Characteristics and identification of enterocins produced by Enterococcus faecium JCM 5804T

AIMS: To screen bacteriocin-producing lactic acid bacteria (LAB) in 52 type and reference strains, which have not previously been studied, with respect to bacteriocins, and to characterize the presence of bacteriocins. METHODS AND RESULTS: Only Enterococcus faecium JCM 5804T showed bacteriocin-like activity. It inhibited the growth of Lactobacillus spp., Enterococcus spp., Clostridium spp., Listeria monocytogenes, and vancomycin resistant Enterococcus (VRE). However, it was not effective against Gram-negative strains, Weisella spp., Leuconostoc spp., Lactococcus spp., or methicillin resistant Staphylococcus aureus (MRSA). The inhibitory activity of Ent. faecium JCM 5804T was inactivated by proteinase K, trypsin, alpha-chymotrypsin, and papain, but not by lysozyme, lipase, catalase, or beta-glucosidase. The inhibitory activity was stable at 100 degrees C for 30 min, and had a pH range from 2 to 10. The molecular weight of the partially purified bacteriocin(s) was approx. 4.5 kDa, according to tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Polymerase chain reaction and direct sequencing methods identified three different types of bacteriocins produced by Ent. faecium JCM 5804T, enterocin A, enterocin B, and enterocin P-like bacteriocin. CONCLUSION: Enterococcus faecium JCM 5804T produced three different types of bacteriocins, and they inhibited LAB and pathogens. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report of enterocin A, enterocin B, and enterocin P-like bacteriocin, detected in Ent. faecium JCM 5804T among LAB type and reference strains.     

Partial characterization of bacteriocins produced by environmental strain Enterococcus faecium EK13

AIMS: The partial characterization of bacteriocins produced by an environmental strain Enterococcus faecium EK13, isolated from cattle dung water. METHODS AND RESULTS: A bacteriocin was partially purified by ammonium sulphate precipitation, followed by a SP-Sepharose column, reverse-phase chromatography and N-terminal region sequenced. The anti-microbial substance produced was found to be a heat-stable polypeptide with molecular mass 4.83 kDa, which was determined by N-terminal amino acid sequencing to be enterocin A. A second substance was specified by PCR as enterocin P. Bacteriocins were stable at 4 and -20 degrees C for long storage periods. The optimum of bacteriocin production was observed in the range of pH 5.0-6.5 at 30 and 37 degrees C. The most active substances are produced by strain EK13 in logarithmic growth phase and bacteriocins are produced after 1 h of fermentation. The highest activity detected in fermentation experiments was 51 200 AU ml(-1) and the most sensitive indicator strain was found to be Listeria innocua LMG 13568. Differences in bacteriocin activity against two indicators could be explained by more than one type of enterocin production by strain EK13, or with different mode of action or in different sensitivity of strains. CONCLUSION: Enterococcus faecium strain EK13 isolated from cattle dung water produces two bacteriocins, enterocin A and P, with an inhibitory effect against the strain of the genera Enterococcus, Leuconostoc, Lactobacillus, Streptococcus, Staphylococcus, Bacillus and Listeria (in different origin). SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococcus faecium EK13 environmental strain is a new producer of enterocin A and P. The E. faecium EK13, isolated from cattle dung water, is presented with the further aim to utilize it for waste treatment by biotechnological processes.     

Characterization of a bacteriocin produced by Enterococcus faecium GM-1 isolated from an infant

