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.     

Lacticin, a bacteriocin produced by Lactobacillus delbrueckii subsp. lactis

Twenty-one strains of Lactobacillus delbrueckii and L. helveticus were tested for bacteriocin production against each other. Lactobacillus delbrueckii subsp. lactis JCM 1106 and 1107 produced an inhibitory agent active against L. delbrueckii subsp. bulgaricus JCM 1002 and NIAI yB-62, L. delbrueckii subsp. lactis JCM 1248 and L. delbrueckii subsp. delbrueckii JCM 1012. Lactobacillus delbrueckii subsp. lactis JCM 1248 inhibited only the growth of L. delbrueckii subsp. bulgaricus NIAI yB-62. These agents were sensitive to proteolytic enzymes and heating (at 60°C for 10min). These agents were considered to be bacteriocins and designated lacticin A and B.     

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.     

Activity of plantaricin SA6, a bacteriocin produced by Lactobacillus plantarum SA6 isolated from fermented sausage

N. REKHIF, A ATRIH AND G. LEFEBVRE. 1995. Plantaricin SA6, a bacteriocin produced by Lactobacillus plantarum SA6, exhibited an inhibitory action against several mesophilic lactobacilli. It was stable at 90–100°C at pH 2–4 and it remained stable in the presence of several organic solvents, urea or β-mercaptoethanol. Plantaricin SA6 bound specifically to the cell surface of only plantaricin SA6-sensitive bacteria. The putative receptors are not destroyed by different hydrolytic enzymes added to the phosphate buffer. Plantaricin SA6 acted as a bactericidal agent lysing sensitive strains, that became more permeable to ortho-nitro-phenol-β-galactoside and lost their intracellular K⁺ ions and u.v.-absorbing materials. Both the adsorption and lethal action of plantaricin SA6 were maximal between pH 4 and 7, but the range of temperature tested (5–37βC) had no effect. Ions (of several salts such as MgCl₂) inhibited the binding of plantaricin SA6 and protected cells against bacteriocin action.     

Characterization of plantaricin KW30, a bacteriocin produced by Lactobacillus plantarum

A protease-sensitive antibacterial substance, produced by a strain of Lactobacillus plantarum isolated from fermented corn, was classified as a bacteriocin and designated plantaricin KW30. The bacteriocin was stable to heat, pH and treatment with surfactants, and unaffected by α-amylase, lipase or lysozyme. Plantaricin KW30 exhibited a bactericidal and non-bacteriolytic mode of action against indicator cells, and inhibitory activity was limited to other lactobacilli. Maximum production was in MRS broth, and coincided with the onset of stationary phase under conditions of low pH and high cell numbers. In a complex medium bacteriocin production was enhanced by the presence of sodium acetate and Tween 80. Curing experiments gave derivatives that no longer produced the bacteriocin but retained immunity to it. These Bac derivatives showed the same plasmid pattern as the parent strain suggesting a chromosomal location for the genes for bacteriocin production.     

Characterization of reutericyclin produced by Lactobacillus reuteri LTH2584

Lactobacillus reuteri LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains of Escherichia coli was inhibited. Reutericyclin exhibited a bactericidal mode of action against Lactobacillus sanfranciscensis, Staphylococcus aureus, and B. subtilis and triggered the lysis of cells of L. sanfranciscensis in a dose-dependent manner. Germination of spores of B. subtilis was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of L. reuteri LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C(8) chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Structural characterization (A. H?ltzel, M. G. G?nzle, G. J. Nicholson, W. P. Hammes, and G. Jung, Angew. Chem. Int. Ed. 39:2766-2768, 2000) revealed that reutericyclin is a novel tetramic acid derivative. The inhibitory activity of culture supernatant of L. reuteri LTH2584 corresponded to that of purified as well as synthetic reutericyclin.     

