Production kinetics of acidophilin 801, a bacteriocin produced by Lactobacillus acidophilus IBB 801

Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801. Studying the relationship between growth and bacteriocin biosynthesis revealed primary metabolite kinetics of bacteriocin production with a peak activity at the end of the exponential growth phase followed by a decrease during the stationary phase. Both microbial growth and bacteriocin production was inhibited by lactic acid. Whereas volumetric bacteriocin production (activity units (AU) ml(-1)) was favoured under pH-controlled conditions, bacteriocin titres rapidly decreased because of strong adsorption of the bacteriocin molecules to the producing cells under less acidic conditions.     

Purification and partial characterization of lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088

Lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088 (NCK88), was purified and characterized. Lactacin F is heat stable, proteinaceous, and inhibitory to other lactobacilli as well as Enterococcus faecalis. The bacteriocin was isolated as a floating pellet from culture supernatants brought to 35 to 40% saturation with ammonium sulfate. Native lactacin F was sized at approximately 180 kDa by gel filtration. Column fractions having lactacin F activity were examined by electron microscopy and contained micelle-like globular particles. Purification by ammonium sulfate precipitation, gel filtration, and high-performance liquid chromatography resulted in a 474-fold increase in specific activity of lactacin F. The purified bacteriocin was identified as a 2.5-kDa peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lactacin F peptide retained activity after extraction from SDS-PAGE gel slices, confirming the identity of the 2.5-kDa peptide. Variants of NCK88 that failed to exhibit lactacin F activity did not produce the 2.5-kDa band. Sequence analysis of purified lactacin F identified 25 N-terminal amino acids containing an arginine residue at the N terminus. Composition analysis indicates that lactacin F may contain as many as 56 amino acid residues.     

Purification and partial characterization of lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088.

Lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088 (NCK88), was purified and characterized. Lactacin F is heat stable, proteinaceous, and inhibitory to other lactobacilli as well as Enterococcus faecalis. The bacteriocin was isolated as a floating pellet from culture supernatants brought to 35 to 40% saturation with ammonium sulfate. Native lactacin F was sized at approximately 180 kDa by gel filtration. Column fractions having lactacin F activity were examined by electron microscopy and contained micelle-like globular particles. Purification by ammonium sulfate precipitation, gel filtration, and high-performance liquid chromatography resulted in a 474-fold increase in specific activity of lactacin F. The purified bacteriocin was identified as a 2.5-kDa peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lactacin F peptide retained activity after extraction from SDS-PAGE gel slices, confirming the identity of the 2.5-kDa peptide. Variants of NCK88 that failed to exhibit lactacin F activity did not produce the 2.5-kDa band. Sequence analysis of purified lactacin F identified 25 N-terminal amino acids containing an arginine residue at the N terminus. Composition analysis indicates that lactacin F may contain as many as 56 amino acid residues.     

Partial purification and characterization of the bacteriocin produced by Lactobacillus acidophilus YIT 0154

One strain of Lactobacillus acidophilus was found to produce a bacteriocin-like substance in the culture filtrate. The substance was produced in a growth-associated manner, showed heat stability at neutral and acidic pH and exhibited antibacterial activity against various species of Lactobacillus including L. acidophilus itself. The molecular weight of the substance was in the range of 6.2-95 kDa. N-terminal amino acid sequence analysis suggests that the substance may belong to class IIb bacteriocin.     

Purification and characterisation of acidocin D20079, a bacteriocin produced by Lactobacillus acidophilus DSM 20079

