International Journal of Food Microbiology 185 (2014) 41–50 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro Identification of beer-spoilage bacteria using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry☆ Anneleen D. Wieme a,b, Freek Spitaels b, Maarten Aerts b, Katrien De Bruyne c, Anita Van Landschoot a,PeterVandammeb,⁎ a Laboratory of Biochemistry and Brewing, Faculty of Bioscience Engineering, Ghent University, Valentin Vaerwyckweg 1, B-9000 Ghent, Belgium b Laboratory of Microbiology, Faculty of Sciences, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium c Applied Maths N.V., Keistraat 120, B-9830 Sint-Martens-Latem, Belgium article info abstract Article history: Applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) Received 10 January 2014 for identification of beer-spoilage bacteria was examined. To achieve this, an extensive identification database Received in revised form 22 April 2014 was constructed comprising more than 4200 mass spectra, including biological and technical replicates derived Accepted 4 May 2014 from 273 acetic acid bacteria (AAB) and lactic acid bacteria (LAB), covering a total of 52 species, grown on at least Available online 29 May 2014 three growth media. Sequence analysis of protein coding genes was used to verify aberrant MALDI-TOF MS iden- tification results and confirmed the earlier misidentification of 34 AAB and LAB strains. In total, 348 isolates were Keywords: MALDI-TOF MS collected from culture media inoculated with 14 spoiled beer and brewery samples. Peak-based numerical anal- MLSA ysis of MALDI-TOF MS spectra allowed a straightforward species identification of 327 (94.0%) isolates. The re- Beer-spoilage bacteria maining isolates clustered separately and were assigned through sequence analysis of protein coding genes Identification either to species not known as beer-spoilage bacteria, and thus not present in the database, or to novel AAB spe- Peak-based cluster analysis cies. An alternative, classifier-based approach for the identification of spoilage bacteria was evaluated by combin- fi Classi er-based analysis ing the identification results obtained through peak-based cluster analysis and sequence analysis of protein coding genes as a standard. In total, 263 out of 348 isolates (75.6%) were correctly identified at species level and 24 isolates (6.9%) were misidentified. In addition, the identification results of 50 isolates (14.4%) were con- sidered unreliable, and 11 isolates (3.2%) could not be identified. The present study demonstrated that MALDI- TOF MS is well-suited for the rapid, high-throughput and accurate identification of bacteria isolated from spoiled beer and brewery samples, which makes the technique appropriate for routine microbial quality control in the brewing industry. © 2014 Elsevier B.V. All rights reserved. 1. Introduction most problematic organisms are lactic acid bacteria (LAB) and acetic acid bacteria (AAB) which are the focus of the present study. The brewing pro- Beer is a beverage with good microbiological stability because it con- cess itself is prone to bacterial growth because of the nutrient-rich envi- tains almost no oxygen and nutrients for bacterial growth. In addition, ronment of wort which comprises fermentable sugars and amino acids, low pH, high CO2-content and the presence of ethanol and antibacterial and vitamins produced by the pitching yeast during fermentation hop compounds ensure microbiological stability (Fernandez and (Vaughan et al., 2005). Low cell numbers (i.e., 1–50 CFU/100–250 mL Simpson, 1995; Haakensen et al., 2009; Pittet et al., 2011; Sakamoto sample) of a spoilage bacterium can cause visible turbidity (Steiner and Konings, 2003; Suzuki et al., 2005). Nevertheless, beer spoilage due et al., 2010), acidity and off-flavours (Priest, 2006). At present, the hop- to bacteria is a common problem in the brewing industry and causes im- resistant lactic acid bacteria Lactobacillus backii, Lactobacillus brevis, portant economic losses worldwide (Menz et al., 2010; Priest, 2006). The Lactobacillus buchneri, Lactobacillus coryniformis, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus parabuchneri, Lactobacillus plantarum, Pediococcus claussenii, Pediococcus damnosus and Pediococcus Abbreviations: MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of- flight mass spectrometry; AAB, acetic acid bacteria; LAB, lactic acid bacteria; SSP, summary inopinatus are generally regarded as the most problematic beer- spectral profile; RAPD, random amplified polymorphic DNA; UPGMA, unweighted pair spoilage microorganisms (Bokulich and Bamforth, 2013; Hutzler et al., group method with arithmetic mean. 2013). The prevalence of