Characteristics and Genetic Determinants of Bacteriocin Activities Produced by Carnobacterium piscicola CP5 Isolated from Cheese

Carnobacterium piscicola CP5, isolated from a French mold-ripened soft cheese, produced a bacteriocin activity named carnocin CP5, which inhibited Carnobacterium, Enterococcus and Listeria spp. strains, and among the Lactobacillus spp. only Lactobacillus delbrueckii spp. [24]. The activity was purified by ammonium sulfate precipitation, anion exchange, and hydrophobic interaction chromatography followed by reverse-phase high-performance liquid chromatography (RP-HPLC). This latter step separated two peaks with anti-listerial activity (CP51 and CP52). Carnocin CP51 was partially sequenced, and the N-terminal part revealed the presence of the “pediocin-like consensus” sequence-Tyr-Gly-Asn-Gly-Val-. Then, a degenerated 24-mer oligonucleotide probe was constructed from the N-terminal sequence and used to detect the structural gene. It was localized on a plasmid of about 40 kb. Cloning of restriction fragments of this one, followed by DNA sequencing, revealed the presence of the second anti-Listeria bacteriocin gene (CP52). By comparing sequences in data banks and confirming results with PCR reactions, carnocin CP51 shared homologies with carnobacteriocin BM1, and carnocin CP52 was similar to carnobacteriocin B2, both produced by C. piscicola LV17 [2]. However, carnobacteriocin A from C. piscicola LV17 gene was lacking in C. piscicola CP5, and the two microorganisms have been isolated from different ecological environments: C. piscicola CP5 and C. piscicola LV17 were isolated from soft cheese and vacuum-packed meat respectively. This fact could allow different application perspectives for C. piscicola CP5. Received: 16 April 1997 / Accepted: 9 May 1997     

Carnocin KZ213 produced by<Emphasis Type="Italic"> Carnobacterium piscicola</Emphasis> 213 is adsorbed onto cells during growth. Its biosynthesis is regulated by temperature,pH and medium composition

Carnocin KZ213 is an antilisterial bacteriocin produced by Carnobacterium piscicola 213. The effects of pH and temperature were studied during batch fermentation in MRS* medium (modified MRS without ammonium citrate or sodium acetate). The optimal pH for growth is between 6 and 7. The maximum bacteriocin productivity in the supernatant occurs at pH 7. Operating at controlled pH increases the volumetric activity of the free bacteriocin by 8- to 16-fold, compared with uncontrolled pH. No bacteriocin production is observed below pH 6.5. Temperature has a dramatic effect on carnocin KZ213 production. Growth is optimal at 25 °C and 30 °C, although no bacteriocin production is detected at 30 °C. Also, bacteriocin production is observed at 25 °C in MRS*, but not in complex APT broth, where growth is optimal. The presence of glucose as a carbon and/or energy source is important for carnocin KZ213 synthesis. Hence, bacteriocin synthesis is regulated by temperature, carbon source and medium composition. Quantification studies of bacteriocin adsorbed onto producer cells show that the majority of the carnocin KZ213 secreted is adsorbed onto the producer cells during growth. Only 15% of the total bacteriocin produced is detected in the cell-free supernatant at the end of growth.     

Mesenterocin 52, a bacteriocin produced by Leuconostoc mesenteroides ssp. mesenteroides FR 52

F. MATHIEU, I.S. SUWANDHI, N. REKHIF, J.B. MILLIERE AND G. LEFEBVRE. 1993. One hundred and sixty-five isolates of Leuconostoc spp. were tested for bacteriocin production. Only one strain, Leuc. mesenteroides ssp. mesenteroides FR 52, isolated from a raw milk, produced a bacteriocin which was named Mesenterocin 52. This bacteriocin inhibited other Leuconostoc strains and several strains of Enterococcus and Listeria spp. No activity was found against lactococci and lactobacilli. The antibacterial spectrum differed from that of previously described Leuconostoc bacteriocins. Mesenterocin 52 was secreted into the medium during the growth phase. It was inactivated with protease treatments. At pH 7.0 it had a relative stability after heating at 100C (15 min), but it had a greater stability at pH 4.5 than at pH 7.0 after 6 h at 80C. The apparent molecular mass was estimated to be less than 10 kDa by ultrafiltration. Mesenterocin 52 showed a bactericidal effect on Leuconostoc paramesenteroides DSM 20288.     

