Indian beans, also known as Green beans (Phaseolus vulgaris), were used as a source for the isolation of bacteriocinogenic LAB. The fresh young beans were washed, cut into small pieces and incubated in sterile 5% NaCl solution at room temperature to enrich resident microflora. Samples drawn at different time intervals were pour-plated on MRS agar (Hi Media, Mumbai; India). After sufficient growth, the plates having well separated colonies were overlaid with indicator bacterium Pediococcus acidilactici NRRL B1153 or Listeria monocytogenes Scott-A and allowed to incubate further. Colonies of LAB obtained from bean-fermentation that exhibited a zone of inhibition (>10 mm diameter) against the lawn of the indicator bacterium were picked aseptically and subjected for further studies.

Antimicrobial compound produced in the culture filtrate (CF) was characterized by agar well diffusion assay as described by Geis et al., (1983). One of the potent cultures identified based on its wider zone of inhibition (> 10 mm diameter) against the indicator B1153, was B3. It was subsequently used to study the antimicrobial spectrum against the series of indicator bacteria. Characterization of the antimicrobial compound was carried out as described by Halami et al. (2005).

The isolate B3 was subjected for taxonomic identification as suggested previously (Lee et al., 1999); and as described in Bergey's manual of Systematic Bacteriology (Mundt, 1986). The 16S rRNA gene was PCR amplified as demonstrated previously (Halami et al., 2005) following standard techniques of molecular biology (Sambrook and Russell, 2001). Nucleotide sequences of the PCR product was determined and deposited in GenBank (Acc No. EU439292).

Antibiotic sensitivity pattern of the culture B3 was studied using an antibiotic sensitivity disc (Hi Media). Plasmid profiling of the test culture was performed by the method described by Anderson and McKay (Anderson and Mckay, 1983).

Antimicrobial compound produced by culture B3 was concentrated by extraction with chloroform following the method of Burianek and Yousuf (2000). Bacteriocin preparation was subsequently analyzed by Tricine SDS-PAGE (16.5T & 6C acrylamide) as described by Schagger and von Jagow (1987). Direct detection of antimicrobial compound was carried out as described by Bhunia et al. (1994)

As many as 10 different bean samples procured from the market of the city of Mysore were subjected to screening for bacteriocinogenic cultures. The strain B3 was one of the potent cultures obtained among atleast 200 zone producing cultures against the indicator bacteria Ped. acidilactici B1153. One of the representative plates was also overlaid with List. monocytogenes Scott-A. The number of zone producers observed were almost same when two parallel plates of the same sample was overlaid with B1153 and Scott-A respectively. Hence, we successfully replaced the use of pathogenic strain Scott-A by B1153 as indicator for isolation of anti-listerial bacteriocin producers. Use of lactic culture as a bacteriocin indicator eliminated the effect of zone production due to acids, which is usually seen against pathogenic strains. The culture B3 was selected for further studies based on its antimicrobial activity in comparison to pediocin PA-1, and standard nisin preparations (Fig 1).

Figure 1

Figure 1: Comparative CF activity of native bean isolate against indicator B1153

1, CF of Ped. acidilactici K7 (Halami et al., 2005); 2, Lc. lactis CFR-B3 and 3, standard nisin preparation (1 IU/ml) obtained from Aplin and Barret (UK).

Antimicrobial spectrum indicated that the CF of B3 was potent against the species of pathogenic bacteria such as Listeria and Bacillus as well as spoilage bacteria like Leuconostoc sp and Enterococcus sp. The antimicrobial compound was able to get inactivated by proteolytic enzymes and was stable at 100oC for 10 min. However, activity was lost upon autoclaving at 121oC for 20 min. The antimicrobial compound was active in a wide rang of pH (2 to 10). These results suggest that the antimicrobial compound is very similar to class IIa bacteriocins that have the properties of anti-listerial activity, heat-stability and activity over a wide pH range.

The culture B3 was unable to utilize citrate and was able to produce ammonia from arginine. Negative reaction for starch and gelatin hydrolysis were also observed. Optimum growth at 32oC, pH 6.5 as well as NaCl concentration of upto 4% was observed. The culture was able to utilize several plant sugars such as xylose, arabinose, sucrose etc. BLASTn search of the 16S rRNA gene sequences (582 bp) suggested >99% sequence homology with Lactococcus lactis (Acc no. EF589778), followed by several other species of Lactococcus lactis of vegetable and dairy origin including the genome sequence of Lc. lactis IL1403. With the microbiological characteristics of B3, in combination with 16S rRNA gene sequence data, we identified the native isolate as Lactococcus lactis CFR-B3, and deposited in the culture collection of this dept.

The plasmid profile of CFR-B3 indicated that it harbors at least one low molecular weight (MW) and two high MW plasmids (Fig 2).

Figure 2

Figure 2: Agarose (0.8%) gel analysis of plasmid DNA isolated from CFR-B 3, lane 1. Arrow indicates different native plasmids and position of the chromosomal DNA band), M, ? DNA 911 digest (MBI, Fermentas).

One of the high MW plasmid resolved above the chromosomal DNA. The size of the smaller plasmid was approximately 7 kb. The plasmids found in CFR-B3 act as a marker in identification and are probably involved in bacteriocin production and carbohydrate fermentation. Tricine gel analysis.

Figure 3

Figure 3: Bioassay of bacteriocin preparation by Tricine SDS-PAGE. a, silver staining and b, activity assay overlaid with indicator B1153. lane 1, chloroform extracted sample and 2, trypsin treated chloroform extract. M, protein mol wt marker, medium range (Bangalore Genei, India). Arrow indicates zone producing protein bands.

revealed that bacteriocin of ~5 kDa exhibiting zone of inhibition against the indicator. Bacteriocin activity was abolished upon treatment with trypsin (Fig 3, lane 2b).

Antibiotic sensitivity test (Table 1) suggested that the strain was sensitive to a majority of antibiotics. That includes netilmycin, ciprofloxacin, lomefloaxcin and cefuroxime that characteristically deferred from Ped. acidilactici K7, a previously characterized pediocin PA-1 producer isolated from cucumber. Antibiotic sensitivity data suggested a desirable level of antibiogram that is generally acceptable for food applications.

Figure 4

Table 1: Comparative antibiotic sensitivity pattern of B3 and K7.

Bacterial cultures used, K7; Pediococcus acidilactici K7 and B3,

Lactococcus lactis CFR B3. *R: Resistant; S: Sensitive; #unit concentration used