strictly aerobic spoilage bacteria has decreased ☆ The Genbank accession numbers for sequences generated in this study are KF910090- because of improved process technology resulting in lower oxygen levels KF910095 for the dnaK sequences, KF910096-KF910112 for the rpoB sequences, during filling (Vaughan et al., 2005). However, in aerobic environments, KF910113-KF910156 for the pheS sequences and HG329605 for the groEL sequence. ⁎ Corresponding author. Tel.: +32 9 264 51 13; fax: +32 9 264 50 92. acetic acid bacteria, such as Acetobacter spp. and Gluconobacter spp., can E-mail address: [email protected] (P. Vandamme). prevail and form biofilms in which other spoilage bacteria are protected http://dx.doi.org/10.1016/j.ijfoodmicro.2014.05.003 0168-1605/© 2014 Elsevier B.V. All rights reserved. 42 A.D. Wieme et al. / International Journal of Food Microbiology 185 (2014) 41–50 (Storgårds et al., 2006; Timke et al., 2004). During the last two decades, strains examined proved to be misclassified; therefore, the names of anaerobic Gram negative bacteria like Pectinatus, Megasphaera and such misidentified strains will be written in square brackets below. In Zymophilus species have gained importance as spoilage bacteria in the order to construct the identification database Acetobacter strains were brewing industry (Matoulkova et al., 2012b; Paradh et al., 2011; Priest, grown on five different media: acetic acid medium (AAM) agar 2006; Vaughan et al., 2005). (Lisdiyanti et al., 2001); deoxycholate–mannitol–sorbitol (DMS) medi- In order to anticipate or to prevent further spoilage, it is necessary to um (Guiraud, 1998); GY medium (5.0% D-glucose, 1.0% yeast extract detect and identify detrimental bacteria in a fast and easy manner. Cur- and 1.5% agar); GYAE medium (GY medium supplemented with 1.0% rently, beer-spoilage bacteria are primarily detected by plating on selec- acetic acid and 2.0% ethanol) and YPM medium (0.5% yeast extract, tive isolation media (Fischborn, 2009; Matoulkova et al., 2012a; Suzuki 0.3% bacteriological peptone, 2.5% D-mannitol, and 1.5% agar). et al., 2008; Taskila et al., 2009, 2010), an approach that is time- Gluconobacter, Gluconacetobacter and Komagataeibacter strains were consuming and neither sensitive nor specific(Back, 2005; Juvonen cultured on the same media, with the exception of GYAE medium et al., 2010). Also, subsequent characterization and identification of which was replaced by reinforced AE (RAE) medium (4.0% D-glucose, spoilage bacteria is commonly carried out via phenotypic tests (Gram 1.0% yeast extract, 1.0% acetic acid, 2.0% ethanol, 1.0% bacteriological staining, cell morphology and sugar metabolism), but these tests are peptone, 0.34% Na2HPO4.2H2O and 0.15% citric acid, a layer of 1.0% agar again time-consuming and lack specificity (Back, 2005). Consequently, is topped with a layer of medium containing 2.0% agar) (Sokollek et al., quality control requires lengthy storage of beer, which increases brewing 1998). Lactobacillus and Pediococcus strains were cultured on three dif- costs and demand for storage capacity (Beimfohr and Snaidr, 2004). ferent media: de Man, Rogosa and Sharpe (MRS) medium (Oxoid), Many culture-dependent and culture-independent detection and/or Nachweismedium für bierschädliche Bakterien (NBB) (1:1 lager pilsner identification techniques for beer-spoilage bacteria have been devel- beer:water) medium (Conda Pronadisa) and Raka-Ray medium (Oxoid). oped. These include species-specific oligonucleotide probes (Beimfohr and Snaidr, 2004; Huhtamella et al., 2007; Weber et al., 2008), PCR- 2.2. Sample preparation and MALDI-TOF MS data acquisition based methods (Asano et al., 2008, 2009; Haakensen et al., 2008; Haakensen and Ziola, 2008; Iijima et al., 2008; Juvonen et al., 2008; After resuscitating lyophilized and cryopreserved cultures, strains Pittet et al., 2010), random amplified polymorphic DNA (RAPD) finger- were subcultured twice on each growth medium and cell extracts were printing (Fujii et al., 2005; Hayashi et al., 2001), ribotyping (Barney prepared from the subsequent 3rd, 4th and the 5th generation. Hence, et al., 2001; Koivula et al., 2006; Motoyama et al., 1998; Takeuchi variation induced during the adaptation to the growth medium was ex- et al., 2005), sodium dodecyl sulphate–polyacrylamide gel electropho- cluded and three biological replicates (i.e., three generations) were in- resis of whole-cell proteins (Dowhanick et al., 1990; Satokari et al., cluded. Five milligrams of wet cells taken from 1 to 10 single colonies 2000; Yansanjav et al., 2004) and the use of monoclonal antibodies was suspended into 300 μL Milli-Q water after which 900 μL pure ethanol (March et al., 2005;