Characterization, Production, and Purification of Carnocin H, a Bacteriocin Produced by Carnobacterium 377

Carnocin H, a bacteriocin produced by a Carnobacterium sp., inhibited lactic acid bacteria, clostridia, enterococci, and some Staphylococcus aureus strains. Some strains of Listeria and Pediococcus were also sensitive to carnocin H. The bacteriocin was produced during the late stationary growth phase. Carnocin H was purified by cation exchange chromatography and reverse phase chromatography. Amino acid sequence and composition indicate that carnocin H is a novel bacteriocin belonging to the class II bacteriocins. The bacteriocin consists of approximately 75 amino acid residues with a highly cationic N-terminal containing six succeeding lysines. Activity, as measured by agar diffusion zones, was reduced at increased pH values, levels of indicator bacteria, NaCl, agar, and soy oil.     

Fermenting Cucumber, a Potential Source for the Isolation of Pediocin-Like Bacteriocin Producers

Summary A strain of Pediococcus acidilactici CFR K7 isolated from cucumber, produced an antimicrobial peptide which acted against Leuconostoc mesenteroides, selected strains of Lactobacillus spp., Pediococcus spp. and Enterococcus spp. The partially purified bacteriocin had molecular weight of ~4.6 kDa, heat stability in a range of 40–121 °C and was active over a wide range of pH (2.0–9.0). This bacteriocin possessed strong antilisterial activity and was susceptible to proteolytic enzymes. Southern hybridization using the PCR-generated pedA probe established that the gene for the bacteriocin was plasmid-borne as in the case of pediocin PA-1. Nucleotide sequence of the pedAB gene indicated 100% homology to a pediocin AcH/PA-1. Certain bacteriocinogenic strains isolated from naturally fermented cucumber were tested by colony hybridization using the pedA gene probe. Nine out of twenty colonies reacted with the probe indicating their ability to produce the pediocin-like bacteriocin. These nine colonies were further tested for their antimicrobial spectrum, proteolytic inactivation and plasmid profile. It was found that a few of them were active against Bacillus cereus, Micrococcus luteus and Listeria monocytogenes. Their proteolytic inactivation showed that the antimicrobial compound was susceptible to proteinase K. Colony hybridization could thus enable rapid detection of pediocin and pediocin-like bacteriocin producers among a population of bacteriocinogenic strains.     

Effect of pH and the bacteriocin carnocin 54 on growth and cell morphology of two Leuconostoc strains

Leuconostoc carnosum LA54A produces carnocin 54, a bacteriocin inhibitory to Listeria and closely related lactic acid bacteria. The effects of the pH of cell-free LA54 culture supernatants on the antibacterial activity of carnocin 54 was assessed using Leuc. mesenteroides DSM 20343 and TA10C as indicator strains. Carnocin 54 showed greatest activity against both strains at pH 4.5. At pH 6.5, activity was reduced, especially against Leuc. mesenteroides TA10C. Scanning electron microscopy showed irregular and rough surfaces on bacteriocin-treated cells at both pH values.     

Detection and partial characterization of a bacteriocin produced by Carnobacterium piscicola 213

BLIS 213, is a bacteriocin-like inhibitory substance produced by Carnobacterium piscicola 213. It is active against Carnobacterium, Enterococcus and Listeria spp. No activity was observed against tested Lactobacillus, Lactococcus, Leuconostoc and Pediococcus strains, nor against Gram-negative bacteria. The BLIS 213 activity was inactivated by several proteolytic enzymes. It was heat resistant (121°C for 20 min), and stable over a pH range of 2–8. Activity was determined by a dilution micromethod; it was increased after SDS treatment. A mutant strain which lacks bacteriocin production was isolated and designated as Carnobacterium piscicola 213a. It had the same phenotypic and biochemical properties as the parent strain, and was not sensitive to bacteriocin activity. The apparent molecular weight of the bacteriocin in the crude extract was greater than 10 kDa. It was about 6 kDa after SDS-PAGE of a partially purified bacteriocin by adsorption on producer cells. The isoelectric point of the BLIS 213 was around 9.3. Received 21 January 1997/ Accepted in revised form 25 April 1997     

Identification and partial characterization of tochicin, a bacteriocin produced by Bacillus thuringiensis subsp tochigiensis