Bacteriocins are natural antimicrobial agents produced by food fermentative bacteria. Lactobacillus acidophilus DSM 20079 produces a small bacteriocin, with a molecular mass of 6.6 kDa, designated acidocin D20079. This antimicrobial peptide was extremely heat-stable (30 min at 121 degrees C) and was active over a wide pH range. It was found to be sensitive to proteolytic enzymes (trypsin, ficin, pepsin, papain, and proteinase K). Acidocin D20079 has a narrow inhibitory spectrum restricted to the genus Lactobacillus which includes L. sakei NCDO 2714, an organism known to cause anaerobic spoilage of vacuum-packaged meat. Maximum production of acidocin D20079 in MRS broth was detected at pH 6.0, and the peptide was purified by ammonium sulphate precipitation followed by sequential cation exchange and hydrophobic interaction chromatography. Purified acidocin D20079 spontaneously formed spherulite crystals during dialysis. As the N-terminus was found to be blocked for sequencing, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used to determine a partial sequence, and the molecular mass of the bacteriocin in the formed crystals (6.6 kDa). Estimates of the molecular weight of the partially purified peptide, using tricine-SDS-PAGE, in which bacteriocin activity was confirmed by overlayer techniques were in accordance with this value.     

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.     

Purification and some properties of acidocin 8912, a novel bacteriocin produced by Lactobacillus acidophilus TK8912.

Acidocin 8912, a bacteriocin produced by Lactobacillus acidophilus TK8912, was purified by ammonium sulfate fractionation and successive chromatographies on CM-cellulose, Sephadex G-50, Sephadex G-25, and reversed-phase HPLC on Aquapore RP-300. The purified acidocin 8912 migrated as a single band on SDS-PAGE. The molecular weight was estimated to be 5200 by SDS-PAGE, and 5400 by HPLC gel filtration on TSKgel G3000PWXL. Both the amino acid composition and the N-terminal amino acid sequence analysis indicated that acidocin 8912 was a peptide composed of presumably 50 amino acids containing a Lys residue at the N-terminus. The purified acidocin 8912 showed a bactericidal effect on sensitive cells but not a bacteriolytic effect.     

Production and physicochemical characterization of acidocin D20079, a bacteriocin produced by Lactobacillus acidophilus DSM 20079

Lactobacillus acidophilus DSM 20079 is the producer of a novel bacteriocin termed acidocin D20079. In this paper, a partial sequence of this peptide is determined, together with data on its secondary structure. A modification of the MRS-growth medium (replacing the detergent Tween 80 with oleic acid), was shown to improve the production level of the peptide by one order of magnitude, as well as to stabilize the activity level. Addition of a detergent (Tween 20, less interfering in mass spectrometric analysis), was however necessary for solubilization of the purified acidocin D20079. Digestion of the peptide followed by de-novo sequencing of generated fragments, allowed determination of a partial sequence consisting of 39 of the totally estimated 65 residues. Acidocin D20079 has a high content of glycine residues, hydrophobic residues, and acidic residues. No modified amino acids were found. Edman degradation, and C-terminal sequencing failed, suggesting that the peptide may be cyclic, and a novel member of class IIc bacteriocins. Circular dichroism spectroscopy and secondary structure prediction showed random coil conformation in aqueous solution, but secondary structure was induced in the presence of sodium-dodecyl sulfate. The data could be fitted assuming 2–13% of the residues to be in α-helix and 23–27% of the residues to be in β-strand conformation. This indicates that a membrane/membrane-mimicking hydrocarbon–water interface induces an active conformation.     

Partial characterization of a bacteriocin produced by Lactobacillus helveticus

AIMS: To investigate the antimicrobial activity of a strain of Lactobacillus helveticus. METHODS AND RESULTS: The culture supernatant fluid Lact. helveticus G51 showed antimicrobial activity against thermophilic strains of Lactobacillus. Purification of the active compound was achieved after gel filtration and ion exchange chromatography. As revealed by SDS-PAGE, active fractions were relatively homogeneous, showing a protein with a molecular mass of 12.5 kDa. The antimicrobial compound was heat labile, inactivated by proteolytic enzymes and had a bactericidal mode of action. CONCLUSION: The antimicrobial activity expressed by Lact. helveticus G51 was correlated with the production of a bacteriocin with properties that were different to other helveticins. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has provided further data on Lact. helveticus bacteriocins. The strong activity of the bacteriocin towards various thermophilic lactobacilli warrants further investigation for its potential to obtain attenuated cultures for the enhancement of the cheese-ripening process.     

