Identification of a Bacteriocinogenic Lactic Acid Bacteria Isolated from Raw Cow's Milk and Partial Characterization of its Antilisterial Bacteriocin

Introduction: There is a demand for safe, healthy and minimally processed foods, and this stimulates the search for new antimicrobial agents, such as the bacteriocins of lactic acid bacteria. Bacteriocins are peptides ribosomally synthesized by bacteria and can be degraded by digestive enzymes, representing a potentially safe alternative to classical food preservatives.

Purpose: To study a milk isolate bacteriocin-producing lactic acid bacterium strain (LAB 126) by phenotypical and molecular identification and partial physico-chemical characterization of the antimicrobial peptide produced.

Methods: LAB 126 was isolated in Brazil from raw cow’s milk and it was identified at genus level by Gram stain, catalase test, gas production from glucose, growth in regular MRS broth at 10 and 45 °C, and in 2, 4 and 6.5% NaCl MRS broth. Molecular identification by sequencing of 16S rDNA is under progress. Antagonistic activity was tested against Listeria monocytogenes and other food related bacteria, using the spot-on-the-lawn agar assay. A modified well-diffusion agar assay was used to evaluate the sensitivity of the inhibitor to proteases (type XIV from Streptomyces griseus, proteinase K and chymotrypsin). To exclude bacterial inhibition due to acid production, filter-sterilized neutralized supernatant from overnight LAB 126 MRS broth culture was spotted (10µl) on BHI agar plate, previously overlaid with soft BHI agar seeded with the indicator strain L. monocytogenes (ca. 10⁵ CFU/ml). The bacteriocin produced by LAB 126 was purified from a neutralized cell-free supernatant of a BHI broth culture grown at 25 °C/24 h (crude extract) using Amberlite^® XAD-16 resin (Sigma-Aldrich, EUA), followed by solid phase extraction – SPE (HF Mega Bond Elut C18 10 g-60 ml, Varian, EUA). Extracts containing bacteriocin were analysed by tricine SDS-PAGE and antimicrobial activity in extracts was measured by critical dilution assay using L. monocytogenes as indicator strain and expressed as arbitrary units per ml (AU/ml).

Results: LAB 126 was identified as Lactococcus sp. by phenotypical tests and produced an inhibitory compound susceptible to degradation by proteases, indicating it was a bacteriocin. The bacteriocin was active against L. monocytogenes, Listeria innocua and other lactic acid bacteria tested. Crude extract from BHI culture of Lactococcus sp. 126 presented an activity of 6,400 AU/ml against L. monocytogenes. The yield of the partial purification carried out with XAD-SPE was calculated as 27%, by comparing total activities from crude and final extracts. Results from SDS-PAGE assays showed the molecular weight of the bacteriocin is in the range of 3.5 to 8.5 kDa.

Significance: The isolation of bacteriocin-producing LAB and the characterization of novel bacteriocins can contribute for improving food safety by providing new alternatives for food biopreservation approaches.