The influence of pH and growth rate on production of the bacteriocin, bavaricin MN, in batch and continuous fermentations

Bavaricin MN, a bacteriocin produced by Lactobacillus bavaricus MN, reached titres of 2000 AU ml^-1 in APT broth maintained at pH 6.0, 30°C in a batch fermenter. Levels of bavaricin MN at pH 5.5 and 6.5 were lower despite comparable levels of producer cells. The addition of 3.0 g l^-1 beef extract to APT broth resulted in increases in both the growth rate of the culture and the production of bavaracin MN. The titre of bavaricin MN in batch fermenters controlled at pH 6.0 in APT broth plus 3 g l^-1 beef extract reached 3200 AU ml^-1 at 30°C. This level was reduced to 800 AU ml^-1 by 76 h. Glucose-limited continuous culture of Lact. bavaricus MN under the same conditions resulted in an increase in the titre of bavaricin MN to 6400 AU ml^-1. This level was maintained, independent of growth rate, for 345 h. Growth rates of 0.205, 0.118, 0.169 and 0.058 h^-1 were examined.     

Biopreservation of Brined Shrimp (Pandalus borealis) by Bacteriocins from Lactic Acid Bacteria

In brined shrimp (ca. 3% NaCl), the effects of three different lactic acid bacteria bacteriocins (crude [6.54 x 10(sup10) U of bacteriocin activity {BU}/g] and purified [8.13 x 10(sup23) BU/g] nisin Z, carnocin UI49 [2.32 x 10(sup4) BU/g], and crude bavaricin A [2.78 BU/g]) on bacterial growth and shelf life were compared with those of a benzoate-sorbate solution (0.1% each [wt/wt]) and a control with no preservatives. The shelf life of shrimp subjected to the control treatment was found to be 10 days. Carnocin UI49 did not extend the shelf life, while crude bavaricin A (a cell-free supernatant of Lactobacillus bavaricus MI 401) resulted in a shelf life of 16 days, as opposed to 31 days with nisin Z for both its crude and purified forms. The benzoate-sorbate solution preserved the brined shrimp for the whole storage period (59 days). In the control, carnocin UI49, and crude bavaricin A treatments, a gram-positive flora dominated towards the end of the storage period while in the nisin Z treatment a gram-negative flora was more pronounced.     

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.     

A novel antimicrobial activity of a Paenibacillus polymyxa strain isolated from regional fermented sausages

A strain isolated from Argentinean regional fermented sausages was found to produce and secrete a compound that inhibited growth of Lactobacillus strains used as indicators. It was characterized as Paenibacillus polymyxa (P13). The antimicrobial activity, named polyxin, was obtained from culture supernatant fluid of late stationary phase and was inhibitory to actively growing cells. It was effective against a wide range of Gram-positive and Gram-negative bacterial species tested including food-borne pathogens. Bacteriocin-like properties such as proteinaceous nature (sensitive to proteases), insensitivity to organic solvents and chelators, stability to heat (up to 10 min at 90 °C), and acidic pH but instability in alkaline conditions, were determined. A molecular mass of 10 kDa was estimated by molecular gel filtration.     

Antibacterial activity of Lactobacillus sake isolated from meat.

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.     

Antibacterial activity of Lactobacillus sake isolated from meat

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.     

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.     

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 Partial Characterization of Novel Bacteriocin L23 Produced by <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">fermentum L</Emphasis>23

Lactobacillus fermentum strain L23 produced a small bacteriocin, designated bacteriocin L23, with an estimated molecular mass of < 7000 Da. Isolation, purification, and partial characterization of bacteriocin L23 are described. It displayed a wide inhibitory spectrum including both Gram-negative and Gram-positive pathogenic strains and two species of Candida. The antibacterial activity of cell-free culture supernatant fluid was not affected by catalase or urease but was abolished by the proteolytic enzymes trypsin and protease VI. Bacteriocin L23 was heat stable (60 min at 100°C) and showed inhibitory activity over a wide pH range (4.0 to 7.0). The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin sample was prepared by a process of ammonium sulfate precipitation, gel filtration, thin-layer chromatography, bioautography, and reversed-phase HPLC.     