AIM: To partially characterize the bacteriocin produced by the GM-1 strain of Enterococcus faecium, isolated from the faeces of a newborn human infant. METHODS AND RESULTS: The bacteriocin produced by E. faecium GM-1 showed a broad spectrum of activity against indicator strains of Escherichia coli, Staphylococcus aureus, Vibrio spp., Salmonella typhimurium, Listeria monocytogenes, Lactobacillus acidophilus, and Streptococcus thermophilus. Treatment of the GM-1 bacteriocin with proteolytic enzymes reduced its inhibitory activities. The bacteriocin was stable at 100 degrees C for 20 min and displayed inhibitory activity at neutral pH. The optimal production of bacteriocin from E. faecium GM-1 was obtained when the culture conditions were pH 6.0-6.5 and 35-40 degrees C. The inhibitory activity of the bacteriocin was not substantially changed by the use of different carbon sources in the media, except when galactose was substituted for glucose. The use of a sole nitrogen source caused a decrease in inhibitory activity. A bacteriocin gene similar to enterocin P was identified from the total DNA of E. faecium GM-1 by PCR and direct sequencing methods. CONCLUSION: E. faecium GM-1, which was isolated from the faeces of a newborn baby, produces an enterocin P-like bacteriocin with inhibitory activity against Gram-positive and Gram-negative bacteria, including food-borne pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: E. faecium GM-1, isolated from infant faeces, produces a new bacteriocin that is similar to enterocin P. This bacteriocin is heat stable and has a broad antibacterial spectrum that includes both Gram-positive and Gram-negative bacteria.     

Antilisterial activity of lactic acid bacteria isolated from rigouta, a traditional Tunisian cheese

AIMS: Screening for lactic acid bacteria (LAB) producing bacteriocins and other antimicrobial compounds is of a great significance for the dairy industry to improve food safety. METHODS AND RESULTS: Six-hundred strains of LAB isolated from 'rigouta', a Tunisian fermented cheese, were tested for antilisterial activity. Eight bacteriocinogenic strains were selected and analysed. Seven of these strains were identified as Lactococcus lactis and produced nisin Z as demonstrated by mass spectrometry analysis of the purified antibacterial compound. Polymerase chain reaction experiments using nisin gene-specific primers confirmed the presence of nisin operon. Plasmid profiles analysis suggests the presence of, at least, three different strains in this group. MMT05, the eighth strain of this antilisterial collection was identified, at molecular level, as Enterococcus faecalis. The purified bacteriocin produced by this strain showed a molecular mass of 10 201.33 +/- 0.85 Da. This new member of class III bacteriocins was termed enterocin MMT05. CONCLUSIONS: Seven lactococcal strains producing nisin Z were selected and could be useful as bio-preservative starter cultures. Additional experiments are needed to evaluate the promising strain MMT05 as bio-preservative as Enterococci could exert detrimental or beneficial role in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Only a few antibacterial strains isolated from traditional African dairy products were described. The new eight strains described herein contribute to the knowledge of this poorly studied environment and constitute promising strains for fermented food safety.     

Bacteriocin production, plasmid content and plasmid location of enterocin P structural gene in enterococci isolated from food sources

AIMS: To characterize bacteriocin production, antimicrobial spectrum and plasmid content in bacteriocinogenic enterococci from foods. METHODS AND RESULTS: Bacteriocinogenic Enterococcus faecium (14 isolates) and Enterococcus faecalis (three isolates) showed two different patterns of bacteriocin production in liquid broth: exponential-phase and stationary-phase production. Bacteriocin concentrates from all enterococci were inactivated by trypsin, but seldom by heat (100-117 degrees C), extremes of pH (2.0 to 9.0) or reducing agents (such as dithiothreitol). All bacteriocin concentrates were active against Listeria innocua and Listeria monocytogenes, and most were also active against many Ent. faecalis and Ent. faecium isolates. Enterococci clustered in three main groups according to their plasmid content (which included plasmids from 2.0 to 53 kb). Several isolates from different foods showed almost identical plasmid profiles. The enterocin P structural gene (entP) was detected by hybridization on plasmids of c. 19, 26 and/or 35-38 kb. CONCLUSIONS: Enterococci from food show different patterns of bacteriocin production and different plasmid content in spite of carrying similar bacteriocin-encoding genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the diversity of bacteriocinogenic enterococci from food sources carrying apparently similar enterocin genes.     

