Characterization of psychrotrophic bacterial communities in modified atmosphere-packed meat with terminal restriction fragment length polymorphism T.T. Nieminen a, ⁎, E. Vihavainen a , A. Paloranta a , J. Lehto a , L. Paulin b , P. Auvinen b , M. Solismaa a , K.J. Björkroth a a Department of Food Hygiene and Environmental Health, PO Box 66, FI-00014, University of Helsinki, Finland b DNA Sequencing and Genomics Laboratory, PO Box 56, FI-00014, University of Helsinki, Finland abstract article info Article history: Received 13 July 2010 Received in revised form 30 September 2010 Accepted 17 October 2010 Keywords: Meat Modified atmosphere Spoilage Lactic acid bacteria Polymorphism Restriction fragment length Characterization of psychrotrophic lactic acid bacteria (LAB) and Brochothrix thermosphacta communities is needed to understand the microbial ecology of spoilage of modified atmosphere-packed (MAP) meats. To overcome the limitations of the currently used methods for the characterization of psychrotrophic bacterial communities in meat, we developed a culture-independent, 16S rRNA gene-targeted terminal restriction fragment length polymorphism (T-RFLP) method. An identification library consisting of 100 Gram-positive and 30 Gram-negative meat-associated bacterial strains was set up to identify the terminal restriction fragments derived from the communities. The taxonomic resolution level of the T-RFLP method was in between genus and species within the investigated LAB strains and within family and genus within the investigated Gram-negative strains. The established library was applied to identify the members of bacterial communities in MAP minced meat at the end of the shelf life. The T-RFLP results and plate counts on Man- Rogosa-Sharpe, Violet Red Bile Glucose, and Streptomycin sulfate thallium acetate actidione agars indicated that LAB and B. thermosphacta predominated in meat. The bacterial taxa associated with the T-RFLP results were compared to those identified among plate-grown LAB isolates by numerical ribopattern analysis. Both methods agreed that Leuconostoc spp. and Carnobacterium spp. prevailed in the LAB community in minced meat followed by Lactobacillus algidus, Lactococcus spp. and Weissella spp. Colony identification revealed that Leuconostoc gasicomitatum, L. gelidum, Carnobacterium divergens and C. maltaromaticum were the predominant LAB species. The T-RFLP results were shown to correlate with viable counts of Leuconostoc spp. and B. thermosphacta. The T-RFLP method was found to be a useful tool enabling rapid and high- throughput characterization of psychrotrophic bacteria prevailing in MAP meat. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Psychrotrophic lactic acid bacteria (LAB) and Brochothrix thermo- sphacta are typical spoilage organisms of various cold-stored, modified atmosphere-packed (MAP) raw meats due to their ability to grow at low temperatures under CO 2 -rich atmosphere (Borch et al., 1996; Samelis, 2006) . The LAB most often detected in meat belong to the genera Carnobacterium, Lactobacillus, Leuconostoc and Weissella (Nychas et al., 2007). LAB show varying potential to cause spoilage (Laursen et al., 2006; Samelis 2006; Schillinger et al., 2006; Schirmer et al., 2009), e.g. by discoloration (Vihavainen and Björkroth, 2007), slime production (Korkeala et al., 1988) or production of off-odorous compounds (Egan, 1983). Hence, characterization of LAB communi- ties is needed to understand the microbial spoilage of MAP meat. Currently, psychrotrophic bacterial communities in meat are typically characterized by identification of colonies plated on solid medium. The method provides a quantitative estimate of the cultivable community structure and enables high taxonomic resolu- tion of the isolates chosen for identification. However, colony identification is time-consuming, expensive and laborious and thus unsuitable for characterization of large number of communities. Tens of colonies typically chosen for identifications may not be a representative sample of the community. Furthermore, selectivity of growth medium may distort the results. To overcome the limitations, psychrotrophic bacterial communities in meat have been characterized culture independently using 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE) (Bright- well et al., 2009; Cocolin et al., 2007; Ercolini et al., 2006). The method requires simple equipment but suffers from a number of technical drawbacks, including gel-to-gel variation, labor intensity and co- International Journal of Food Microbiology 144 (2011) 360–366 Abbreviations: DGGE, Denaturing gradient gel electrophoresis; LAB, Lactic acid bacteria; MAP, Modified atmosphere-packed; MRS, Man-Rogosa-Sharpe; STAA, Streptomycin sulfate thallium acetate actidione; T-RFLP, Terminal restriction fragment length polymorphism; TRF, Terminal restriction fragment; VRBG, Violet Red Bile Glucose. ⁎ Corresponding author. Tel.: +358 9 191 57458; fax: +358 9 191 57101. E-mail address: timo.t.nieminen@helsinki.fi (T.T. Nieminen). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.10.018 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro