Fed-batch enhancement of jenseniin G, a bacteriocin produced by Propionibacterium thoenii (jensenii) P126.

Jenseniin G is an antibotulinal bacteriocin (antimicrobial peptide) produced by the dairy culture, Propionibacterium thoenii (jensenii) P126. Activity from crude jenseniin G preparations isolated from static cultures was not detected in unconcentrated cultures before day 7. Activity was not detectable until the spent culture medium was concentrated 50-100 fold. Maximum activity (21 AU/ml) was observed in concentrated supernates at day 9. The production of bacteriocin jenseniin G was increased in fed-batch fermentations for 14 d at 32 degrees C in sodium lactate broth (NLB) containing 1.2% sodium lactate. Viable cell numbers in static and fed-batch cultures reached 1.2 and 5.4x10(9), respectively, during late exponential/early stationary phase (3 d). Concentrations of viable cells in fed-batch fermentations remained constant throughout the incubation period; those in static fermentations dropped after day 6 to a final concentration of 1.5x10(7). During fed-batch fermentations, jenseniin G was directly detected at day 5. In fed-batch fermentations, maximum activity in concentrated supernates (384 AU/ml) on day 12 provided an 18 fold increase over yields in static cultures and in fermenter without pH control, and 2.4 fold increase over yields in fermenter at controlled pH at 6.4. Fed-batch fermentation shows promise as a method to obtain high concentrations of industrially significant bacteriocins from dairy propionibacteria.

Hydroxyapatite composites designed for antibiotic drug delivery and bone reconstruction: a caprine model.

The purpose of this study was to investigate the feasibility of fabricating a drug delivery system that serves a dual function, to eradicate infection as well as to provide a scaffold for osseous integration. Two porous composite systems were fabricated using hydroxyapatite (HA) as the carrier for gentamicin sulfate (GS), an aminoglycoside antibiotic. Structural and mechanical properties of porous HA-GS composites were characterized and the in vitro release behavior of GS from fabricated composites was monitored and compared with the well-known polymethylmethacrylate (PMMA)-GS delivery system. Scanning electron microscopy revealed a macropore range of 150 to 200 microm and 100 to 190 microm for the sintered and unsintered HA-GS composites, respectively. The effect of GS inclusion on bone apposition and ingrowth was assessed using a caprine model. Plugs 10 mm x 6 mm of cylindrical tricalcium phosphate, sintered HA, and sintered HA-GS were implanted in the femoral diaphysis for a period of 6 weeks. Data collected during the in vitro study showed that GS can successfully be incorporated into HA and used as a drug delivery system to eradicate Staphylococcus aureus. In vivo data confirmed that the inclusion of GS within a ceramic matrix did not stimulate or inhibit osteointegration or bone apposition. In conclusion, the fabricated sintered HA-GS composite may be beneficial in the treatment of infected osseous sites as a drug delivery system.

Growth and survival of Listeria monocytogenes in vacuum-packaged ground beef inoculated with Lactobacillus alimentarius FloraCarn L-2.

A culture of the psychotrophic strain FloraCarn L-2 of Lactobacillus alimentarius was added to ground beef (pH 5.4) inoculated with two isolates of Listeria monocytogenes able to grow in refrigerated ground beef. The ground beef was vacuum-packaged and stored for 9 weeks at 4 degrees C. Populations of inoculated L. monocytogenes initially were 6.3 to 6.4 log10 CFU/g and increased to 7.4 log10 CFU/g in ground beef with no added lactobacilli. Addition of L. alimentarius L-2 or its antibiotic-resistant mutant SRL-2 reduced the final populations of L. monocytogenes to 4.3 or 4.1 log10 CFU/g, respectively. L. alimentarius L-2 did not produce bacteriocins or hydrogen peroxide in vitro. The antilisterial effect of L. alimentarius observed in laboratory media and ground beef is attributed to lactic acid (ca. 50 mM) produced by growing cultures.

Identification and purification of a protein that induces production of the Lactobacillus acidophilus bacteriocin lactacin B.

Lactacin B is a heat-stable bacteriocin produced by Lactobacillus acidophilus N2 that is active against closely related lactobacilli, including Lactobacillus delbrueckii subsp. lactis (formerly Lactobacillus leichmannii) ATCC 4797. Pure producer cultures propagated in MRS broth (initial pH 6.5) contain no lactacin B; it is detected only in cultures maintained at pH 5.0 to 6.0 and produced optimally at pH 6.0 S. F. Barefoot and T. R. Klaenhammer, Antimicrob. Agents Chemother. 26:328-334, 1984). Associative growth of producer and indicator, L. delbrueckii subsp. lactis ATCC 4797, resulted in production of an inhibitor identical to lactacin B. Associative growth increased lactacin B production from nondetectable levels (< 100 activity units [AU]/ml) to between 3,200 and 6,400 AU/ml in MRS broth (initial pH 6.5) and resulted in early but equal production of lactacin B (approximately 25,600 AU/ml) in broth maintained at pH 6.0. Indicator cells, but not spent culture filtrates, induced lactacin B production. Indicator cells disrupted by a French pressure cell yielded cell-free filtrates containing inducing activity. Chromatofocusing and gel filtration high-performance liquid chromatography of cell-free filtrates yielded a protein with a pI of 4.1 and a molecular size of approximately 58 kDa that induced lactacin B production. Analytical isoelectric focusing yielded a single protein band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels contained a 28-kDa protein suggesting a two-subunit structure. Protein sequencing identified an N-terminal serine and 18 additional amino acids. To our knowledge, there are not previous descriptions of proteins that induce bacteriocin production in lactic acid bacteria.

Antibiosis revisited: bacteriocins produced by dairy starter cultures.

