María I. Martínez

Pediocin PA-1, a wide-spectrum bacteriocin from lactic acid bacteria

Referee: Dr. Helen Dodd, Food Safety Science Division, Institute of Food Research, Norwich Research Park, Colney NR4 7UA, Norwich, United Kingdom Pediocin PA-1 is a broad-spectrum lactic acid bacteria bacteriocin that shows a particularly strong activity against Listeria monocytogenes, a foodborne pathogen of special concern among the food industries. This antimicrobial peptide is the most extensively studied class IIa (or pediocin family) bacteriocin, and it has been sufficiently well characterized to be used as a food biopreservative. This review focuses on the progress that have been made in the elucidation of its structure, mode of action, and biosynthesis, and includes an overview of its applications in food systems. The aspects that need further research are also addressed. In the future, protein engineering, genetic engineering and/or chemical synthesis may lead to the development of new antimicrobial peptides with improved properties, based on some features of the pediocin PA-1 molecule.

Heterologous production of bacteriocins by lactic acid bacteria.

Over the last two decades, bacteriocins produced by lactic acid bacteria (LAB) have been the subject of considerable research and industrial interest due to their potential as food biopreservatives. The development of heterologous expression systems for such antimicrobial compounds may offer a number of advantages over native systems, such as facilitating the control of bacteriocin gene expression or achieving higher production levels. In addition, the heterologous production by food-grade LAB offers an attractive method for overcoming some of the adverse situations that may affect the effectiveness of some bacteriocins in food systems. Construction of multibacteriocinogenic strains or acquisition of antimicrobial properties by industrial strains are further objectives that can be achieved through the use of heterologous gene expression systems. The development of new biotechnological tools and recent advances in LAB genetics account for the escalating number of studies dealing with heterologous production of bacteriocins by such hosts. This paper reviews the literature published on the subject and compares the different experimental strategies that have been used up to the present for this purpose.

Antibodies to a synthetic 1-9-N-terminal amino acid fragment of mature pediocin PA-1: sensitivity and specificity for pediocin PA-1 and cross-reactivity against Class IIa bacteriocins

Polyclonal antibodies specific for pediocin PA-1 (PedA1) were generated by immunization of rabbits with a chemically synthesized 1-9-N-terminal amino acid fragment of this bacteriocin (PH1) conjugated to the carrier protein keyhole limpet haemocyanin (KLH). The PH1 fragment holds a highly conserved amino acid sequence with closely related Class IIa bacteriocins. The sensitivity and specificity of the PH1-KLH-generated rabbit polyclonal antibodies were evaluated by the development of various ELISAs, such as a non-competitive indirect ELISA (NCI-ELISA), a competitive indirect ELISA (CI-ELISA), a competitive direct ELISA (CD-ELISA) and a sandwich ELISA (S-ELISA), and by protein slot-blotting and Western blotting. NCI- and CI-ELISA were valuable for detecting the existence of PedA1-specific antibodies in the sera of immunized rabbits. The limit of detection of PedA1 in MRS medium was found to be 0.5 microg ml(-1) in NCI-ELISA, while CI-ELISA on plates coated with purified PedA1 increased the affinity of the PH1-KLH-generated antibodies for PedA1; the limit of detection of PedA1 was less than 0.01 microg ml(-1) and 50% binding inhibition was achieved with 0.1 microg PedA1 ml(-1). Similarly, the limits of detection of PedA1 in MRS medium were found to be 5 microg ml(-1) by protein slot-blotting and 0.01 microg ml(-1) by Western blotting. Most importantly, PH1-KLH-generated polyclonal antibodies detected the presence of PedA1 in the supernatants of the producing strains of Pediococcus acidilactici 347, Z102, A172, X13 and P20, with no reactivity or negligible immunoreactivity with the supernatants of other lactic acid bacteria producing or not producing closely related or different bacteriocins. The approaches taken for the selection of the bacteriocin peptide fragment, the generation of antibodies and the development of immunoassays could prove useful for the generation and evaluation of antibodies of adequate specificity for other bacteriocins of interest in the food industry.

Research note: unsuitability of the MRS medium for the screening of hydrogen peroxide-producing lactic acid bacteria.

The effect of MRS broth on the stability of hydrogen peroxide (H2O2) has been studied. Known concentrations (1–100 μg ml−1) of H2O2 were prepared in distilled water, phosphate buffer (pH 7·0) and MRS broth (pH 6·2 and 3·9). H2O2 was very stable in aqueous and buffer solutions but it was rapidly degraded in MRS broth (pH 3·9). The presence of H2O2 in MRS broth (pH 6·2) could not be detected.

Generation of polyclonal antibodies against a chemically synthesized N‐terminal fragment of the bacteriocin pediocin PA‐1

Six mice were immunized intraperitoneally (i.p.) with a chemically synthesized 9‐mer fragment (PH1) designed from the N‐terminal part of the bacteriocin pediocin PA‐1 and conjugated to keyhole limpet haemocyanin (KLH). After three doses of the immunogen had been administered, serum‐specific antibodies were detected by a competitive direct ELISA. Myeloma cells were injected i.p. into mice in order to obtain ascites polyclonal antibodies. Although four mice developed ascites, only mouse 2 had detectable specific antibodies in the ascites fluid. The serum and ascites antibodies were specific for PH1 but they did not recognize the whole pediocin PA‐1 molecule. This is the first attempt to generate antibodies against bacteriocins with a chemically synthesized oligopeptide as immunogen. This approach still remains attractive for detection, quantification, mode of action studies and purification of bacteriocins, especially those for which the purification process is difficult or inefficient at present.

Use of Genetic and Immunological Probes for Pediocin PA-1 Gene Detection and Quantification of Bacteriocin Production in Pediococcus acidilactici Strains of Meat Origin

PCR amplification analysis with pediocin PA-1 (pedA1) specific primers, a gene probe covering the pedA1 structural gene, and an immunological probe based on anti-peptide antibodies with predetermined specificity for pedA1, have been evaluated for pedA1 structural gene detection and quantification of bacteriocin production in Pediococcus acidilactici strains of meat origin. PCR products were obtained from colonies and purified plasmid DNA. The gene probe was used in dot-blot and colony-blot hybridization analysis. The production of pedA1 was evaluated by a non-competitive indirect ELISA (NCI-ELISA), a competive indirect ELISA (CI-ELISA), a competitive direct ELISA (CD-ELISA), and by immunoblotting. The approaches taken in the generation and performance of the reported probes could be useful for the rapid detection and identification of other bacteriocin-producing lactic acid bacteria (LAB) from many substrates and reservoirs and for quantification of bacteriocin production in food and environmental isolates and heterologous hosts.