Durancin L28-1A, a new bacteriocin from Enterococcus durans L28-1, isolated from soil

AIMS: To isolate, characterize and identify bacteriocins from lactic acid bacteria in soil. METHODS AND RESULTS: Thirty-four acid-producing bacteria were isolated from 87 soil samples. Antibacterial activities were detected, and one strain, L28-1 produced a bacteriocin that was active against some Gram-positive bacteria. L28-1 was identified as Enterococcus durans by 16S rDNA sequence analysis and API50CHL. This bacteriocin did not lose its activity after autoclaving (121 degrees C for 15 min), but was inactivated by protease K. The bacteriocin was purified by hydrophobic column chromatography, and Sep-Pak C(18). Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the partially purified bacteriocin contained numerous protein bands. Two bands that displayed antibacterial activities were c. 3.4 and 2.5 kDa in size. In this work, the 3.4-kDa bacteriocin was analysed with N-terminal amino acid and DNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results indicated that the 3.4-kDa bacteriocin of Ent. durans L28-1 is a new natural enterocin variant. CONCLUSIONS: Enterococcus durans L28-1 produced a new bacteriocin. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a novel bacteriocin that is produced by Ent. durans that has potential for use as a food preservative.     

Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces

A total of 92 enterococci, isolated from the faeces of minipigs subjected to an in vivo feeding trial, were screened for the production of antimicrobial substances. Bacteriocin production was confirmed for seven strains, of which four were identified as Enterococcus faecalis and three as Enterococcus faecium, on the basis of physiological and biochemical characteristics. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by alpha-chymotrypsin, proteinase K, trypsin, pronase, pepsin and papain, but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS-PAGE, was approximately 3.4 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B.     

Bacteriocin T8, a novel class IIa sec-dependent bacteriocin produced by Enterococcus faecium T8, isolated from vaginal secretions of children infected with human immunodeficiency virus

Enterococcus faecium T8, isolated from vaginal secretions of children with human immunodeficiency virus, produces a class IIa sec-dependent bacteriocin that is structurally different from three other class IIa sec-dependent bacteriocins, i.e., enterocin P and an enterocin P-like bacteriocin, produced by Enterococcus faecium, and bacteriocin 31, produced by Enterococcus faecalis, and from a class III bacteriocin produced by E. faecalis. The genes encoding the bacteriocin, immunity protein, mobilization protein, and relaxase nuclease are located on a 7-kb plasmid. Bacteriocin T8 has a molecular mass of 5.1 kDa based on its DNA sequence, similar to the 5.0 kDa recorded for bacteriocin 31 but larger than the 4.6 kDa reported for enterocin P. At the amino acid level, bacteriocin T8 is 69% homologous to bacteriocin 31 and 47% homologous to enterocin P. Bacteriocin T8 is active against E. faecalis isolated from patients diagnosed with vaginosis, against Lactobacillus sakei, and against a Propionibacterium sp. The peptide is heat stable (60 min at 100 degrees C) and remains active in phosphate buffer from pH 4.0 to 10.0. The mode of activity is bactericidal, as determined with E. faecalis.     

In vitro study on bacteriocin production of Enterococci associated with chickens

In recent years, the approach of using innovative strategies such as probiotics or bacteriocins for the prevention or treatment of bacterial infections has come into focus. The present study was undertaken to check in vitro ability of Enterococci-isolated from the gastrointestinal tract of chickens-to produce a bacteriocin-like substance and to describe some further probiotic properties in five selected Enterococcus faecium strains. All strains (n=17) were found to produce bacteriocin-like substances against 14 out of 20 indicator bacteria of animal, food or environmental origin. Selected E. faecium strains expressed sufficient survival by pH 3.0 after 3h, in the presence of 1% bile after 24h and they were sensitive to most of antimicrobials tested. All tested strains adhere to the human, canine and porcine intestinal mucus (between 1.5% and 9.2%). However, better adhesion ability was observed for the canine mucus. PCR detection of enterocin structural genes determined presence of enterocins A and P genes in all selected strains. Characterization of bacteriocin substance in detail was performed in E. faecium EF55. The EF55 strain produced a bacteriocin-like substance (during the late logarithmic and early stationary growth phase) with inhibitory activity mostly against Gram-positive bacteria (100-51,200 AU/mL) including Listeria monocytogenes. Proteinaceous character of the bacteriocin substance was confirmed (its inhibitory activity was lost after its treatment with proteases), it was found to be stable after heating (100 degrees C 10 min) and during 12 months storage at -20 degrees C. The highest inhibitory activity of bacteriocin produced by EF55 strain (growing in MRS) broth was achieved between pH 7.0 and 9.0.     