Detection of bacteriocins of lactic acid bacteria isolated from foods and comparison with pediocin and nisin

A total of 663 533 colonies from 72 dairy and meat sources showed a detection rate of 0·2% for bacteriocin producers using direct plating techniques. A further 83 000 colonies from 40 fish and vegetable sources showed a detection rate of 3·4% for bacteriocin producers using selective enrichment procedures. A collection of seven purified isolates showing a different host spectrum of bacteriocin activity and with the ability to produce bacteriocins in broth culture were compared with nisin and pediocin (with respect to their inhibitory activity, determined by the critical dilution method), against various indicator bacteria in agar and broth. The sensitivity of Listeria species to various bacteriocins was influenced by the agar and broth test systems used. A Lactobacillus curvatus strain was found to be the most suitable indicator for quantitating antimicrobial effects of all the bacteriocins investigated in both agar and broth test systems. The bacteriocin-producing isolates were characterized by biochemical reactions and DNA restriction enzyme profiles and taxonomic identification revealed species of Lactobacillus , Carnobacterium and Lactococcus assigned on the basis of 16S rDNA sequences.     

Bactericidal and non-cytotoxic activity of bacteriocin produced by Lacticaseibacillus paracasei F9-02 and evaluation of its tolerance to various physico-chemical conditions

This study aims to explore novel lactic acid bacteria (LAB) from breast-fed infants' faeces towards characterizing their antimicrobial compound, bacteriocin. The LAB, Lacticaseibacillus paracasei F9-02 showed strong antimicrobial activity against clinical pathogens. Their proteinaceous nature was confirmed as the activity was completely abolished when treated with proteinaceous enzymes and retained during neutral pH and catalase treatment. The purified bacteriocin showed antimicrobial activity at the minimum inhibitory concentration (MIC) value of 7.56 μg/ml against vancomycin-resistant Enterococcus sp. [vancomycin-resistant enterococcal (VRE)], and methicillin-resistant Staphylococcus aureus (MRSA), 15.13 μg/ml against Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype typhi and 30.25 μg/ml against Shigella flexneri. Present study also proved the bactericidal, non-cytotoxic and non-hemolytic nature of bacteriocin. Additionally, bacteriocin retained their stability under various physico-chemical conditions, broad range of pH (2–10), temperature (40–121°C), enzymes (amylase, lipase and lysozyme), surfactants [Tween-20, 80, 100 and sodium dodecyl sulfate (SDS)], metal ions (CaCl₂, FeSO₄, ZnSO₄, MgSO₄, MnSO₄, CuCl₂) and NaCl (2%–8%). The molecular weight of bacteriocin (~28 kDa) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), functional and active groups were assessed by Fourier Transform-Infrared (FT-IR). To our knowledge, this is the first study reporting L. paracasei from breast-fed infants' faeces and assessing their antimicrobial compound, bacteriocin. The study results furnish the essential features to confirm the therapeutic potential of L. paracasei F9-02 bacteriocin.     

Reutericin 6, a new bacteriocin produced by Lactobacillus reuteri LA 6

Lactobacillus reuteri LA 6, isolated from infant faeces, produced an antimicrobial agent active against Lactobacillus acidophilus JCM 2125, Lactobacillus delbrueckii subsp. bulgaricus JCM 1002 and Lactobacillus delbrueckii subsp. lactis JCM 1148 and JCM 1248. The agent was sensitive to proteolytic enzymes and retained activity after heating at 100°C for 20 min. This agent was a bacteriocin and has been designated as reutericin 6. Reutericin 6 exceeds 200 kDa as determined by ultrafiltration studies. Activity against sensitive cells was both bacteriocidal and bacteriolytic.     

Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens

Aims: To purify and characterize an antimicrobial protein (bacteriocin) isolated from the dairy product‐derived Bacillus amyloliquefaciens. Methods and Results: An unknown bacterial species cultured from the Yogu Farm? probiotic dairy beverage was identified through 16S ribosomal RNA analysis as B. amyloliquefaciens, a phylogenetically close relative of Bacillus subtilis. The cell‐free supernatant (CFS) of overnight cultures was active against Listeria monocytogenes and also against clinical isolates of Gardnerella vaginalis and Streptococcus agalactiae. At the same time, several isolates of vaginal probiotic Lactobacilli were resistant to the CFS. The nature of the compound causing inhibitory activity was confirmed as proteinaceous by enzymatic digestion. The protein was isolated using ammonium sulfate precipitation, and further purified via column chromatography. PCR analysis was conducted to determine relatedness to other bacteriocins produced by Bacillus spp. Conclusion: The antimicrobial protein isolated from B. amyloliquefaciens was shown to be subtilosin, a bacteriocin previously reported as produced only by B. subtilis. Significance and Impact of the Study: This is the first report of intra‐species horizontal gene transfer for subtilosin and the first fully characterized bacteriocin isolated from B. amyloliquefaciens. Finally, this is the first report on subtilosin’s activity against bacterial vaginosis‐associated pathogens.     