Bacillus thuringiensis subsp tochigiensis HD868 was identified as a bacteriocin producer which exhibited a bactericidal effect against closely related species. This bacteriocin designated as tochicin, was partially purified by 75% ammonium sulfate precipitation followed by subsequent dialysis. This partially purified tochicin showed a narrow antibacterial spectrum of activity against most of 20 typical B. thuringiensis strains and a strain of B. cereus, but not against other bacteria and yeasts tested. The antibacterial activity of tochicin on sensitive indicator cells disappeared completely by proteinase K treatment (1 mg ml⁻¹), which indicates its proteinaceous nature. Tochicin was very stable throughout the range of pH 3.0–9.0 and was relatively heat-stable at 90°C, but bacteriocin activity was not detected after boiling for 30 min. The relationship between cell growth and bacteriocin production was studied in a semi-defined medium. Tochicin activity was detected at the mid-log growth phase, reached the maximum at the early stationary phase, but decreased after the stationary phase. Direct detection of tochicin activity on sodium dodecyl sulfate-polyacrylamide gel suggested it has an apparent molecular mass of about 10.5 kDa. Tochicin exhibited a bactericidal activity against B. thuringiensis subsp thompsoni HD522 in phosphate buffer (pH 7.0). Received 02 December 1996/ Accepted in revised form 25 August 1997     

Large scale purification protocol for carnocin KZ 213 from Carnobacterium piscicola

Large scale purification of a class IIa bacteriocin has been developed to recover pure carnocin KZ 213 produced by Carnobacterium piscicola 213. Most previous protocols reported in the literature for the purification of small peptides have used reversed phase chromatography but scale-up is difficult. The first step of this new protocol is hydrophobic interaction chromatography, the second and third steps are cation exchange chromatography. The protocol leads to a complete recovery of carnocin KZ 213 with 95% purity and to a concentration factor of 83. From 10 l culture supernatant, 5.8 mg carnocin KZ 213 have been produced with a specific activity of 8,500 UA g⁻¹. The protocol is easy to implement for larger volumes.     

Purification and characterization of a new bacteriocin isolated from a Carnobacterium sp.

A bacteriocin-producing Carnobacterium sp. was isolated from fish. The bacteriocin, termed carnocin UI49, was purified to homogeneity by a four-step purification procedure, including hydrophobic interaction chromatography and reverse-phase chromatography. Carnocin UI49 has a bactericidal mode of action. It was shown to be heat tolerant and stable between pH 2 and 8. At pH above 8, carnocin UI49 was rapidly inactivated. Amino acid analysis revealed a composition of about 35 to 37 amino acids in addition to an unidentified peak which migrates at the position of lanthionine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggests a molecular weight of about 4,500 to 5,000. Mass spectrometry gave a molecular weight of 4,635, which is about 1,000 larger than that calculated from the amino acid analysis data. Performic acid oxidation of carnocin UI49, followed by amino acid hydrolysis, revealed the presence of cysteic acid. The sequence of the first seven amino acid residues was determined to be N-Gly-Ser-Glu-Ile-Gln-Pro-Arg. After the seventh amino acid, carnocin UI49 was not available for further Edman degradation. The results suggest that carnocin UI49 belongs to the class of bacteriocins termed lantibiotics.     

Purification and partial characterization of bacteriocin produced by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> ssp. <Emphasis Type="Italic">lactis</Emphasis> LL171

Lactic acid bacteria (LAB) are possessing ability to synthesize antimicrobial compounds (like bacteriocin) during their growth. In this regard, novel bacteriocin compound secreting capability of LAB isolated from Tulum Cheese in Turkey was demonstrated. The synthesized bacteriocin was purified by ammonium sulphate precipitation, dialysis and gel filtration. The molecular weight (≈3.4 kDa) of obtained bacteriocin was confirmed by SDS-PAGE, which revealed single peptide band. Molecular identification of LAB strain isolated from Tulum Cheese was conducted using 16S rDNA gene sequencing as Lactococcus lactis ssp. lactis LL171. The amino acid sequences (KKIDTRTGKTMEKTEKKIELSLKNMKTAT) of the bacteriocin from Lactococcus lactis ssp. lactis LL171 was found unique and novel than reported bacteriocins. Further, the bacteriocin was possessed the thermostable property and active at wide range of pH values from 1 to 11. Thus, bacteriocin reported in this study has the potential applications property as food preservative agent.     

