M Kumar, N Ghosh, S Srivastava
antimicrobial spectrum, bacteriocin, biochemical and molecular identification, characterization, enterococcus.
M Kumar, N Ghosh, S Srivastava. Production and characterization of a bacteriocin produced by Enterococcus faecium LR/6. The Internet Journal of Microbiology. 2009 Volume 8 Number 1.
A soil isolate of Lactic acid bacteria (LAB), strain LR/6 was identified as
Among Lactic acid bacteria, members of the genus
Lactic acid bacteria have been used in the production of a variety of dairy, vegetables, meat and fermented foods from many centuries. In addition to the contribution to the typical sensory characteristics of these foods (Fox and Wallace, 1997), LAB exerts strong antagonistic activity against many food-contaminating microorganisms as a result of the production of organic acids, hydrogen peroxide, diacetyl, inhibitory enzymes and bacteriocins (Piard and Desmazeaud, 1992). Bacteriocins produced by LAB are of great interest to the food fermentation industry as natural preservatives because of their ability to inhibit the growth of many food spoilage and pathogenic bacteria including
The bacteriocins of LAB have been classified into four classes on the basis of common, mainly structural, characteristics (Klaenhammer, 1993). Most of the bacteriocins isolated so far belong to classes I or II. Class II bacteriocins have emerged in recent years as the most promising bacteriocin candidates for food preservation, as they display overall better performance, in terms of biological activity and physico-chemical properties, than most bacteriocins from other classes (Nes and Holo, 2000, Garneau
This paper reports the identification of a bacteriocin producing strain LR/6 by phenotypic, biochemical and genetic method. In addition, production conditions, physico-chemical properties, and mode of action of the crude bacteriocin are described.
Materials and Methods
Bacterial strains, culture media, growth conditions and chemicals
Strain LR/6 isolated from soil was routinely propagated in normal TGYE medium (5 g.L-1 tryptone, 1 g.L-1 glucose, 3 g.L-1 yeast extract, and pH 7.0) medium at 37ºC and 200 rpm in an incubator shaker (Kuhner, Switzerland) as described earlier (Tiwari and Srivastava, 2008). For the purpose of production of bacteriocin, an optimized TGYE medium (20 g.L-1 glucose, 20 g.L-1 yeast extract, 15 g.L-1 tryptone, 1.0 g.L-1 tween 80, 3.0 g.L-1 triammonium citrate, 11.3 g.L-1 sodium acetate, 3.0 g.L-1 K2HPO4, 0.5 g.L-1 MgSO4, 0.2 g.L-1 MnSO4, pH 7.0) at 37°C and 200 rpm.
Phenotypic and genotypic identification of the strain
Cells of strain LR/6 were examined by light microscopy to determine their morphology and tested for Gram-staining reaction and catalase activity. Other identification tests included, the ability to grow at 4, 10 and 45ºC, on media containing 1 to 10% NaCl, growth at different pH (4.4 to 10.0), on different carbon sources and hydrolysis of arginine (Table 1). Carbohydrate utilization profiling was also done by API 50CHL system (BioMerieus, Lyon, France).
For the identification based on 16S rDNA sequence. DNA from the strain was isolated from 1.5 mL of log phase culture using Bacterial Genomic DNA kit (Sigma-Aldrich, USA). By using the set of universal primers pA (5' AGA GTT TGA TCC TGG CTC AG 3') and pE (5' CCG TCA ATT CCT TTG AGT TT 3') (Beasley and Saris, 2004), the region of the 16S rRNA gene was amplified by 29 cycles of PCR (consisting of 30 s at 94ºC, 60 s at 55ºC, and 90 s at 72ºC, with a final 120 s extension step at 72ºC) with purified chromosomal DNA from strain as template. The amplification assay comprised ~50 ng of template DNA, along with master mix that included reaction buffer, dNTPs, magnesium chloride and Taq DNA polymerase (BIOTOOLS, Spain), and 25 pmol of each oligonucleotide primers in a final volume of 25 µL. PCR assay was carried out on a GeneAmpR PCR system 2700 (Applied Biosystems, USA). The amplified 900-bp fragment was resolved on 1% horizontal agarose gel, purified by means of the Gel elution kit (mdi, India), and sequenced using an automated gene sequencer (ABI PRISM 310). The sequence so obtained was aligned and compared with known sequences of 16S rRNA gene of different lactic acid bacteria strains from the database using BLAST (www.ncbi.nlm.nih.gov. /BLAST/).
