Characterization of two bacteriocins produced by Pediococcus acidilactici isolated from “ Alheira ” , a fermented sausage uced in Portugal traditionally prod a , 1 b , 2 b , 2 a , 1

Lactic acid bacteria were isolated from “Alheira” sausages that have been sampled from different regions in Portugal. The sausages were produced according to different recipes and with traditional starter cultures. Two isolates (HA-6111-2 and HA-5692-3) from different sausages were identified as strains of Pediococcus acidilactici. Each strain produces a bacteriocin, designated as bacHA-6111-2 and bacHA-5692-3. Both bacteriocins are produced at low levels after 18 h of growth in MRS broth (3200 AU/ml against Enterococcus faecium HKLHS and 1600 AU/ml against Listeria innocua N27). BacHA-6111-2 and bacHA-5692-3 are between 3.5 kDa and 6.5 kDa in size, as determined by tricine-SDS-PAGE. Complete inactivation or significant reduction in antimicrobial activity was observed after treatment of cell-free supernatants with proteinase K, pronase and trypsin. No change in activity was recorded when treated with catalase. Both bacteriocins are sensitive to treatment with Triton X-114 and Triton X-100, but resistant to Tween 20, Tween 80, SDS, Oxbile, NaCl, urea and EDTA. The bacteriocins remained stable after 2 h at pH 6.0. A decrease in antibacterial activity was recorded after 60 min at 100 °C. After 60 min at 80 °C, 60 °C and 25 °C the antibacterial activity against L. innocua N27 decreased by 25%. Addition of bacHA-6111-2 and bacHA-5692-3 (1600 AU/ml) to a mid-log (5-h-old) culture of L. innocua N27 inhibited growth for 7 h. Addition of the bacteriocins (3200 AU/ml) to a mid-log (5-h-old) culture of E. faecium HKLHS repressed cell growth. The bacteriocins did not adhere to the surface of the producer cells. Both strains contain a 1044 bp DNA fragment corresponding in size to that recorded for pediocin PA-1. Sequencing of the fragments from both bacteriocins revealed homology to large sections of pedA (188 bp), pedB (338 bp) and pedC (524 bp) of pediocin PA-1 and the bacteriocins are considered similar to pediocin PA-1. Fermented meat products are part of the daily diet in rural areas of Portugal and have become very popular in urban centres. “Alheira” is a traditional fermented meat product typical of the Northern regions (Trás-os-Montes) in Portugal. ⁎ Corresponding author. Tel. : +27 21 808 5849; fax: +27 21 808 5846. E-mail addresses: lmtd@sun.ac. za (L.M.T. Dicks), paula@esb.ucp. pt (P. Teixeira). 1 Tel. : +351 225 580 001; fax: +351 225 580 111. 2 Tel. : +27 21 808 5849; fax: +27 21 808 5846. The specific characteristics of the product depend on the raw materials used, the agro-ecosystem of the production area, and the traditional production methods used. “Alheira” is produced from a combination of pork meat, pork lard, poultry, wheat bread and olive oil mixed with salt, garlic and spices. The paste is stuffed in casings made from animal products and is smoked for a maximum of 8 days. “Alheira” contains mainly lactic acid bacteria andMicrococcaceae. Some pathogens, such as Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus are occasionally present (Ferreira et al., 2006). Selected strains of lactic acid bacteria, used as starter cultures, may inhibit spoilage microorganisms by production