap-GC-MS), and iii) metabolomics-based method of data processing using the XCMS package of R software and multivariate analysis. This approach was applied to eleven species: five lactic acid bacteria (Leuconostoc lactis, Lactobacillus sakei, Lactobacillus paracasei, Lactobacillus fermentum, and Lactobacillus helveticus), four actinobacteria (Bra- chybacterium articum, Brachybacterium tyrofermentans, Brevibacterium aurantiacum, and Microbacterium gubbeenense), Propionibacterium freudenreichii, and Hafnia alvei. All the strains grew, with maximal populations ranging from 7.4 to 9.2 log (CFU/mL). In total, 52 volatile aroma compounds were identified, of which 49 varied significantly in abundance between bacteria. Principal component analysis of volatile profiles differentiated species by their ability to produce ethyl esters (associated with Brachybacteria), sulfur compounds and branched-chain alcohols (H. alvei), branched-chain acids (H. alvei, P. freudenreichii and L. paracasei), diacetyl and related carbonyl compounds (M. gubbeenense and L. paracasei), among others. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Characterization of microorganisms for their production of odor-active volatile compounds and evaluation of their utility as ripening cultures in cheese manufacture is an ongoing scientific challenge in dairy microbiology. New strains isolated from dairy or non-dairy environments should be evaluated for their aromatic potential, since flavor is a very important characteristic from the consumer's point of view (Niimi et al., 2014). The formation of flavor compounds in cheese results from numerous metabolic reactions and is largely influenced by microbial diversity and the complex dynamics of growth and metabolism during cheese ripening (Hassan et al., 2013; Steele et al., 2013). The microbiota of traditional Protected Designation of Origin (PDO) raw milk cheeses depends on the microbial community, which naturally arises from raw milk and natural whey culture, and from the environment, and contributes to specific intense flavor of raw milk cheeses (Gatti et al., 2014; Neviani et al., 2013; Ordiales et al., 2013). However, in cheese manufacturing, there is a continual need to modulate cheese flavor via the addition of selected new strains with aroma potential, particularly in the case of cheeses made from pasteurized milk. For example, there is currently a demand to diversify the rather mild flavor of some semi-hard cheeses. Therefore, efficient aroma screening approaches are required to evaluate diverse spe- cies of microorganisms. Most studies have targeted a few groups of bacteria, mainly lactic acid bacteria (LAB) such as Lactobacillus, Lactococcus, and Leuconostoc, or propionibacteria (De Bok et al., * Corresponding author. INRA, UMR1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France. Tel.: þ33 223 485 337; fax: þ33 223 485 350. ** Corresponding author. Present address: Department of Dairy Science, Faculty of Agriculture University of Zagreb, Sveto simunska 25, 10 000 Zagreb, Croatia. Tel.: þ385 1239 3646. E-mail addresses: tpogacic@agr.hr (T. Poga ci c), anne.thierry@rennes.inra.fr (A. Thierry). Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm http://dx.doi.org/10.1016/j.fm.2014.07.018 0740-0020/© 2014 Elsevier Ltd. All rights reserved. Food Microbiology 46 (2015) 145e153