Purification and characterization of plantaricin Y,a novel bacteriocin produced by Lactobacillus plantarum 510

Lactobacillus plantarum 510, previously isolated from a koshu vineyard in Japan, was found to produce a bacteriocin-like inhibitory substance which was purified and characterized. Mass spectrometry analysis showed that the mass of this bacteriocin is 4,296.65 Da. A partial sequence, NH₂- SSSLLNTAWRKFG, was obtained by N-terminal amino acid sequence analysis. A BLAST search revealed that this is a unique sequence; this peptide is thus a novel bacteriocin produced by Lactobacillus plantarum 510 and was termed plantaricin Y. Plantaricin Y shows strong inhibitory activity against Listeria monocytogenes BCRC 14845, but no activity against other pathogens tested. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was completely inactivated by protease K. Furthermore, trypsin-digested bacteriocin product fragments retained activity against L. monocytogenes BCRC 14845 and exhibited a different inhibitory spectrum.     

Acidophilucin A, a new heat-labile bacteriocin produced by Lactobacillus acidophilus LAPT 1060

Lactobacillus acidophilus LAPT 1060, isolated from infant faeces, produced an antimicrobial agent active against six strains of Lactobacillus delbrueckii subsp. bulgaricus and six strains of Lactobacillus helveticus . The agent was sensitive to proteolytic enzymes and heat (10 min at 60°C) and is a bacteriocin and designated acidophilucin A.     

Purification and characterization of curvaticin L442, a bacteriocin produced by Lactobacillus curvatus L442

Lactobacillus curvatus L442, isolated from Greek traditional fermented sausage prepared without the addition of starters, produces a bacteriocin, curvaticin L442, which is active against the pathogen Listeria monocytogenes. The bacteriocin was purified by 50% ammonium sulphate precipitation, cation exchange, reverse phase and gel filtration chromatography. Partial N-terminal sequence analysis using Edman degradation revealed 30 amino acid residues, revealing high homology with the amino acid sequence of sakacin P. Curvaticin L442 is active at pH values between 4.0 and 9.0 and it retains activity even after incubation for 5 min at 121 °C with 1 atm of overpressure. Proteolytic enzymes and α-amylase inactivated this curvaticin, while the effect of lipase was not severe.     

Mode of action of acidocin 8912, a bacteriocin produced by Lactobacillus acidophilus TK8912

K.A. HOEKSTRA AND R.J.L. PAULTON. 1996. Disc agar diffusion testing was performed on 547 isolates (two common pathogens) to determine if the site of isolation influenced the antimicrobial susceptibility results for a given bacterium. The most statistically significant results ( P < 0.05) included cephalothin (ear) against Staphylococcus aureus and cephalothin (ear), lincomycin (ear), trimethoprim sulpha (ear), and amoxycilin and clavulanic acid (nose) against Staph. intermedius. Although the impact of these results (empirical treatment) is unknown, it is hypothesized that the site of isolation of Staph. aureus and Staph. intermedius may influence the choice of antimicrobial therapy in the dog and cat.     

Purification and partial characterization of a bacteriocin produced by Eikenella corrodens

Aims: The purpose of this study was to purify and characterize a bacteriocin produced by Eikenella corrodens A32E2. Methods and Results: Peptostreptococcus anaerobius ATCC27337 was used as indicator strain in antagonistic assays for bacteriocin‐producing E. corrodens A32E2. Protein extraction was influenced by pH and buffer composition. The protein was active in the pH range 6–8. Inhibitory activity was lost by both heating and treatment with proteolytic enzymes and decreased with organic solvents. The substance is rather unstable but maintains 100% of its activity after being exposed to acetone and when stored at ?70°C. The antagonistic substance was first precipitated by ammonium sulfate and further partially purified by Mono‐Q FPLC and C‐18 HPLC. Mass spectrometry analysis showed that the molecular mass was 23 625 Da, and the sequence obtained for the N‐terminus was: Met‐Asn‐Phe‐Asp‐Glu‐Lys‐Val‐Gly‐Lys‐Val‐X‐Phe‐Lys‐Val‐Gly‐Asp. Conclusions: The evidence presented in this study supports the idea that an antagonistic substance produced by E. corrodens A32E2 isolated from a periodontal diseased site is a novel bacteriocin, which we designate corrodecin. Significance and Impact of the Study:  We anticipated that corrodecin might play an important role at the periodontal site. This compound could also be attractive in biotechnological applications as an interesting tool for oral ecosystem control.     