Production and Characterization of Nisin-Like Peptide Produced by a Strain of Lactococcus lactis Isolated from Fermented Milk

An isolate of Lactococcus lactis from fermented milk was found to produce a bacteriocin peptide. The isolate could grow in a medium with an initial pH of 11.0, in which it produced the bacteriocin extracellularly at the highest level. The level of the bacteriocin in the medium increased in parallel to the bacterial growth and reached its peak during the late exponential phase; thereafter it plateaued. The bacteriocin had a broad antibacterial spectrum similar to that of nisin and inhibited several related species of lactic acid bacteria and other Gram-positive bacteria. The inhibitory activity of the bacteriocin was found to be stable over a wide range of pH and temperature. The molecular weight of the peptide was judged to be 2.5 kDa by SDS-polyacrylamide gel electrophoresis.     

<Emphasis Type="Italic">Enterococcus faecium</Emphasis> isolated from honey synthesized bacteriocin-like substances active against different <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> strains

Four Enterococcus faecium strains, isolated from honeycombs (C1 and M2d strains) and feral combs (Mori1 and M1b strains) secreted antimicrobial substances active against fourteen different Listeria spp. strains. The antimicrobial compound(s) present in the cell free supernatant were highly thermostable (121°C for 15 min) and inactivated by proteolytic enzymes, but not by α-amylase and lipase, thus suggesting a peptidic nature. Since the structural bacteriocin gene determinants of enterocins A and B were PCR amplified from the four E. faecium isolates, only the bacteriocin produced by strain C1 was further characterized: it showed a broad band of approximately 4.0–7.0 kDa in SDS-PAGE and was bactericidal (4 log decrease) against L. monocytogenes 99/287. L. monocytogenes 99/287R, a clone spontaneously resistant to the enterocin produced by E. avium DSMZ17511 (ex PA1), was not inhibited by the enterocin-like compounds produced by strain C1. However, it was inhibited in mixed culture fermentations by E. faecium C1 and a bacteriostatic effect was observed. The bacteriocin-producer Enterococcus strains were not haemolytic; gelatinase negative and sensitive to vancomycin and other clinically relevant antibiotics.     

Divergicin M35-Chitosan Film: Development and Characterization

Chitosan films loaded with bacteriocin were examined by FTIR spectroscopy, tested for color, puncture strength, water vapor permeability, and as antimicrobials of Listeria innocua HPB13. Divergicin M35, a bacteriocin produced by Carnobacterium divergens, was incorporated into films made with chitosan of molecular mass 2 kDa, 20 kDa, or 100 kDa and de-acetylated either 87% or 95%. Only 100 kDa chitosan yielded films that could be peeled and handled easily. The higher degree of de-acetylation increased the total color factor (ΔE) of bacteriocin-loaded films, their permeability, and puncture strength. Incorporation of divergicin M35 into the films increased amide I peak intensity but otherwise did not induce significant structural change. The FTIR spectra of divergicin M35 shed from the films did not differ from those of the original free bacteriocin, except in overall peak intensity. The release of active divergicin M35 from the film was faster into the buffer than into tryptic soy broth and peaked at 10–12 h in both cases. Chitosan 95% de-acetylated and loaded with divergicin M35 was the most active, producing a six-log drop in Listeria innocua HPB13 viable count within 24 h. These results suggest that the biocompatible and biodegradable films developed here have the potential for application as antimicrobials of Listeria spp. in foods, especially ready-to-eat, minimally processed products.

     

Production and characterization of a low‐molecular‐weight bacteriocin from Bacillus licheniformis MKU3

Aims: Enhancing production and characterization of a low‐molecular‐weight bacteriocin from Bacillus licheniformis MKU3. Methods and Results: The culture supernatant of B. licheniformis MKU3 exhibited bacteriocin‐like activity against Gram‐positive and ‐negative bacteria and different fungi and yeast. SDS–PAGE analysis of the extracellular proteins of B. licheniformis MKU3 revealed a bacteriocin‐like protein with a molecular mass of 1·5 kDa. This bacteriocin activity was found to be stable under a pH range of 3·0–10·0 and at temperatures up to 100°C for 60 min, but inactivated by proteinase K, trypsin or pronase E. An experimental fractional factorial design for optimization of production medium resulted in a maximum activity of bacteriocin (11 000 AU ml^?1) by B. licheniformis MKU3. Conclusions: A low‐molecular‐weight bacteriocin‐like protein from B. licheniformis MKU3 exhibited a wide spectrum of antimicrobial activity against several Gram‐positive bacteria, several fungi and yeast. A 3·6‐fold increase in the production of bacteriocin was achieved using the culture medium optimized through a fractional factorial design. Significance and Impact of the Study: A bacteriocin with wide spectrum of activity against Gram‐positive bacterial pathogens, filamentous fungi and yeast suggested its potential clinical use. Statistical method facilitated optimization of cultural medium for the improved production of bacteriocin.     

