Monitoring of the multiple bacteriocin production by Enterococcus faecium NKR-5-3 through a developed liquid chromatography and mass spectrometry-based quantification system Rodney H. Perez, 1 Kohei Himeno, 1 Naoki Ishibashi, 1 Yoshimitsu Masuda, 1 Takeshi Zendo, 1 Koji Fujita, 1 Pongtep Wilaipun, 2 Vichien Leelawatcharamas, 3 Jiro Nakayama, 1 and Kenji Sonomoto 1 , 4, * Laboratory of Microbial Technology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School,Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, 1 Department of Fishery Products, Faculty of Fisheries, Kasetsart University, Chatuchak, Bangkok 10900, Thailand, 2 Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Chatuchak, Bangkok 10900, Thailand, 3 and Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 4 Received 24 April 2012; accepted 5 June 2012 Available online 3 July 2012 Enterococcus faecium NKR-5-3 produces four antimicrobial peptides referred here as enterocins NKR-5-3A, B, C and D. A two-step electrospray ionization-liquid chromatography and mass spectrometry (ESI-LC/MS)-based quantification system was developed to monitor its multiple bacteriocin production profiles, which is essential in understanding the complex production regulation mechanism of strain NKR-5-3. The developed ESI-LC/MS-based quantification system can easily monitor the multiple bacteriocin production of this strain. Using the developed system, the production of enterocin NKR-5-3B was found to be not as variable as those of the other enterocins in different cultivation media. Production of enterocin NKR-5-3B was also found to have a wider optimum incubation temperature (20e30  C) than enterocins NKR-5-3A, C and D (25  C). Furthermore, at least 2 nM of the bacteriocin-like inducing peptide, enterocin NKR- 5-3D, regulated the production of NKR-5-3 enterocins except enterocin NKR-5-3B. These findings taken together suggest that enterocin NKR-5-3B has an independent production regulation mechanism from the other NKR-5-3 enterocins. The developed system could effectively pin-point the production profiles of the multiple bacteriocins of E. faecium NKR-5-3 under different fermentation conditions. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Lactic acid bacteria; Multiple bacteriocin; Bacteriocin quantification; Electrospray ionization-liquid chromatography and mass spectrometry (ESI-LC/MS); Induction] Lactic acid bacteria (LAB) are ubiquitous in nature. They exist in various ecological niches including a wide array of fermented food products. LAB have a long history of application in fermented foods because of their beneficial influence on nutritional, organoleptic, and shelf-life on foods (1). Among the beneficial attributes of LAB, its ability to produce antimicrobial peptidesebacteriocins, has attracted particular attention both in food and pharmaceutical industries due to its potential use as natural food preservative and therapeutic antibiotics (2e4). Bacteriocins are ribosomally synthesized antibacterial peptides that possess antagonistic activity toward closely related strains, while its producer cells are immune to their own bacteriocins (1). Unlike common antibiotics, LAB bacteriocins are generally considered food-grade due to its typical association in food fermentation. The U.S. Food and Drug Administration (FDA) classified LAB and its metabolites as generally regarded as safe (GRAS) as human food ingredient (5). This distinction has given bacteriocin a critical advantage over common antibiotics in the legal stand point in its use in food applications. Moreover, the U.S. FDA’s approval of the use of nisin in pasteurized processed cheese spreads on 1988 has established a legal precedent in the U.S. for the use of bacteriocins as food preservative (4). However, in order to materialize the full potential of new bacte- riocins as food preservatives, it is important first to understand the biology of these bacteriocins, and, in particular, to elucidate its structureefunction relationships, production, immunity, regulation and its mode of antimicrobial action (6). Enterococcus faecium NKR-5-3, isolated from a Thai fermented fish e Pla-ra, produces multiple bacteriocins, enterocins NKR-5-3A, Z, B, C and a bacteriocin-like inducing peptide, enterocin NKR-5-3D (7,8). Enterocin NKR-5-3A and Z forms a two-peptide bacteriocin that showed 100% and 95% homologies to the peptides of a known two-peptide bacteriocin brochocin-C (brochocins A and B respec- tively) (9), although enterocin NKR-5-3Z was not detected in the supernatant, its putative structure gene was found in the genome of strain NKR-5-3 (8). Enterocin NKR-5-3B is believed to be a novel bacteriocin, based on the fact that its molecular mass does not resemble to any reported bacteriocin, however its structure has not yet been elucidated since initial attempts to obtain its amino acid sequence by Edman Degradation were unsuccessful, indicating that * Corresponding author at: Laboratory of Microbial Technology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel./fax: þ81 92 642 3019. E-mail address: sonomoto@agr.kyushu-u.ac.jp (K. Sonomoto). www.elsevier.com/locate/jbiosc Journal of Bioscience and Bioengineering VOL. 114 No. 5, 490e496, 2012 1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.06.003