Characterization and purification of acidocin CH5, a bacteriocin produced by Lactobacillus acidophilus CH5

AIMS: To characterize and to purify a bacteriocin produced by Lactobacillus acidophilus strain with its activity restricted to Gram-positive bacteria. METHODS AND RESULTS: Native acidocin CH5, a bacteriocin produced by L. acidophilus CH5 an isolate from a dairy starter culture forms in MRS (Oxoid, Basingstoke, UK) broth high-molecular weight aggregates which can dissociate into smaller units (retained by 5 kDa membrane) with higher activity. Acidocin CH5 was purified using combinations of chromatographic methods based on hydrophobic and cation exchange principles and the N-terminal region was sequenced. CONCLUSIONS: Based on our results it is evident that acidocin CH5 belongs, according to bacteriocin classification, to the class II bacteriocins with identical N-terminal amino acid sequence described in the literature previously. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has provided further data on bacteriocin acidocin CH5 from class II with wide spectrum of antimicrobial activity atypical for bacteriocins produced by L. acidophilus sharing the same homology.     

Isolation, partial characterization and mode of action of acidocin J1229, a bacteriocin produced by Lactobacillus acidophilus JCM 1229

Lactobacillus acidophilus JCM 1229 produces a heat-stable bacteriocin, designated as acidocin J1229, that has a narrow inhibitory spectrum. Production of acidocin J1229 in MRS broth was pH dependent, with maximum activity detected in broth culture maintained at pH 5:0. Acidocin J1229 was purified by ammonium sulphate precipitation and sequential cation exchange and reversed-phase chromatographies. The sequence of the first 24 amino acid residues of the N terminus of acidocin J1229 was determined. The molecular mass of acidocin J1229 as determined by mass spectrometry was 6301 Da. Acidocin J1229 showed a bactericidal effect but not a bacteriolytic effect on sensitive cells. Acidocin J1229 dissipated the membrane potential and the pH gradient in sensitive cells, which affected such proton motive force-dependent processes as amino acid transport. Acidocin J1229 also caused an efflux of glutamate, previously taken up via a unidirectional ATP-driven transport system. Secondary structure prediction revealed the presence of an amphiphilic a-helix region that could form hydrophilic pores. These results suggest that acidocin J1229 is a pore-forming peptide that creates cell membrane channels through the 'barrel-stave'mechanism.     

Characterization and purification of acidocin 1B, a bacteriocin produced by Lactobacillus acidophilus GP1B

In the present study, acidocin 1B, a bacteriocin produced by Lactobacillus acidophilus GP1B, exhibited profound inhibitory activity against a variety of LAB and pathogens, including Gram-negative bacteria, and its mode of action was to destabilize the cell wall, thereby resulting in bactericidal lysis. Acidocin 1B was found to be heat stable, because it lost no activity when it was heated up to 95 degrees C for 60 min. It retained approximately 67% of the initial activity after storage for 30 days at 4 degrees C, and 50% of its initial activity after 30 days at 25 degrees C and 37 degrees C. The molecular mass of acidocin 1B was estimated to be 4214.65 Da by mass spectrometry. Plasmid curing results indicated that a plasmid, designated as pLA1B, seemed to be responsible for both acidocin 1B production and host immunity, and that the pLA1B could be transformed into competent cells of L. acidophilus ATCC 43121 by electroporation. Our findings indicate that the acidocin 1B and its producer strain may have potential value as a biopreservative in food systems.