Identification and Characteristics of Nisin Z-Producing <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> Isolated from Kimchi

We isolated bacteriocin-producing Lactococcus lactis subsp. lactis from Kimchi. The bacteriocin inhibited strains of Clostridium perfringens, C. difficile, Listeria monocytogenes, vancomycin-resistant Enterococcus, and one out of four methicillin-resistant Staphylococcus aureus strains, as well as some closely related lactic acid bacteria. In tricine-SDS-PAGE, the bacteriocin migrated with an apparent molecular weight of about 4 kDa to the same location as nisin A and crude nisin Z. The gene encoding this bacteriocin was found to be identical to that of nisin Z with direct PCR sequence methods. The inhibitory activity was stable against heat and pH, but it was lost at 100°C for 1 h and at 121°C for 15 min. The bacteriocin was inactivated by proteolytic enzymes, but was not affected by lysozyme, lipase, catalase, or β-glucosidase. There were some differences in characteristics from those of nisins described previously. Received: 21 June 2002 / Accepted: 22 July 2002     

Effect of Medium Components on Bacteriocin Production by Lactobacillus Pentosus ST151BR,a Strain Isolated from Beer Produced by the Fermentation of Maize,Barley and Soy Flour

Lactobacillus pentosus ST151BR, isolated from home-brewed beer, produces a 3.0 kDa antibacterial peptide (bacteriocin ST151BR) active against Lactobacillus casei, Lactobacillus sakei, Pseudomonas aeruginosa, Enterococcus faecalis and Escherichia coli. Treatment with Proteinase K or Pronase resulted in loss of activity. Bacteriocin levels of 6400 AU/ml were recorded in MRSbb (De Man-Rogosa-Sharpe broth without Tween 80) at pH 5.5, 6.0 and 6.5. The same growth conditions at pH 4.5 yielded only 1600 AU/ml bacteriocin. Inclusion of Tween 80 in the growth medium reduced bacteriocin production by more than 50%. Growth in the presence of tryptone or tryptone plus meat extract stimulated bacteriocin production, whereas much lower activity was recorded when the bacteria were grown in the presence of meat extract, yeast extract, tryptone plus yeast extract, meat extract plus yeast extract, or a combination of tryptone, meat extract and yeast extract. MRSbb supplemented with maltose, lactose or mannose (2.0%, w/v) yielded bacteriocin levels of 6400 AU/ml. Sucrose or fructose at these concentrations reduced the activity by 50 and 75%, respectively. Growth in the presence of 4.0%(w/v) glucose resulted in 50% activity loss. Glycerol levels as low as 0.1%(w/v) repressed bacteriocin production. Addition of cyanocobalamin, ascorbic acid, thiamine and thioctic acid (1.0 mg/l) to the growth medium did not lead to an increase in bacteriocin production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of growth conditions on production and activity of mesenterocin 5 by a strain of Leuconostoc mesenteroides

The effects of various parameters on production and activity of mesenterocin 5, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides UL5, were investigated. Titres of bacteriocin and minimum inhibitory concentration values were determined by a critical dilution micromethod, using a sensitive strain of Listeria ivanovii as an indicator. Production of the antimicrobial compound was optimal at 37 and 40°C after 9 h of incubation, and was maximized in an aerobic fermentor maintained at pH 5.0. Tween 80 was a major factor in increasing mesenteroxin 5 production and specific production. Large quantities of bacteriocin could be obtained in whey and in whey permeate supplemented with yeast extract in the presence of the surfactant (0.1%). Most of the Listeria strains tested including L. monocytogenes were highly sensitive to the bacteriocin in the pH range 5.5 to 6.0 and at a temperature of 20 to 25°C.     

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.     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.