Characterization of Bacteriocins Produced by Lactobacillus brevis NM 24 and L.fermentum NM 332 Isolated from Green Olives in Iran
N Mojgani, G Sabiri, M Ashtiani, M Torshizi
antagonistic activity, bacteriocin, food preservative, green olives, lactobacillus
N Mojgani, G Sabiri, M Ashtiani, M Torshizi. Characterization of Bacteriocins Produced by Lactobacillus brevis NM 24 and L.fermentum NM 332 Isolated from Green Olives in Iran. The Internet Journal of Microbiology. 2008 Volume 6 Number 2.
The aim of the study was to isolate and characterize bacteriocin producing Lactic Acid Bacteria (LAB) from local olive samples and to exploit their potential as biopreservative. The two lactobacillus strains namely
Lactic acid bacteria
In recent years food safety has become an important international concern. Great attention is being drawn towards application of the bacteriocins from lactic acid bacteria (LAB). Much interest have developed in the bacteriocins isolated from LAB as most of the bacteria in this group are reported as GRAS (generally regarded as safe) microorganisms and their bacteriocins are considered innocuous due to proteolytic degradation in the gastrointestinal (GI) tract. Only few reports are available which indicate bacteriocin production in LAB isolated from olives (Todorov and Dicks, 2005), and hence it appeared interesting to screen bacteriocin producing potential of LAB strains isolated from green olives in Iran. In this attempt, two bacteriocin producing lactobacillus strains were isolated from local green olive samples and characterized for exploiting their potential as suitable candidate for future application as a safe and efficacious biological preservative.
Materials And Methods
I. Bacterial strains and culture media
A number of green olive samples procured from local market were analyzed for the presence of Lactic Acid Bacteria. Initially, the seeds were removed from the olives and then crushed, homogenized with kitchen blender, and inoculated in the deMan Ragosa and Sharpe (MRS) broth for enrichment of resident LAB. From appropriate 10-fold dilutions, isolation of bacteria was carried out on MRS agar by incubation anaerobically at 37°C for 48 h. The cultures were purified by repeated streaking. Strains were identified to genus level by gram staining (morphological characteristics) and catalase test. The potentially interesting isolates were later identified to species level by biochemical tests, carbohydrate fermentation pattern using the AP1 50CH strips (AP1 Systems, Biomerieux Sa, France).
All other bacteria used as indicator organisms for sensitivity tests were propagated in brain-heart-infusion broth (BHI, Difco) and trypticase soya broth (HiMedia, India) at 37°C for 24 hrs. For agar medium 1.6% w/v of granulated agar-agar was added to broth medium, while 0.7% semisolid medium was used. The isolate was maintained as frozen stock culture at -20°C in MRS broth with 5% glycerol and propagated twice before use in experiments.
II. Bacteriocin Screening
All the isolated lactobacillus strains were screened for their antibacterial potential by the well diffusion and spot on lawn assay described earlier (Schillinger and Lucke, 1989; Harris et al., 1989; Aly and Abo-Amer, 2007). The two isolates identified as
To pertinacious nature of the antagonistic agent was evaluated by treating the crude bacteriocin samples to various enzymes. Enzymes (all obtained from Sigma) used were lipase, pronase E , pepsin, catalase, trypsin, lysozyme and proteinase K. 500 µl samples (fraction a) from both the isolates were incubated with 1 mg of each enzyme per ml at 37ºC except for samples containing trypsin and catalase, which were incubated at 25ºC. The remaining activity was determined after 1, 2, 4 and 12 hrs by well diffusion assay. Prior to being assayed for bacteriocin activity, preparations containing trypsin were treated with trypsin inhibitor (Sigma) according to the manufacturer’s instructions (Wanda et
The antimicrobial activity of the bacteriocin was defined as the reciprocal of the highest dilution showing inhibition of the indicator lawn and was expressed in arbitrary units per ml (AU ml-1)
III. Partial Purification and molecular size estimation of Bacteriocin Samples
The filtered supernatant fluid (fraction b) collected from both producer strains was concentrated to one tenth of its original volume by PEG and Vacuum evaporation, followed by precipitation assays.