Further confirmation of the result was done using species-specific primers of
Nucleotide sequence accession number
The 16 S rDNA sequence of strain LR/6 so obtained was submitted to GeneBank (NCBI) under the Accession number EU366176.
Growth and bacteriocin production
An overnight grown culture of strain LR/6 (~106 CFU.mL-1) was used to inoculate the optimized TGYE medium and was incubated at 37ºC and 200 rpm over a period of 24 h. Growth was measured periodically in terms of A630 and viable cells were counted by plating an appropriate dilution on TGYE agar plates. Bacteriocin production was quantified in terms of AU.mL-1 by microtitre plate assay method as described later. The change in pH was also recorded.
Preparation of Bacteriocin sample
Bacteriocin activity assay
The antimicrobial activity of the bacteriocin was routinely determined by the agar-well diffusion assay (AWDA) method (Tagg
Bacteriocin activity was quantified by a microtitre plate assay (Holo
Effect of β-glycerophosohate and catalase treatment
In order to neutralize the effect of lactic acid, the crude bacteriocin was treated with sodium β-glycerophosphate (1.7% final concentration). The mixture was incubated at 37ºC for 2 h and antimicrobial activity was determined. Similarly, one sample was treated with catalase (final concentration 5 mg.mL-1) so as to see involvement of hydrogen peroxide.
Physico-chemical properties of crude bacteriocin
Crude bacteriocin was treated to a range of pH, temperature and storage at different temperatures so as to see the effect of the same on its activity. To check the pH response, the crude bacteriocin was adjusted to different pH ranging from 2.0 to 8.0 in 1: 1 ratio of different buffer solutions (HCl-KCl 50 mM, pH 2.0 and 4.0; phosphate buffer 50 mM pH 6.0, 7.0 and 8.0) and incubated for 4 h at 37ºC. Then the samples were sterilized by filtration through 0.2 µm membrane and antimicrobial activity was determined in terms of AU.mL-1. Negative controls consisted of buffers of different pH used, and untreated crude bacteriocin was used as positive control.
To evaluate the heat stability the crude bacteriocin was incubated at 60, 90 and 100ºC for 30 min and one sample was autoclaved (121ºC, 15 psi, 15 min). The effect of extended storage at different temperature 37ºC, room temperature (ranging from 10 to 30 ºC), 4ºC and (-) 20ºC was assessed by regularly monitoring its activity upto one year. A positive control, consisting of freshly prepared crude bacteriocin was tested each time in parallel.
Sensitivity to hydrolytic enzymes, surfactants and organic solvents
To demonstrate the proteinaceous nature of the antimicrobial compound, it was treated with different proteolytic enzymes: proteinase K, pepsin, papain, α-chymotrypsin, and protease (Sigma-Aldrich, USA) at a final concentration of 1mg.mL-1, and incubated at 37ºC for 2 h. The crude bacteriocins in buffer without enzymes as well as the enzymes in buffer solutions were used as control. The samples were tested for antimicrobial activity as described above. The crude bacteriocin was also treated with α-amylase and lipase, each at 1mg.mL-1 final concentration, to show its effect on the activity.
The surfactants used were SDS, Tween 80, Tween 20, Triton X-100 and urea, which were added to the crude bacteriocin at a final concentration of 1% (v/v) and incubated at 37ºC for 5 h. Surfactants at 1% in TGYE broth were used as controls. Activity in all samples was determined as above. Surfactants (Sigma, USA) were prepared as 10% aqueous solution and filter sterilized before use.
The crude bacteriocin was mixed with various organic solvents (ethanol, methanol, isopropanol, acetone, ethyl acetate and toluene) at a final concentration of 50% (v/v). After incubation for 2 h at 37ºC, the organic solvents were evaporated and residual antimicrobial activity was determined as described earlier.