Development of a PCR-based assay for rapid detection of class IIa bacteriocin genes

Aims: We have developed a PCR‐based assay using custom designed panel of primers which allows rapid detection of class IIa bacteriocin‐coding genes. To demonstrate the applicability of the developed assay, the method was applied on 40 metagenomic DNA preparations isolated from native microbiota of Polish artisanal cheeses produced in the Tatra Mountains. Methods and Results: The developed assay was designed on the basis of a large scale alignment of class IIa bacteriocin‐coding genes. A panel of seven primer pairs with confirmed ability to detect class IIa bacteriocin‐coding sequences was obtained. The following study has revealed a superb bacteriocinogenic potential of all forty analysed cheese samples. Conclusions: The majority of obtained sequences were lactic acid bacteria (LAB) related, although some sequences showed significant similarity to bacteriocin‐coding sequences present in non‐LAB bacteriocin producers. The results suggest that several potentially new bacteriocin‐coding sequences were found. Significance and Impact of the Study: The developed assay can be extremely helpful in establishing whether isolates from the environment of interest have a potential of synthesizing antilisterial class IIa bacteriocins. Application of the approach may represent a useful tool contributing to ecological studies looking for valuable probiotic, bacteriocinogenic microbiota developing in foods.     

Isolation and biochemical characterisation of enterocins produced by enterococci from different sources

AIMS: Comparison of enterocins produced by six Enterococcus faecium strains and one Ent. faecalis strain isolated from different origin with regard to their microbiological and biochemical characteristics in view of their technological potential and practical use. METHODS AND RESULTS: The seven enterococci were sensitive to the glycopeptide antibiotics vancomycin and teicoplanin and did not show haemolytic activity. The absence of the glycopeptide-resistant genotypes and the genes involved in the production of the lantibiotic cytolysin was confirmed by PCR. The enterocins were active towards Listeria innocua and other lactic acid bacteria. Their temperature stability was dependent on the pH and their activity was higher at acidic pH. A bactericidal and bacteriolytic effect was shown. PCR analyses revealed that the gene of enterocin A was present in the genome of Ent. faecium CCM 4231, Ent. faecium 306 I.2.20 and Ent. faecalis Y; both enterocin A and B genes were present in the genome of Ent. faecium LMG 11423T, Ent. faecium RZS C5 and Ent. faecium RZS C13. Enterocin P was detected in the genome of Ent. faecium RZS C5 and Ent. faecium RZS C13. No signal was found for Ent. faecium SF 68. Enterocins from Ent. faecium RZS C5, Ent. faecium RZS C13 and Ent. faecium SF 68 were purified to homogeneity. CONCLUSIONS: Ent. faecium RZS C5 and Ent. faecium RZS C13 produced an enterocin with a molecular mass of 5460 and 5477 Da, respectively, which was in the range of that of enterocin B. The amino acid sequence analysis of the enterocin from Ent. faecium RZS C13 revealed 24 N-terminal residues, which were identical to those of enterocin B. The enterocin from Ent. faecium SF 68 had a molecular mass of 4488 Da, which did not correspond to any enterocin known so far. SIGNIFICANCE AND IMPACT OF THE STUDY: The number of characterized enterocins is increasing. As this type of work is tedious and time-consuming, it may be interesting to include PCR as a first step to know if the Enterococcus strain in study produces either a known or a new enterocin. Also, it is important to check the absence of cytolysin and resistance to vancomycin for a further application of the Enterococcus strain in food or health applications.     

