Mondal and Aruna

Other bacteriocins such as pediocin, may also have

ability to ferment glucose and mannitol. Catalase

potential applications in foods, though they are not

activity was tested by spotting colonies with 3%

currently approved as antimicrobial food additives

hydrogen peroxide and oxidase test was performed

(Naghmouchi et al ., 2007).

using oxidase disc (Ravi et al ., 2011; Dhewa, 2011).

Fresh fruits harbor various microorganisms, some

Maintenance of LAB cultures

of them may be pathogenic and spoilage that are

Isolates and the indicator strains were streaked and

capable of growing at room temperature. Therefore,

re-streaked on MRS agar medium and Nutrient agar

it is important to seek biopreservatives that could

medium (containing 0.6% yeast extract), respectively

control these microorganisms. Since the isolation

at frequent intervals of time. The stock cultures were

and screening of microorganisms from natural

preserved in a refrigerator at 4 o C (Sharma et al ., 2006,

sources has always been the most powerful means

Cherif et al 2001).

for obtaining useful and genetically stable strains

for industrially important products (Ibourahema et

Preparation of culture supernatant

al ., 2008). The present study involved the isolation,

identification of LAB from fermented green gram

The strains were grown in MRS broth at 37 o C for

batter and biopreservative property of bacteriocin

48 hrs. The broth was centrifuged at 10,000 rpm for

10 min and the bacterial cells were separated out

with different concentrations in apple juice and

(Maldonado et al., 2003). The cell free supernatants of

coconut water. Prior to arriving to certain level

all the four isolates were used as crude bacteriocins.

of concentrations to be used in the apple juice and

coconut water, experiments were carried out in our

Determination of antimicrobial activity of

laboratory to decide the appropriate concentrations

bacteriocin

of bacteriocin.

The bacteriocin was used for testing inhibitory

Materials and Methods

activity against indicator organisms. The indicator

organisms include Gram positive bacteria, Bacillus

Sample preparation

subtilis, Staphylococcus aureus and Gram negative

bacteria Pseudomonas aerosginosa, Escherichia coli ,

Green gram was collected, cleaned, soaked in water

Proteus vulgaris and Klebsiella species .

for 8 hrs and batter was prepared. The batter was

allowed to ferment at room temperature for overnight

Agar well diffusion method

(O/N).

Antimicrobial

activity

of

bacteriocin

against

Isolation and identification of bacteriocin producing

pathogenic microorganisms was determined by well

LAB species

diffusion method at pH 5 and pH 7. The pH of the

bacteriocin was adjusted to pH 5 and pH 7, using

Serial dilutions were performed upto 10 -7 and plated

1N HCl and 1M NaOH. Agar plates were inoculated

on the MRS agar and incubated at 37 o C for 24 hrs.

with 100 ul of each indicator microorganisms after

After incubation, pure culture was made on MRS agar

growing them in a nutrient broth and diluting

and tested for bacteriocin production (Federal, 1988).

appropriately. The inhibitory activity against all

Then, the strains were Gram stained and examined

pathogenic microorganisms was tested on nutrient

microscopically. The Gram staining and microscopic

agar .Wells (6 mm) were cut in agar plate and 100 ul

examination of LAB isolates at regular intervals

of cell free culture supernatant (crude bacteriocin) of

was performed according to the method described

the isolated strains was added into each well. Plates

(Dhewa et al ., 2010b). Based on Bergey’s Manual of

were incubated at 37 o C for 24 hrs. The antimicrobial

Systematic Bacteriology (Kandler and Weiss, 1986;

activity was determined by measuring the diameter

Cleveland et al ., 2001) strains were tested for their

of the inhibition zone around the wells.

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