Microbial analysis of Malaysian tempeh, and characterization of two bacteriocins produced by isolates of Enterococcus faecium M.R.F. Moreno 1 , J.J. Leisner 2 , L.K. Tee 3 , C. Ley 2 , S. Radu 4 , G. Rusul 3 , M. Vancanneyt 5 and L. De Vuyst 1 1 Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing (IMDO), Department of Applied Biological Sciences, Vrije Universiteit Brussel (VUB), Brussels, Belgium, 2 Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark, 3 Department of Food Science, 4 Department of Biotechnology, Universiti Putra Malaysia, Serdang, Selangor DE, Malayasia, and 5 BCCM LMG Bacteria Collection, Laboratory of Microbiology, Universiteit Gent, Gent, Belgium 2001/41: received 2 April 2001, revised 17 May 2001 and accepted 23 July 2001 M.R.F. MORENO, J.J. LEISNER, L.K. TEE, C. LEY, S. RADU, G. RUSUL, M. VANCANNEYT AND L. DE VUYST. 2002. Aims: Isolation of bacteriocinogenic lactic acid bacteria (LAB) from the Malaysian mould- fermented product tempeh and characterization of the produced bacteriocin(s). Methods and Results: LAB were present in high numbers in ®nal products as well as during processing. Isolates, Enterococcus faecium B1 and E. faecium B2 (E. faecium LMG 19827 and E. faecium LMG 19828, respectively) inhibited Gram-positive indicators, including Listeria monocytogenes. Partially puri®ed bacteriocins showed a proteinaceous nature. Activity was stable after heat-treatment except at alkaline pH values. Both strains displayed a bacteriostatic mode of action. Bacteriocin production was associated with late exponential/early stationary growth. Molecular mass, calculated by SDS-PAGE, was 3á4 kDa for B1 bacteriocin, and 3á4 kDa and 5á8 kDa for B2 bacteriocins. PCR screening of enterocin-coding genes revealed three ampli®ed fragments in total genomic DNA that may correspond with PCR signals for enterocin P, enterocin L50A and enterocin L50B. Both B1 and B2 contained a 42-kb plasmid. No differences in bacteriocinogenic capacity were found between wild type strains and plasmid- cured strains. Conclusions: It was possible to isolate bacteriocinogenic E. faecium active against various Gram-positive bacteria from ®nal products of tempeh. Signi®cance and Impact of the Study: A ®rst step in applying biopreservation to fermented South-east Asian foods is to obtain bacteriocinogenic LAB from this source. Such isolates may also be used for biopreservation of mould-fermented foods in general, including various types of mould-ripened cheese. INTRODUCTION Many traditional South-east Asian foods are fermented before consumption and naturally occurring lactic acid bacteria (LAB) are among the micro-organisms that often play an important role in the fermentation process. Tempeh is such a fermented product, which in Malaysia is made typically from soybeans in a cottage industry process (Steinkraus 1996). Traditional manufacture of tempeh includes two major steps, ®rst a soaking process, and secondly a mould fermentation initiated by addition of a mould starter culture (Nout and Rombouts 1990; Steinkraus 1996). It is well known that inhibition of various pathogenic micro-organisms in the mould fermentation step can be obtained by an ef®cient acid production by indigenous LAB during the soaking process, as shown for laboratory-made tempeh (Nout et al. 1987, 1988). Correspondence to: J.J. Leisner, Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Stigbùjlen 4, DK-1870 Frederiksberg C, Denmark (e-mail: jjl@kvl.dk). ã 2002 The Society for Applied Microbiology Journal of Applied Microbiology 2002, 92, 147±157