Characterization and antimicrobial spectrum of bacteriocins produced by lactic acid bacteria isolated from traditional Bulgarian dairy products

Aims:  To isolate bacteriocin-producing lactic acid bacteria (LAB) with high wide spectrum antibacterial activity and to characterize their inhibitory peptides.
Method and Results:  Seven LAB strains [ Lactobacillus casei ssp. rhamnosus (PC5), Lactobacillus delbrueckii ssp. bulgaricus (BB18), Lactococcus lactis ssp. lactis (BCM5, BK15), Enterococcus faecium (MH3), Lactobacillus plantarum (BR12), Lactobacillus casei ssp. casei (BCZ2)], isolated from authentic Bulgarian dairy products were capable of producing bacteriocins, inhibiting the widest range of pathogenic bacteria. The bacteriocins were resistant to heating at 121°C for 15 min, stable at pH 2–10, sensitive to protease, insensitive to α-amylase and lipase. Two of bacteriocins produced by Lact. bulgaricus BB18 (bulgaricin BB18) and E. faecium MH3 (enterocin MH3) were purified and the molecular masses were determined. The N -terminal amino acid sequence of bulgaricin BB18 did not show strong homology to other known bacteriocins.
Conclusions:  Lactobacillus bulgaricus BB18 and E. faecium MH3 produce two novel bacteriocins highly similar to the pediocin-like nonlantibiotics.
Significance and Impact of the Study:  The two bacteriocins are potential antimicrobial agents and, in conjunction with their producers, may have use in applications to contribute a positive effect on the balance of intestinal microflora. Furthermore, bulgaricin BB18 strongly inhibits Helicobacter pylori .     

Use of bacteriocinogenic lactic acid bacteria to inhibit spontaneous nisin-resistant mutants of Listeria monocytogenes Scott A

Nisin is a bacteriocin with a broad antibacterial spectrum including strains of Listeria monocytogenes . Populations of L. monocytogenes , however, frequently contain spontaneous nisin-resistant mutants. When a culture of L. monocytogenes Scott A was exposed to nisin concentrations between 10 and 500 IU ml⁻¹, the initial decrease in viable numbers was followed by regrowth of survivors to nisin. Nisin-resistant mutants of L. monocytogenes Scott A were isolated after a single exposure to nisin at 100 IU ml⁻¹ and were shown to be sensitive to the non-nisin bacteriocins, sakacin A and enterocin B, produced by Lactobacillus sake Lb 706 and Enterococcus faecium BFE 900, respectively. The regrowth of L. monocytogenes Scott A following the initial decrease due to exposure to nisin was prevented by nisin-resistant Lact. sake Lb 706–1a and to a somewhat lesser extent, by Ent. faecium BFE 900–6a. Listerial cells surviving nisin action were thus inhibited by the bacteriocin-producing strains that might be used as starter or protective cultures in foods. Growth of a nisin-resistant mutant of L. monocytogenes Scott A (Li3) was also suppressed by the bacteriocinogenic cultures. Use of nisin in combination with a starter culture producing a non-nisin antilisterial bacteriocin may therefore prevent the emergence of nisin-resistant mutants of L. monocytogenes .     

A comparison of factors affecting the production of two bacteriocins from lactic acid bacteria

The effect of tryptone, yeast extract, Tween 80 and initial pH on the production of enterocin 1146 and lactocin D, two bacteriocins produced by lactic acid bacteria, was studied in a basal buffered medium (tryptone-yeast extract-tween, TYT) using factorial experiments and empirical modelling. Production of enterocin 1146 was affected by pH, yeast extract and Tween 80 and to a lesser degree, by the initial pH of the medium. On the basis of the predictions of the models developed, three TYT media (TYT10, TYT11 and TYT30) were designed to maximize bacteriocin production while minimizing the amount of peptides in the medium. Growth and bacteriocin production by Enterococcus faecium DPC 1146 (enterocin 1146), Lactococcus lactis subsp. lactis biovar diacetylactis DPC 3286 (lactocin D) and Lact. lactis subsp. cremoris LMG2130 (lactococcin A) was compared in TYT media and seven other culture media (Elliker lactic broth, M17, M17 dialysate, MRS, tryptose phosphate, tryptone yeast extract broth, yeast glucose Lemco broth). Bacteriocin production in TYT media was comparable with that in M17 and MRS, which had a higher peptide content. TYT30 allowed good production of enterocin 1146 and lactocin D while TYT11 proved acceptable for all the strains tested.     