Antibacterial activity of plantaricin SIK-83, a bacteriocin produced by Lactobacillus plantarum

Lactobacillus plantarum SIK-83 produces a bacteriocin, designated plantaricin SIK-83, which inhibits 66 of 68 lactic acid bacteria from the genera Lactobacillus, Leuconostoc, Pediococcus and Streptococcus. A 500-fold dilution of L. plantarum SIK-83 MRS culture supernatant with phosphate buffer was sufficient to kill 10(5) cells/ml of Pediococcus pentosaceus within 120 s. The killing of a sensitive population followed exponential kinetics. It was shown that the bacteriocin binds specifically to sensitive cells but not to nonsensitive lactic acid bacteria, the producer strain or Gram-negative bacteria. Sensitive cells, after exposure to the bacteriocin, could be rescued by treatment with proteolytic enzymes. In buffer, plantaricin SIK-83 was adsorbed to the cell surface almost immediately, and morphological lesions were observed within 2 h after the cells were exposed to the bacteriocin. The lethal mode of action appeared to be due to damage to the cell membrane, resulting in cell lysis, which was detected by electron microscopy and by determination of released intracellular components.     

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.     

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.     

Plantaricin D, a bacteriocin produced by Lactobacillus plantarum BFE 905 from ready-to-eat salad

Lactobacillus plantarum BFE 905 isolated from 'Waldorf' salad produced a bacteriocin termed plantaricin D which was active against Lact. sake and Listeria monocytogenes strains. Plantaricin D was heat stable, retaining activity after heating at 121 °C. The bacteriocin was inactivated by α-chymotrypsin, trypsin, pepsin and proteinase K, but not by papain and other non-proteolytic enzymes tested. Plantaricin D was stable at pH values ranging from 2·0 to 10·0. The bacteriocin inhibited growth of L. monocytogenes in automated turbidity assays. Although Lact. plantarum BFE 905 harboured plasmids ranging in size from 3 to 55 kilobase pairs, loss of bacteriocin production could not be correlated with plasmid loss. A role for bacteriocin-producing Lact. plantarum of vegetable origin in assuring the safety of vegetable foods is suggested.     

Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801

Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801, with an estimated molecular mass of less than 6.5 kDa. It displays a narrow inhibitory spectrum (only related lactobacilli but including the Gram-negative pathogenic bacteria Escherichia coli Row and Salmonella panama 1467) with a bactericidal activity. The antimicrobial activity of cell-free culture supernatant fluid was insensitive to catalase but sensitive to proteolytic enzymes such as trypsin, proteinase K and pronase, heat-stable (30 min at 121 degrees C), and maintained in a wide pH range. The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin was isolated as a floating pellicle after ammonium sulphate precipitation (40% saturation) and partially purified by extraction/precipitation with chloroform/methanol (2/1, v/v). Further purification to homogeneity was performed by reversed phase Fast Performance Liquid Chromatography. The amino acid composition was determined. Amino acid sequencing revealed that the N-terminal end was blocked.     

Inhibition of Listeria monocytogenes by bavaricin A, a bacteriocin produced by Lactobacillus bavaricus MI401

Two hundred and forty-five strains of Listeria monocytogenes belonging to 33 different electrophoretic types, grouped by multilocus enzyme electrophoresis, were screened for sensitivity to bavaricin A produced by Lactobacillus bavaricus MI401. Three strains were resistant. Resistance was found to be neither restricted to particular electrophoretic types nor to certain sources of origin. The resistant strains belonged to three different electrophoretic types (ET 1, ET 5 and ET 14) and were isolated from a human case of listeriosis, smoked salmon and pig faeces, respectively.     

Isolation, purification and partial characterization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum

Lactobacillus plantarum 423, isolated from sorghum beer, produces a bacteriocin (plantaricin 423) which is inhibitory to several food spoilage bacteria and food-borne pathogens, including Bacillus cereus , Clostridium sporogenes , Enterococcus faecalis , Listeria spp. and Staphylococcus spp. Plantaricin 423 is resistant to treatment at 80 °C, but loses 50% of its activity after 60 min at 100 °C and 75% of its activity after autoclaving (121 °C, 15 min). Plantaricin 423 remains active after incubation at pH 1–10 and is inactivated when treated with pepsin, papain, α-chymotrypsin, trypsin and Proteinase K. Plantaricin 423 was partially purified and its size estimated at 3·5 kDa, as determined by tricine-SDS-PAGE. The mechanism of activity of plantaricin 423 is weakly bactericidal, as determined against Oenococcus oeni (previously Leuconostoc oenos ). High DNA homology was obtained between the plasmid DNA of strain 423 and the pediocin PA-1 operon of Pediococcus acidilactici PAC 1·0, suggesting that plantaricin 423 is plasmid-encoded and related to the pediocin gene cluster.     