Potential of Lactobacillus gasseri isolated from infant faeces to produce bacteriocin

Thirty strains of Lactobacillus gasseri isolated from infant faeces were examined for production of an antimicrobial agent against 10 strains of the seven species of the genus Lactobacillus. Each of the strains inhibited the growth of at least one of the indicator strains. The agent was sensitive to proteolytic enzymes and stable for 20 min at 120°C. It is a bacteriocin designated as gassericin A.     

Partial purification and characterization of bacteriocin from Yersinia kristensenii

A raw milk bacterial isolate, identified as Yersinia kristensenii was found to produce a bacteriocin which was inhibitory to Yersinia enterocolitica but not to other selected species of Yersinia or Gram-negative bacteria. Maximum production of bacteriocin was obtained when the organism was grown in shake culture at 28°C. Mitomycin C at a concentration of 0.5 μg ml^-1 induced bacteriocin production. The bacteriocin was partially purified and characterized by ammonium sulphate fractionation and gel filtration. The bacteriocin was completely inactivated when treated with proteolytic enzymes (trypsin and chymotrypsin). Bacteriocin activity was heat-resistant and it retained some of its activity after 5 min at boiling temperature. A total of 15 bacteriocin sensitive-suspected food isolates were further identified biochemically as Yersinia enterocolitica and a non-sensitive isolate was identified as Yersinia intermedia.     

Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum.

Lactic acid bacteria were isolated from Spanish dry-fermented sausages and screened for bacteriocin production. About 10% of the isolates produced antimicrobial substances when grown on solid media, but only 2% produced detectable activity in liquid media. Strain L50, identified as Pediococcus acidilactici, showed the strongest inhibitory activity and was active against members of all of the gram-positive genera tested. The strain produced a heat-stable bacteriocin when grown at 8 to 32 degrees C but not at 45 degrees C. The bacteriocin was purified to homogeneity. Its mass was determined to be 5,250.11 +/- 0.30 by electrospray mass spectrometry. The N terminus of the bacteriocin was blocked for sequencing by Edman degradation, but a partial sequence of 42 amino acids was obtained after cleavage of the peptide by cyanogen bromide. The sequence showed no similarity to those of other bacteriocins. Pediocin L50 appears to contain modified amino acids but not lanthionine or methyl-lanthionine.     

Antimicrobial compounds produced by probiotic Lactobacillus brevis isolated from dairy products

A total of 38 lactic acid bacteria, belonging to Lactobacillus, isolated from 24 samples of traditional Egyptian dairy products, were screened for antimicrobial activity against different Gram-positive and Gram-negative bacteria. A strain of Lactobacillus brevis showed the best inhibitory activity when tested by well diffusion assay. The antibacterial activity was pronounced between early logarithmic and early stationary phases. The strain produced a heat-stable antimicrobial compound showing no reduction in activity after heat treatment from 60 to 100°C for 15 and 30 min. Since it was inactivated by proteolytic enzymes, it is considered to be proteinaceous in nature and, therefore, referred to as a bacteriocin-like substance. This compound was also active over a wide pH range (pH 2–6). The antimicrobial compound was partially purified by 40% ammonium sulfate precipitation. Lactobacillus brevis was tested for its in vitro antibiotics susceptibility, tolerance to bile salts, resistance to low pH values, acidifying activity, proteolytic activity, and haemolytic activity. The results showed the potential of L. brevis strain as a probiotic culture, and hence it can be utilized in the manufacturing of pharmaceuticals and dietary supplements.     

Prevalence of bacteriocin activities and bacteriocin-encoding genes in enterococcal clinical isolates in Iran

The aim of the project was to isolate and characterize bacteriocin-producing enterococci, as well as determine the prevalence of enterocin structural genes in 187 enterococcal clinical isolates from the northwest of Iran. The isolates were screened for antibacterial activity against 15 different indicator strains. The proteinaceous nature of the antimicrobial substances was confirmed by sensitivity to proteinase K; their stability to heat treatment was tested at 60 °C and 100 °C for 20 and 10 min, respectively. The PCR method was applied to detect previously identified enterocin genes. Our results showed that 38 (20.3%) of the enterococcal isolates were considered to be potential bacteriocinogenic strains. Furthermore, genes encoding diverse bacteriocin are highly distributed among clinical enterococci, and the strains with multi-bacteriocin genes displayed high antimicrobial activity. Enterocin A, enterolysin A, and enterocin L50A/B were the most abundant structural genes detected in bacteriocinogenic strains. This work is the first survey on the prevalence of bacteriocin genes among clinical enterococci in Iran that has isolated a strain with high antimicrobial activity and sensitivity to clinically relevant antibiotics.     

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 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.