Partial characterization of bacteriocins produced by human Lactococcus lactis and Pediococccus acidilactici isolates

AIMS: The aim of this study was to isolate bacteriocin-producing lactic acid bacteria (LAB) from human intestine. METHODS AND RESULTS: A total of 111 LAB were isolated from human adult stool and screened for their bacteriocin production. Neutralized cell-free supernatants from Lactococcus lactis subsp. lactis MM19 and Pediococcus acidilactici MM33 showed antimicrobial activity. The antimicrobials in the supernatant from a culture of L. lactis inhibited Enterococcus faecium, various species of Lactobacillus and Staphylococcus aureus; while those in the supernatant from a culture of P. acidilactici inhibited Enterococcus spp., some lactobacilli and various serotypes of Listeria monocytogenes. The antimicrobial metabolites were heat-stable and were active over a pH range of 2-10. The antimicrobial activities of the supernatants of both bacteria were inhibited by many proteases but not by catalase. The plate overlay assay allowed an approximation of size between 3.5 and 6 kDa for both antimicrobial substances. CONCLUSIONS: As the antagonistic factor(s) produced by L. lactis MM19 and P. acidilactici MM33 were sensitive to proteolytic enzymes, it could be hypothesized that bacteriocins were involved in the inhibitory activities. Inhibition spectrum and biochemical analysis showed that these bacteria produced two distinct bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: We are the first to isolate bacteriocin-producing strains of Pediococcus and Lactococcus from human intestine. These strains might be useful for control of enteric pathogens.     

Native and heterologous production of bacteriocins from gram-positive microorganisms

In nature, microorganisms can present several mechanisms for setting intercommunication and defense. One of these mechanisms is related to the production of bacteriocins, which are peptides with antimicrobial activity. Bacteriocins can be found in Gram-positive and Gram-negative bacteria. Nevertheless, bacteriocins produced by Gram-positive bacteria are of particular interest due to the industrial use of several strains that belong to this group, especially lactic acid bacteria (LAB), which have the status of generally recognized as safe (GRAS) microorganisms. In this work, we will review recent tendencies in the field of invention and state of art related to bacteriocin production by Gram-positive microorganism. Hundred-eight patents related to Gram-positive bacteriocin producers have been disclosed since 1965, from which 57% are related bacteriocins derived from Lactococcus, Lactobacillus, Streptococcus, and Pediococcus strains. Surprisingly, patents regarding heterologous bacteriocins production were mainly presented just in the last decade. Although the major application of bacteriocins is concerned to food industry to control spoilage and foodborne bacteria, during the last years bacteriocin applications have been displacing to the diagnosis and treatment of cancer, and plant disease resistance and growth promotion.     

Purification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag

AIMS: The aim of this study was to isolate and identify bacteriocin-producing lactic acid bacteria (LAB) issued from Mongolian airag (traditional fermented mare's milk), and to purify and characterize bacteriocins produced by these LAB. METHODS AND RESULTS: Identification of the bacteria (Enterococcus durans) was carried out on the basis of its morphological, biochemical characteristics and carbohydrate fermentation profile and by API50CH kit and 16S rDNA analyses. The pH-neutral cell-free supernatant of this bacterium inhibited the growth of several Lactobacillus spp. and food-borne pathogens including Escherichia coli, Staphylococcus aureus and Listeria innocua. The antimicrobial agent (enterocin A5-11) was heat stable and was not sensitive to acid and alkaline conditions (pH 2-10), but was sensitive to several proteolytic enzymes. Its inhibitory activity was completely eliminated after treatment with proteinase K and alpha-chymotrypsin. The activity was however not completely inactivated by other proteases including trypsin and pepsin. Three-step purification procedure with high recovery yields was developed to separate two bacteriocins. The applied procedure allowed the recovery of 16% and 64% of enterocins A5-11A and A5-11B, respectively, present in the culture supernatant with purity higher than 99%. SDS-PAGE analyses revealed that enterocin A5-11 has a molecular mass of 5000 Da and mass spectrometry analyses demonstrates molecular masses of 5206 and 5218 Da for fractions A and B, respectively. Amino acid analyses of both enterocins indicated significant quantitative difference in their contents in threonine, alanine, isoleucine and leucine. Their N-termini were blocked hampering straightforward Edman degradation. CONCLUSIONS: Bacteriocins A5-11A and B from Ent. durans belong to the class II of bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: Judging from molecular masses, amino acid composition and spectrum of activities, bacteriocins A5-11A and B from Ent. durans show high degree of similarity with enterocins L50A and L50B isolated from Enterococcus faecium (Cintas et al. 1998, 2000) and with enterocin I produced by Ent. faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation (Floriano et al. 1998).     