Well before the existence of starter bacteria was recognized, their activities were instrumental in preserving dairy foods. During growth in fermented products, dairy starters, including lactobacilli, lactococci, leuconostocs, streptococci, and propionibacteria, produce inhibitory metabolites. Inhibitors include broad-spectrum antagonists, organic acids, diacetyl, and hydrogen peroxide. Some starters also produce bacteriocins or bactericidal proteins active against species that usually are related closely to the producer culture. Several bacteriocins have been biochemically and genetically characterized. Evaluating properties of the Lactobacillus acidophilus bacteriocin, lactacin B, led to a new purification protocol. Purified lactacin B migrates in SDS-PAGE as a single 8100-Da band with inhibitory activity after Coomassie blue staining. Production of lactacin B is enhanced by cultivation of the producer with the sensitive indicator, Lactobacillus delbrueckii ssp. lactis 4797; understanding this interaction may increase knowledge of production of bacteriocins in heterogeneous cultures. Bacteriocins have been recently identified in dairy propionibacteria. Jenseniin G, a bacteriocin produced by Propionibacterium jensenii P126, has narrow activity; propionicin PLG-1 produced by Propionibacterium thoenii P127 inhibits propionibacteria, some fungi, Campylobacter jejuni, and additional pathogens. Better understanding of these antagonists may lead to targeted biocontrol of spoilage flora and foodborne pathogens.

Effects of the flrA regulatory locus on biosynthesis and excretion of amino acids in Escherichia coli B/r.

We have partially characterized phenotypic effects of an unusual amino acid regulatory locus, flrA, in E. coli B/r that alters the expression of the ilv and leu operons [Kline, E.L (1972) J. Bacteriol. 110:1127-1134]. This study demonstrated that a primary effect of the flrA7 mutation in haploid strains was overproduction of valine. In diploid strains (FflrA+/flrA7) this mutation resulted in excretion of valine, isoleucine, leucine, aspartate, threonine, glutamate, histidine and lysine. Increased excretion of amino acids by mutant strains might be explained by a membrane alteration or by flrA encoding a positive regulatory factor that affects the ilv operon and has pleiotropic effects on other amino acid operons.

Jenseniin G, a heat-stable bacteriocin produced by Propionibacterium jensenii P126.

The genus Propionibacterium includes cutaneous species typically found on human skin and the dairy or classical species (Propionibacterium freudenreichii, P. jensenii, P. thoenii, and P. acidipropionici) used industrially for the production of Swiss cheese and propionic acid. Grinstead (1989, M.S. thesis, Iowa State University, Ames) has previously observed that some dairy propionibacteria inhibit other species in the classical grouping. We further investigated the inhibitor(s) produced by P. jensenii P126 (ATCC 4872). An antagonist(s) from anaerobic agar cultures of P126 strongly inhibited two closely related strains of propionibacteria, P. acidipropionici P5 and P. jensenii P54, and Lactobacillus bulgaricus NCDO 1489, Lactobacillus delbrueckii subsp. lactis ATCC 4797, Lactococcus cremoris NCDO 799, and Lactococcus lactis subsp. lactis C2. The inhibitor, designated jenseniin G, was active at pH 7.0; inactivated by treatment with pronase E, proteinase K, and type 14 protease; insensitive to catalase; and stable to freezing, cold storage (4 degrees C, 3 days), and heat (100 degrees C, 15 min). Classification of the inhibitor as a bacteriocin is supported by its proteinaceous nature and its bactericidal activity against L. delbrueckii subsp. lactis ATCC 4797. The lack of detectable plasmids suggests a chromosomal location for the determinant(s) of jenseniin G.

Purification and characterization of the Lactobacillus acidophilus bacteriocin lactacin B.

Parameters for production and purification of a bacteriocin produced by Lactobacillus acidophilus N2 are described. Production of lactacin B was pH dependent, with maximum activity detected in broth cultures maintained at pH 6. Lactacin B was purified by ion-exchange chromatography, ultrafiltration, and successive gel filtrations in the presence of 8 M urea and then 0.1% sodium dodecyl sulfate. The molecular weight of lactacin B was ca. 6,000 to 6,500, and the purified compound showed maximum absorbance at 211 nm. The activity of purified lactacin B was bactericidal to sensitive cells and restricted to members of the family Lactobacilliaceae, L. leichmannii, L. bulgaricus, L. helveticus, and L. lactis. Characteristics identified for lactacin B indicated that it was a peptide and confirmed its identity as a bacteriocin.

Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus.

A total of 52 strains of Lactobacillus acidophilus were examined for production of bacteriocins. A majority (63%) demonstrated inhibitory activity against all members of a four-species grouping of Lactobacillus leichmannii, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus lactis. Four L. acidophilus strains with this activity also inhibited Streptococcus faecalis and Lactobacillus fermentum, suggesting a second system of antagonism. Under conditions eliminating the effects of organic acids and hydrogen peroxide, no inhibition of other gram-positive or -negative genera was demonstrated by L. acidophilus. The agent produced by L. acidophilus N2 and responsible for inhibition of L. leichmannii, L. bulgaricus, L. helveticus, and L. lactis was investigated. Ultrafiltration studies indicated a molecular weight of approximately 100,000 for the crude inhibitor. The agent was sensitive to proteolytic enzymes and retained full activity after 60 min at 100 degrees C (pH 5). Activity against sensitive cells was bactericidal but not bacteriolytic. These characteristics identified the inhibitory agent as a bacteriocin, designated lactacin B. Examination of strains of L. acidophilus within the six homology groupings of Johnson et al. (Int. J. Syst. Bacteriol. 30:53-68, 1980) demonstrated that production of the bacteriocin lactacin B could not be used in classification of neotype L. acidophilus strains. However, the usefulness of employing sensitivity to lactacin B in classification of dairy lactobacilli is suggested.