IV. Physico-Chemical Characterization of Partially Purified Bacteriocin Samples
The partially purified bacteriocin samples (fraction d) were characterized with respect to thermal and pH stability, susceptibility to salt and surfactants, stability during storage, treatment with dissociating agents and mitomycin C and UV light induction.
To study the effect of UV light, a 10 ml aliquot of cultured broth was placed in a sterile petri dish and exposed to short – wave UV light (254nm) from a Electric germicidal bulb at a distance of 20 cm. Times of exposure ranged from 0 to 2 min. (Wanda and Bonita, 1991). After each time interval, bacteriocin activity was analyzed.
V. Stability of Bacteriocin during Storage
Purified bacteriocin fractions (d) from both lactobacillus strains was stored at three different temperatures (–20, 4 and 37ºC). After every month of interval the bacteriocin activity was determined by previously described method, till the activity existed.
Initially, two lactobacillus strains demonstrating maximum inhibitory action against other closely related strains, was isolated from green olive samples. The isolates were later identified to species level and studied in detail for their bacteriocin producing ability. The two isolates identified as
Table. 2 depicts the antagonistic effect of the bacteriocins on the growth of other Gram-positive and Gram-negative bacteria used as indicator. The bacteriocins in study were able to inhibit the growth of
A significant increase in yield and purification fold of the bacteriocin in study was observed during different purification stages (Table- 3). The crude supernatant fluid of
Specific Activity (AU/mg) = Total activity of the subsequent purification step/ Total protein of the same step Yield (%) =Total activity of the subsequent purification step/ Total activity in the crude culture supernatant Purification (fold) = Specific activity of the subsequent purification step/ specific activity of the crude culture supernatant
Attempts to size the bacteriocin under denaturing conditions were obscured due to diffuse banding. However under non-denaturing conditions the exact location of the protein giving activity was detected. The bacteriocin NM 332 was resolved as a single band of approximately 8 KDa while that of NM 24 appeared to be 37 KDa. These bands showed zone of clearance when overlaid with the indicator lawn and thus were confirmed to be bacteriocin related.
The effects of heat, pH, organic solvents, salt, and surfactants on bacteriocin activity were determined. The bacteriocin produced by
Table- 6 shows the effect of dissociating agents on bacteriocin activity. Exposure to most of surfactants tested resulted in an increase in the bacteriocin titer (by at least one to two fold dilutions). However, Tween 20 had adverse effect on these bacteriocins and their activity was completely demolished after subjection to this surfactant within only 2 hrs of incubation.
Both the physical and chemical inducing agents adopted for induction of the bacteriocinogenic strains, failed to induce the activity and no increase in titer was seen when the producer cells were subjected to Mitomycin C or UV light. Moreover, the possibility of plasmid encoded bacteriocin production in both the isolates was ruled out as no plasmid was observed in these strains.
Effect of storage on bacteriocin activity indicated full stability of the bacteriocins in study at –20ºC during three years of storage; partial stability for 120 days at 4ºC, while no activity was detected after storage for 30 days at 37ºC.
In the last few decades, tremendous interest has swelled in the potential use of bacteriocins from Lactic Acid Bacteria (LAB). The bacteriocins produced by this group of bacteria are considered potent bio-preservative agents and their application in food is currently the subject of extensive research.
The present investigation highlights the isolation and characterization of bacteriocin producing LAB strains from Iranian green olives. To date, only few bacteriocin producing LAB has been reported in olives (Frantz et al., 1996; Lean et al., 1998; Todorov and Dicks, 2005). We here report the isolation and characterization of two wide spectrum bacteriocin producing LAB strains namely,
According to Fricourt and his co-workers, lactic acid bacteria synthesize bactericidal agents that vary in their spectra of activity (Fricourt et al., 1994). Many of these agents are bacteriocins with a proteinaceous active moiety, while others are non-protein agents (Piard and Desmazeaud, 1991; 1992; Atrih et al., 1993; Lash et al., 1995). During our investigations we recorded the proteinaceous status of the bacteriocins in study, and the antagonistic activity demonstrated by these strains was completely lost when exposed to proteases. The antimicrobial activity of the bacteriocins appeared unrelated to hydrogen peroxide or acidity as their activity was not lost after treatment with catalase or adjustment of pH to 7.0. However, the activity of bacteriocin NM 332 was affected by lipase treatment which might indicates its linkage to a non-protein moiety such as lipids.
