Isolation of anti-listerial bacteriocin producing Lactococcus lactis CFR-B3 from Beans (Phaseolus vulgaris)
P Kiran-kumar, V Badarinath, P Halami
P Kiran-kumar, V Badarinath, P Halami. Isolation of anti-listerial bacteriocin producing Lactococcus lactis CFR-B3 from Beans (Phaseolus vulgaris). The Internet Journal of Microbiology. 2008 Volume 6 Number 1.
Contamination of food samples by
Source of fund: This work was financially supported from the institute project under MLP-0304
Consumption of raw and fresh vegetables is known to be an essential aspect of all healthy food habits. Several culinary vegetables being consumed are known to be nutritious in the human diet. However, the possible spread of pathogenic bacteria in these foods has not viewed seriously. Listeriosis,
Indian beans, apart from its sole use in Indian curry, is being used for the preparation of several culinary dishes that are being served during normal meals. These include salad, vegetable soup, vegetable sandwitch, vegetable rolls, etc. Beside normal practices of minimal processing of the beans by MAP, the pathogenic bacteria can multiply during storage and transport. In order to ensure safety during raw consumption and to minimize the spread of pathogenic bacteria, biopreservation practices can be adopted that include the use of bacteriocinogenic cultures of LAB isolated from the same ecological niche as protective cultures (Kelly
Material and Methods
Isolation of bacteriocin producing strains
Indian beans, also known as Green beans (
Antimicrobial activity assay
Antimicrobial compound produced in the culture filtrate (CF) was characterized by agar well diffusion assay as described by Geis
Biochemical and molecular characterization of the isolate
The isolate B3 was subjected for taxonomic identification as suggested previously (Lee
Antibiotic sensitivity assay
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
Results and Discussion
Isolation of potent bacteriocin producing LAB
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
1, CF of
Antimicrobial spectrum and properties of antimicrobial compound
Antimicrobial spectrum indicated that the CF of B3 was potent against the species of pathogenic bacteria such as
Identification of bacteriocin producing strain
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
Plasmid profiling and Tricine gel analysis
The plasmid profile of CFR-B3 indicated that it harbors at least one low molecular weight (MW) and two high MW plasmids (Fig 2).
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.
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 pattern
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
Bacterial cultures used, K7;
Well characterized cultures with bacteriocinogenic properties isolated from its natural ecological niche finds applications in food industry as a protective culture for Indian culinary purposes to reduce the risk of potential pathogenic bacteria associated with disease-outbreak as well as involved in natural spread of antibiotic resistance.