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.     

Sakacin B, a bacteriocin produced by Lactobacillus sake isolated from Greek dry fermented sausages

When Lactobacillus sake 251, a strain isolated from naturally fermented Greek dry sausage was grown in MRS broth it excreted an antimicrobial factor that differed from organic acids and hydrogen peroxide. The substance was proteinaceous, heat stable and inhibitory towards various lactic acid bacteria of meat origin. This suggested that a narrow spectrum bacteriocin, designated sakacin B, was present in the broth. Sakacin B displayed a bactericidal mode of action on sensitive cells without causing cell lysis. It was secreted during late logarithmic phase and was stable within a pH range 2 to 9. In vitro production of sakacin B by the producer strain in a mixed culture caused a strong biocidal effect on growing indicator cells. Sakacin B was partially purified and found not to contain unusual amino acids. That it was a hydrophobic peptide was confirmed by SDS-PAGE electrophoresis. The molecular weight of sakacin B was estimated to be 6.3 kDa.     

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.     

Identification and characterization of helveticin V-1829, a bacteriocin produced by Lactobacillus helveticus 1829

Lactobacillus helveticus 1829 produced an antimicrobial agent, designated helveticin V-1829, that demonstrated antagonistic activity against closely-related species. The agent was excreted into MRS agar, and was present in the supernatant fluids from both overnight broth and clotted milk cultures. It was heat labile (inactivated by 50°C for 30 min) and was stable over the pH range 2.5 to 6.5. Production of the substance was pH-dependent and maximum yields were obtained in MRS broth cultures maintained at pH 5.5. Helveticin V-1829 was partially purified following growth of the producing strain in a semi-defined MRS medium and precipitating the cell-free filtrate with ammonium sulphate to 30% saturation. The cleared supernatant fluid was then brought to 60% saturation and the resulting precipitate pelleted and dialysed in 0.3 mol/l phosphate buffer. The partially purified inhibitor was sensitive to several proteolytic enzymes, and it was bactericidal in its mode of action against indicator cells of Lact. helveticus 1844 and Lact. delbrueckii subsp. bulgaricus 1489, indicating that it was a bacteriocin. A DNA probe specific for the helveticin J structural gene failed to hybridize to total genomic DNA of Lact. helveticus 1829, indicating that helveticin V-1829 is not significantly related to helveticin J.     

Gassericin A: a circular bacteriocin produced by Lactic acid bacteria Lactobacillus gasseri

During the recent years extensive efforts have been made to find out bacteriocins from lactic acid bacteria (LAB) active against various food spoilage and pathogenic bacteria, and superior stabilities against heat treatments and pH variations. Bacteriocins isolated from LAB have been grouped into four classes. Circular bacteriocins which were earlier grouped among the four groups of bacteriocins, have recently been proposed to be classified into a different class, making it class V bacteriocins. Circular bacteriocins are special molecules, whose precursors must be post translationally modified to join the N to C termini with a head-to-tail peptide bond. Cyclization appears to make them less susceptible to proteolytic cleavage, high temperature and pH, and, therefore, provides enhanced stability as compared to linear bacteriocins. The advantages of circularization are also reflected by the fact that a significant number of macrocyclic natural products have found pharmaceutical applications. Circular bacteriocins were unknown two decades ago, and even to date, only a few circular bacteriocins from a diverse group of Gram positive organisms have been reported. The first example of a circular bacteriocin was enterocin AS-48, produced by Enterococcus faecalis AS-48. Gassereccin A, produced by Lactobacillus gasseri LA39, Reutericin 6 produced by Lactobacillus reuteri LA6 and Circularin A, produced by Clostridium beijerinickii ATCC 25,752, are further examples of this group of antimicrobial peptides. In the present scenario, Gassericin A can be an important tool in the food preservation owing to its properties of high pH and temperature tolerance and the fact that it is produced by LAB L. gasseri, whose many strains are proven probiotic.     

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.