Presence Of Enterocin Research Articles

Characterization of probiotic Enterococcus lactis RS5 and purification of antibiofilm enterocin

Abstract Enterocin R5 is an antibacterial peptide found in the culture supernatant of Enterococcus lactis RS5 with a broad spectrum activity. The RS5 was isolated from a marine fish, Sillago indica. Enterocin R5 inhibited Staphylococcus aureus, Salmonella enterica ser. Typhi, Proteus vulgaris, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Bacillus cereus. Furthermore, bacteriocin gene cluster characterization showed 70–85% amino acid similarity to the class II bacteriocin from Enterococcus species. The bioactive cell-free supernatant was purified by bio-active guided column chromatography followed by SDS-PAGE analysis revealed that the presence of enterocin with a molecular mass of approximately 6 kDa. The probiotic E. lactis RS5 characterization suggest that tolerance to bile salt, low pH and pancreatin digestion. Moreover, enterocin R5 exhibited more than 60% anti-biofilm activity against biofilm forming food spoilage bacteria at 100 μg/ml concentration. Therefore, this enterocin R5 has potential to be used as an alternative antibacterial agent for the treatment of biofilm-associated food spoilage pathogens.

Antilisterial Effect and Influence on Listeria monocytogenes Gene Expression of Enterocin or Enterococcus faecalis in Sliced Dry-Cured Ham Stored at 7°C

In this study, we focused on the effect of an enterocin or an Enterococcus faecalis strain added onto sliced dry-cured ham that was artificially inoculated with Listeria monocytogenes and stored at 7°C. The population of L. monocytogenes and the expression of five genes were monitored throughout the storage period. A persistent and a nonpersistent strain were tested, and both were influenced by the presence of the enterocin; both populations were reduced by more than 2 Log CFU/g after 14 days compared with the control, noninoculated ham. The presence of E. faecalis, a bacteriocin-producing lactic acid bacterium, had a less pronounced effect on the viable counts for both strains. Concerning gene expression, a common trend observed for both strains in the presence of enterocin was the down-regulation of genes tested after 30 min of storage at 7°C. For the remainder of the storage period, the expression fluctuated but was mostly reduced. Similarly, the presence of E. faecalis led to an overall down-regulation of genes. The effect on gene expression of both enterocin and E. faecalis was more pronounced on the nonpersistent L. monocytogenes strain. Although the potential of a bacteriocin and a bacteriocin-producing microorganism to control L. monocytogenes was confirmed, this study highlights that gene expression may be influenced and needs to be evaluated when considering such biopreservation interventions.

Characterization of functional properties of Enterococcus spp. isolated from Turkish white cheese

Enterococcus species was isolated from Turkish white cheese and genotypic characterization of these isolates revealed the presence of 12 distinct strains belonging to 5 different species: Enterococcus faecium, E. faecalis, E. durans, E. gallinarum and E. italicus. All strains showed good survival ability under bile salt conditions. The antibiotic resistance of these strains was low but two strains showed high levels of resistance including resistance to vancomycin. PCR detection of virulence determinant in these isolates revealed that cheese isolate enterococci contained virulence genes common in gut and food originated enterococci and importantly two strains harboured cylB gene related to cytolysin metabolism. No complete hemolytic activity was observed for Enterococcus strains but partial hemolytic activity was observed 8 out of 12 strains. Additionally, all strains showed important levels of antimicrobial activity against food-borne pathogens and PCR screening of genes encoding enterocin A and B indicated the presence of enterocin B gene in all tested strains. The antimicrobial activities of the tested strains were lost after proteolytic enzyme treatments but no alteration was observed at different pHs and after heat treatments. In summary, this study reflected characteristics of Enterococcus strains presented in Turkish white cheese in terms of functional and safety perspectives.

The potential influence of two Enterococcus faecium on the growth of Listeria monocytogenes

Two strains of Enterococcus faecium isolated from meat products were analyzed for the antimicrobial activity, safety and inhibition of Listeria monocytogenes in co-culture experiments. The production of bacteriocin-like substances and the presence of enterocin A, B, P genes (entA, entB, entP) were confirmed in E. faecium B1 whilst E. faecium B2 harbored neither of these characteristics. None of E. faecium strains harbored the virulent determinants studied (esp, hylEfm, IS 16, VanA and VanB) and were sensitive to most common antibiotics. In the co-culture experiment, both E. faecium B1 and E. faecium B2 had an inhibition effect towards L. monocytogenes. However, E. faecium B1 inhibited the growth of L. monocytogenes in the early stages of co-culture, and then probably reduced the cell counts of L. monocytogenes until an undetectable level after four days. Thus the cell counts of L. monocytogenes stayed at the initial inoculum level during the co-culture with E. faecium B2, suggesting that the bacteriocin-like substance played an important role in the inhibition effect. Meanwhile, the pH in co-culture declined lower than that in mono-culture in the early days, indicating the production of acid might participate in the inhibition process. Also, E. faecium B1 could be considered to be used as an effective potential bio-protective culture, so as to improve the safety of meat products.

Enterococcus faecium LM-2, a multi-bacteriocinogenic strain naturally occurring in “Byaslag”, a traditional cheese of Inner Mongolia in China

A bacteriocin-producing strain LM-2 was isolated from “Byaslag”, a traditional cheese in Inner Mongolia of China, and identified as Enterococcus faecium. The strain produced the maximum activity of enterocin (5120 AU ml −1) in MRS medium in the mid-stationary phase of growth (i.e. after 30 h at 37 °C). Purified enterocin LM-2 was obtained by using a two-step procedure consisting of ammonium sulfate precipitation and cation exchange chromatography. Tricine–SDS–PAGE of the purified enteriocin LM-2 contained two active protein bands between 3.5 and 6.4 kDa. It showed a broad spectrum of activity against most of the tested strains in the genera Listeria, Staphylococcus, Bacillus, Clostridium, Salmonella and Pseudomonas. All Listeria strains tested, including L. monocytogenes, were highly sensitive to this bacteriocin. The inhibitory activity of enterocin LM-2 against L. monocytogenes was bactericidal without concomitant cell growth, and the highest level of adsorption (87.5%) to the surface of L. monocytogenes 54002 cells was founded at pH 6.0. This strong inhibitory activity against L. monocytogenes, coupled with its broad pH-resistance and heat-stability indicated its potential use as a food preservative to enhance the safety of fermented foods. The presence of enterocin P and L50-like structural genes in E. faecium LM-2 was established by PCR and direct sequencing, which indicated strain LM-2 may be a new multi-bacteriocin producer strain.

Production of Antibodies against Enterocin B for Immunological Detection and Purification Purposes

Polyclonal antibodies were generated against the bacteriocin enterocin B by conjugating enterocin B (ENT) to bovine serum albumin (BSA) through a bifunctional crosslinking agent. Rabbits immunized with the ENT-BSA conjugates developed antibodies specific for enterocin B. Non-competive indirect (NCI) ELISAs were able to detect the presence of enterocin B in the supernatant of a producer strain and from a partially-purified sample. Competitive indirect (CI) ELISAs demonstrated concentrations of enterocin B ranging from 400-1600 ng ml−1 were required for 50% antibody binding inhibition. An immunoaffinity chromatography column was developed by linking the antibodies to CNBr-activated Sepahrose 4B. The purification method yielded pure enterocin; however, the harsh elution conditions did not render a regeneratable column.