Enzymatic activity of lactic acid bacteria (with antimicrobial properties) isolated from a traditional Spanish cheese Leticia González, Noelia Sacristán, Ricardo Arenas, José M. Fresno, M. Eugenia Tornadijo * Departamento de Higiene y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de León, Campus de Vegazana, 24071 León, Spain article info Article history: Received 13 October 2009 Received in revised form 19 January 2010 Accepted 20 January 2010 Available online 25 January 2010 Keywords: Technological characterization Raw milk cheese Acidifying capacity Lactic acid bacteria Proteolytic activity Esterase abstract Twenty-four strains of lactic acid bacteria (LAB) isolated from a traditional Spanish cheese (Genestoso cheese) were evaluated for their enzymatic activities (acidifying and proteolytic abilities and carboxy- peptidase, aminopeptidase, dipeptidase, caseinolytic and esterase activities), in order to select indige- nous strains of technical interest for the manufacture of cheese. These strains were selected on the basis of their antimicrobial activity relative to five reference strains and were identified as Lactococcus lactis subsp. lactis (thirteen strains), Leuconostoc mesenteroides (two strains), Leuconostoc pseudomesenteroides (one strain), Lactobacillus paracasei (two strains), Lactobacillus plantarum (one strain) and Enterococcus faecalis (five strains). Lactococcus strains were those that showed the greatest degree of acidifying and proteolytic activity. The cell-free extracts (CFE) of L. paracasei exhibited the highest level of aminopeptidase activity. The highest level of caseinolytic activity was shown by the CFE of one strain of L. lactis. High values were also obtained with the CFE of Lactobacillus and of several Leuconostoc. The highest level of dipeptidase activity was found amongst the strains of L. lactis. Carboxypeptidase activity was generally very low or unde- tectable for the majority of strains. The greatest degree of esterolytic activity was detected for Enterococcus. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction The specific characteristics of taste and aroma of craft, or traditionally made, cheese varieties may be attributed to the type of milk, its microbiological quality, the degree of acidification of the curds when moulded, the manufacturing technology or the ripening conditions. If the fact that lactic flora are crucial in liberating the specific compounds involved in the aroma and flavour of cheeses is kept in mind, there is good cause for the interest shown by cheese-makers in the type of starter cultures that must be employed. The use of indigenous lactic cultures, obtained from microorganisms isolated from craft cheeses allows the organoleptic (taste and smell) characteristics of cheese made with pasteurized milk to be brought closer to those of traditional varieties. The enzymatic potential of the strains used in the cheese-making process influences the development of the texture, aroma and flavour characteristics of cheeses. Conse- quently, there is an increasing interest in the design of specific indigenous starter cultures in large-scale manufacturing of traditional-type cheeses, such that they will not change the fundamental properties of the product (Pérez et al., 2003). Proteolysis and lipolysis are biochemical events in flavour development that occur during ripening of most varieties of cheese. There is a lack of detailed information on the peptidolytic enzymes produced by non-starter lactic acid bacteria (NSLAB) (Williams et al., 1998). Moreover, only sparse information is available about the contribution of lactic acid bacteria (LAB) to lipolysis during the ripening of cheeses . LAB strains were isolated from Genestoso cheese, a traditional cheese from the North of Spain. These strains showed antimicrobial activity in a previous study (González et al., 2007) relative to five reference strains: Lactobacillus plantarum CECT 748, Listeria mono- cytogenes CECT 4031, Enterococcus faecalis CECT 481, Clostridium tyrobutyricum CECT 4011 and Staphylococcus aureus CECT 240. The aim of this study was to evaluate the enzymatic activities (acidifying and proteolyitic capacities and aminopepti- dase, dipeptidase, carboxipeptidase, esterase and caseinolytic activities) of twenty-four strains of LAB in order to select strains of technological interest that might be used as starters in the manufacture of cheeses. * Corresponding author. E-mail address: metorr@unileon.es (M. Eugenia Tornadijo). Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm 0740-0020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2010.01.004 Food Microbiology 27 (2010) 592e597