Anti‐listerial activity of bacteriocin‐producing Lactobacillus curvatus CWBI‐B28 and Lactobacillus sakei CWBI‐B1365 on raw beef and poultry meat

Aim: The study aimed to evaluate the effect of the bacteriocins produced by Lactobacillus sakei CWBI‐B1365 and Lactobacillus curvatus CWBI‐B28 on the growth and survival of Listeria monocytogenes in raw beef and poultry meat. Methods and Results: The sakacin P and sakacin G structural genes were identified in Lact. curvatus CWBI‐B28 and Lact. sakei CWBI‐B1365 using PCR amplification, respectively. The effect of the two bacteriocinogenic strains either alone or together, and that of the nonbacteriocin‐producing strain Lact. sakei LMG17302, on the growth of L. monocytogenes was evaluated in beef and poultry meat. In raw beef, the pathogenic bacteria were inhibited by the bacteriocinogenic strains. The bacteriocinogenic strains had no activity in raw chicken meat when inoculated separately, while they showed a clear anti‐Listeria effect when applied together. Conclusion: Sakacin G producing Lact. sakei and sakacin P producing Lact. curvatus may be applied in raw beef to inhibit L. monocytogenes. In poultry meat, the inhibition of L. monocytogenes could only be achieved by a combined application of these bacteriocin‐producing strains. Significance and Impact of the Study: In some meat products, the combined application of different class IIa bacteriocin producing lactic acid bacterium can enhance the anti‐listerial activity.     

Bacteriocins from lactic acid bacteria: production, purification, and food applications

In fermented foods, lactic acid bacteria (LAB) display numerous antimicrobial activities. This is mainly due to the production of organic acids, but also of other compounds, such as bacteriocins and antifungal peptides. Several bacteriocins with industrial potential have been purified and characterized. The kinetics of bacteriocin production by LAB in relation to process factors have been studied in detail through mathematical modeling and positive predictive microbiology. Application of bacteriocin-producing starter cultures in sourdough (to increase competitiveness), in fermented sausage (anti-listerial effect), and in cheese (anti-listerial and anti-clostridial effects), have been studied during in vitro laboratory fermentations as well as on pilot-scale level. The highly promising results of these studies underline the important role that functional, bacteriocinogenic LAB strains may play in the food industry as starter cultures, co-cultures, or bioprotective cultures, to improve food quality and safety. In addition, antimicrobial production by probiotic LAB might play a role during in vivo interactions occurring in the human gastrointestinal tract, hence contributing to gut health.     

Detection and Characterization of Pediocin PA-1/AcH like Bacteriocin Producing Lactic Acid Bacteria

Fifty-five bacteriocinogenic lactic acid bacteria (LAB) isolated from seven different sources. Eight isolates were found to produce pediocin PA-1 like bacteriocin as detected by pedB gene PCR and dot-blot hybridization. The culture filtrate (CF) activity of these isolates exhibited strong antilisterial, antibacterial activity against tested food-borne pathogens and LAB. The identification and genetic diversity among the selected LAB was performed by conventional morphological and molecular tools like RFLP, RAPD, and 16S rDNA gene sequencing. The isolates were identified as, 1 each of Pediococcus acidilactici Cb1, Lactobacillus plantarum Acr2, and Streptococcus equinus AC1, 2 were of P. pentosaceus Cb4 and R38, and other 3 were Enterococcus faecium Acr4, BL1, V3. Partial characterization of the bacteriocins revealed that the peptide was heat-stable, active at acidic to alkaline pH, inactivated by proteolytic enzymes, and had molecular weight around 4.6 kDa and shared the properties of class IIa pediocin-family. The bacteriocin production at different temperatures, pH, and salt concentrations was studied to investigate the optimal condition for application of these isolates as a starter culture or as a biopreservative in either acidic or non-acidic foods.