The bacteriocin produced by L
During the purification procedures, each step resulted in considerable loss of protein concentration while specific activity increased. At 80% saturation with ammonium sulphate highest increase in activity was observed. This agreed with the findings of Ivanova et al. (2000). The Increase in activity could be due to release of active monomers from bacteriocin complexes. During salt precipitation various amount of the protein was fractionated as a surface pellicle, this might be due to the association of bacteriocin molecules with the hydrophobic globular micelle like structure in the supernatant fluid. Similar observations have also been recorded for lactocin S and lactacin F (Muriana and Klaenhammer, 1991).
During ultrafiltration experiments, both the bacteriocins were unable to pass through 5 KDa molecular weight cut-off membranes. A tendency to aggregate with other proteins has been reported in bacteriocins produced by other lactic acid bacteria (Bhunia et al., 1991; Toba et al., 1991), and might have contributed to the reason why the bacteriocins could not pass through the membrane with low molecular weight cut-off. While, slight activity of bacteriocin NM 24 recovered in the filtrate of 30 KDa units, could be attributed to the monomeric form of this bacteriocin. The bounded dimeric form of this bacteriocin was present in the retentate of the respective exclusion units. Moreover, as reported by other researchers the possible involvement of two peptides could not be ruled out (Yang et al., 1992). We could not confirm the presence of two peptides, as single bands giving activity were observed for both the bacteriocins during 15% non-denaturing gel experiments. However, the possibility of smaller proteins not being resolved on this percentage of gel exists. A considerable loss of the bacteriocin activity was observed during ultrafiltration which might be due to absorption of the bacteriocin on the membrane.
The phenomenon of heat stability of LAB bacteriocins have been reported earlier for plantaricin A (Daeschel et al., 1990), plantaricin C19 (Audisio, 1999), plantaricin S (Jimenez-Diaz et al., 1990), plantaricin 149 (Kato et al., 1994), plantaricin SA6 (Ralph et al., 1995), plantaricin 423 (Van-Reenen, 1998), pentocin TV35b (Okkers et al., 1999), lactocin RN 78 (Mojgani and Amirinia, 2007) and a bacteriocin produced by
Like most LAB bacteriocins reported to date (Daeschel et al., 1990; Lade et al., 2006), the activity of the bacteriocin in study appeared pH dependent. The bacteriocin NM 24 exhibited highest activity in acidic pH range of 2 to 6, while almost lost its activity in alkaline pH range. Similar phenomenon of acid stability has also been demonstrated previously in plantaricin, bulgarican, and lactobulgarican (Reddy et al., 1994; Lade et al., 2006). In contradiction to the bacteriocin NM 24 and similar to lactocin RN 78 (Mojgani and Amirinia, 2007), the bacteriocin produced by
Exposure of the bacteriocin samples to surfactants resulted in an increase in the bacteriocin titers. This increase might be due to the effect of surfactant on the permeability of the cell membrane (Graciela et al., 1995). It has also been suggested that the dispersion of the bacteriocin complex into active subunits ultimately results in more lethal hits and consequently enhanced activity is witnessed (Muriana and Klaenhammer, 1991).
The high stability of the bacteriocins in study during prolong storage makes them superior to a number of other reported natural and synthetic bacteriocins. Both the bacteriocins remained fully stable after storage for three years at -20°C, but became non-detectable within 30 days of storage at 37°C, indicating that cold temperature may be the most appropriate preservation technique.
The peculiar antimicrobial characteristics and technological properties and especially heat and storage stability of