Polyclonal antibodies against the bacteriocin propionicin PLG-1 were produced in rabbits at high titer (256,000 to 512,000, as determined by indirect enzyme-linked immunosorbent assay [ELISA]). Anti-PLG-1 antiserum neutralized the antimicrobial activity of PLG-1 preparations in a well diffusion assay. Cross-reacting protein was detected using an indirect ELISA of the culture supernatant from a fed-batch fermentation of the producer strain Propionibacterium thoenii P127 within the first 24 h of incubation, but bacteriocin activity was not detected in the same culture until 217 h of incubation. Culture supernatants from 156 strains of classical dairy propionibacteria were tested by indirect ELISA at 5 and 12 days of incubation for production of cross-reacting protein and by well diffusion assay for bacteriocin activity. Cross-reacting protein was detected in 52 strains: all of the tested strains of P. thoenii, most of the strains of Propionibacterium jensenii, and a minority of the Propionibacterium acidipropionici and Propionibacterium freudenreichii strains. Of these 52 strains, only 4 strains of P. thoenii showed bacteriocin activity in a well diffusion assay. Eight bacteriocin-negative mutants of strain P127 were negative in both ELISA and well diffusion assays. Western blot analysis showed that three protein bands bound anti-PLG-1 antibodies in culture supernatants: a 9.1-kDa band that is assumed to be the PLG-1 monomer and 16.2-and 27.5-kDa bands that may be precursors, multimers, or complexes of PLG-1.The best characterized bacteriocin from the dairy propionibacteria is propionicin PLG-1, which is produced by Propionibacterium thoenii P127 (14). This bacteriocin is moderately heat-stable, sensitive to proteolytic enzymes, and stable at pH 3 to 9 (15). It contains 99 amino acid residues, has a molecular mass of 9,328 Da, and seems unrelated to other bacteriocins from lactic acid bacteria based on a comparison of its N-terminal amino acid sequence to others in the SWISS-PROT data bank (21). Propionicin PLG-1 is rapidly bactericidal against other dairy propionibacteria and lactic acid bacteria (14, 16). Methods for PLG-1 production and purification have been optimized (10,21).The use of immunological methods in bacteriocin research has been limited. Recent attempts to produce bacteriocin-specific antibodies have had varied results (1,3,4,12,17,18). The relatively low molecular mass (Ͻ5,000 Da) of many bacteriocins makes them poorly or nonimmunogenic (22); conjugation of small bacteriocins to carrier proteins can improve their immunogenicity. Immunological techniques based on immunoblotting and enzyme-linked immunosorbent assays (ELISA) can be useful in investigating details of bacteriocin production, structure, and function (17, 18). Production of bacteriocinspecific antibodies has enabled development of sensitive immunoassays for nisin (7,22) and pediocins (1).The objective of this work was to produce polyclonal antibodies against propionicin PLG-1 to enable immunoassay development. These assays were used for...
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