Mode of action
To determine the mode of action, the indicator strain was grown to an exponential stage (~106) then resuspended in normal saline (0.85%) and examined for the inhibition with 200 AU.mL-1 of crude bacteriocin. Samples were withdrawn periodically, and plated on NB-agar medium for determination of CFU.mL-1. Untreated cells served as control.
Determination of antimicrobial spectrum
To determine the antimicrobial spectrum a wide range of bacteria comprising some LAB and food-borne pathogens were tested against the crude bacteriocin. Effect was ascertained both by CFU.mL-1 and agar well diffusion assay (AWDA).
Tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed, as a step gradient gel (4, 10, and 16.5% acrylamide for the stacking, spacer and separating gel, respectively), according to the procedure of Schägger and Von Jagow (1987). Myoglobin fragments (16,950, 14,440, 10,600, 8160, 3480 and 2510 Da) were used as marker proteins (Sigma, USA). Samples were run in duplicate. One part of gel containing the molecular mass markers and sample was stained with Coomassie Brilliant Blue R-250 (CBB-R-250), to visualize the protein. The other lane, containing only sample, was extensively washed with regular changes of sterile MilliQ water for overnight. For direct detection of antimicrobial activity, the gel was overlayed with soft agar (0.8%) seeded with indicator strain
Each result is expressed as mean along with respective Standard error of mean. Each data point is the average of three repeated measurements from two independent replicates.
Results and Discussion
Strain LR/6 was identified to be Gram-positive, coccoid in shape, and catalase-negative. The strain was able to grow between 30ºC to 45ºC, in a pH range of 4.4-10.0 even in the presence of 1 to 6.5% NaCl, and produced ammonia from arginine (Table 1).
The carbohydrate utilization pattern determined by API 50CHL analysis showed that strain LR/6 could utilize a large number of sugars except xylose and two sugar alcohols
(Table 1). Based on the observed characteristics, the isolate was tentatively classified as belonging to the genus
Strain LR/6 when grown in optimized TGYE medium at 37ºC, showed a typical sigmoidal growth response and associated bacteriocin production (Figure 2) Antimicrobial activity could be detected in the culture filtrate after 4 h of incubation, (the mid-log phase) with maximum accumulation in stationary phase (18 h). Thereafter, the bacteriocin activity remained constant till 24 h. The pH of the medium was dropped from neutral to 3.8 during this period. The antimicrobial activity of the culture supernatant was not affected by treatment with β-glycerophosphate or catalase suggesting that neither acid nor H2O2 production are responsible for the same. Alternatively, it may also indicate that
The crude bacteriocin of strain LR/6 was stable over a wide range of pH (2.0-8.0). The maximum activity was obtained between pH 2.0 to 6.0, and a loss of ~20%, was observed at pH 7.0 and 8.0. Of the other bacteriocins isolated from
To determine the chemical nature of the antimicrobial compound, the effect of some proteolytic enzymes (proteinase K, pepsin, papain, α-chymotrypsin and protease) was studied. The active compound was found to be sensitive to these enzymes, but α-amylase, lipase did not affect, indicating that the inhibitory material is proteinaceous in nature. Similar results have been reported for the bacteriocins from
The effect of crude bacteriocin on the cell viability of indicator strain,
Figure 3: Bactericidal effect of crude bacteriocin on
In order to study the antimicrobial host range, several LAB strains and food-borne pathogenic bacteria comprising both Gram-positive and Gram-negative members were tested. As is clear from the results highly efficient activity was observed against
As described in Material and Methods, the crude bacteriocin of strain LR/6 was resolved on the SDS-PAGE. The electrophoretogram of the gel stained with Coomassie Brilliant Blue R-250 showed a diffused band of protein corresponding to a molecular mass of ~6.0 kDa (Figure 4). When the gel was overlaid with the indicator bacteria a large inhibitory zone corresponding to this band could be observed. The molecular size of LR/6 bacteriocin is close to that produced by
This work was financially supported by the Council of Scientific and Industrial Research (CSIR), and Department of Biotechnology, India. The facilities provided to the Department of Genetics, by University Grant Commission under SAP and by Department of Science and Technology, Government of India under FIST programme is thankfully acknowledged. MK was supported by a UGC fellowship.