Antibacterial activity produced by Lactococcus lactis ssp. lactis CECT 539 in different culture media N. Pérez Guerra, R. Agregán Pérez, E. Alonso González and A. Torrado Agrasar Nutrition and Bromatology Group, Department of Analytical and Food Chemistry, Food Science and Technology Faculty, Ourense Campus, University of Vigo, As Lagoas s/n, E-32004, Ourense, Spain. E-mail: nelsonpg@uvigo.es. Phone: +34 988 387053. Fax: +34 988 387001 In this work, the antagonistic activity of a cell-free supernatant (CFS) from a culture of Lactococcus lactis subsp. lactis CECT 539 was firstly characterised by determining its antibacterial spectrum and potency against related lactic acid and pathogenic bacteria by employing the agar well-diffusion method. The antagonistic activity produced by the nisin- producing strain was similar to those obtained with a solution of 1 g/L of Nisaplin and a CFS from a culture of Pediococcus acidilactici NRRL B-5624, a pediocin producing-strain. Subsequently, a photometric bioassay in culture tubes was used to quantify the individual antibacterial activity against Carnobacterium piscicola CECT 4020, of different metabolites (nisin, lactic acid, acetic acid, butane-2,3-diol and ethanol) present in CFS obtained from three different realkalised fed-batch cultures of L. lactis CECT 539. In this study, nisin was found to be the most potent antibacterial product while butane-2,3-diol did not exhibited a significant inhibitory effect. Keywords Nisin; Lactoccoccus lactis; Pediococcus acidilactici; antibacterial products; cell-free supernatant; inhibition. 1. Introduction Chemical preservatives (e.g. nitrite, nitrate and sodium benzoate, SO 2 ) and heat treatment have been usually used to control the growth of undesirable microorganisms in foods. However, the serious risk that some of these preservatives have to the health and safety of consumers, as well as the growing consumer rejection of heat-treated foods have focused the attention on the production of natural (less-processed) foods. This approach includes the vacuum packaging and storage at low temperatures, among other alternatives. However, these foods may contain pathogenic microorganisms capable of growing even under these conditions [1–3]. The use of ionizing radiation can destroy these organisms, but this method often causes some flavour changes in foods and, in addition, radiation does not protect food from contamination after treatment [4]. One solution to this dilemma could be the use of antimicrobial metabolites (e.g. bacteriocins) produced by lactic acid bacteria (LAB). This procedure may reduce the addition of chemical preservatives, as well as the thermal processing of low-acid foods with high water activity, resulting in less-processed food products that preserve their initial characteristics, especially those related with the texture and flavour [5, 6]. Bacteriocins are ribosomally synthesised antibacterial peptides with a narrow (against related species) or a broad (against unrelated species) antibacterial activity spectrum [7, 8]. Combination of bacteriocins with other stress-inducing processes, including freezing, acid treatment, chelating agents, high hydrostatic pressure and electroporation, has found to be a way to control the growth of Gram negative or resistant Gram-positive bacteria [9, 10]. Nisin, produced by Lactococcus lactis, has been the most studied bacteriocin to date and has been authorised for food preservation in different countries [11, 12]. This bacteriocin exhibits antibacterial activity towards different important spoilage and pathogenic microorganisms such as Listeria monocytogenes and Bacillus cereus and also the outgrowth of spores of bacilli and clostridia [13–15]. During the fermentation process, nisin and other antimicrobial substances such as lactic, acetic and propionic acids accumulate in the culture media. Therefore, the antibacterial activity of cell-free supernatants (CFS) from these culture media towards any indicator strain could be due to the synergistic effect of nisin and the other antimicrobial compounds [16]. For this reason, to quantify the individual antibacterial activity and the concentration of nisin in the CFS is necessary to determine the individual activity of each antimicrobial compound. In the present work, the antibacterial spectrum and potency of a cell-free supernatant obtained from a Lactococcus lactis CECT 539 batch culture in MRS broth was compared to those of the cell-free supernatant from a Pediocccus acidilactici NRRL B-5624 (a pediocin-producing strain) culture in MRS broth and a solution of 1 g/L of Nisaplin. Subsequently, the contribution to the overall antibacterial activity of nisin and other antimicrobial products (lactic acid, acetic acid, ethanol and butane-2,3-diol) produced in three different realkalised fed-batch cultures of strain CECT 539 [16], was estimated by using response surface methodology and empirical modelling. Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.) © FORMATEX 2013 ____________________________________________________________________________________________ 815