Selvajeyanthi and Hemashenpagam
positive role in mental health. Milk is one of the natural
habitats and rice source of LAB, (Delavenne et al., 2012,
Wouters et al, 2002 and Misganaw wassie and Teketay
wassie, 2016, Mokoena et al., 2016).
The LAB in milk and milk products enhance bioavail-
ability of nutrients and act as a preservative(Misganaw
wassie and Teketay wassie, 2016). Fermented and func-
tional foods and the products are crucial to the human
health(oktay Yerlikaya, 2014).Potential probiotic isolates
of Lactobacillus rhamnosus and L. plantarum were pre-
sent in indigenous goat milk (Setyawardani et al., 2011).
The probiotic L.yoghurt supplementation to worldwide
waterborne diarrhea causing Giardia infected mice
reduced the severity of Giardia infection (Geeta shukla
et al., 2010). Lactic acid bacteria produce antimicro-
bial compounds, vitamins or useful enzymes which
could help in promotion of food industry (Ashmaig
et al., 2009). Identify potent attributes to meet out cur-
rent demands of the functional food industry (Sub-
hashini, 2014).
MATERIALS AND METHODS
36 samples of raw fresh milks were collected from lac-
tating cow, buffalo, goat and sheep in the rural area
surrounding of Tirupur & Erode District. Samples were
collected using sterile centrifuge tubes and stored in
an icebox until delivery of the laboratory for analysis.
Till the analysis samples were kept in 4°C(refrigerator).
About 1ml of milk sample was mixed with 9ml of saline
[8.5g / L] to make an initial dilution [10
-1
]. The suspen-
sion was used for making suitable serial dilutions up to
10
-8
. Enumeration of LAB was determined using MRS
(Man de Rogosa Sharpe) agar and M17 agar medium
by pour plate [1ml in 15ml medium] incubated at 37 C
for 24-48 hours. After incubation colonies were chosen
based on their morphology on MRS (pH-5.7) agar plate.
The typical LAB were randomly picked up and puri ed
for further work. Simple tests such as gram staining,
catalase test, motility and sugar fermentation test were
performed for isolates.
The isolates grown in freshly prepared liquid media
and incubated overnight. After incubation the cells were
taken and then gram staining procedure was performed.
The gram reaction of the isolates was determined by
light microscopy. Catalase enzyme produced by many
microorganisms that breaks down the H
2
O
2
into water
and oxygen that releases O
2
gas bubbles. The forma-
tion of gas bubbles indicates the presence of catalase
enzyme.
2H
2
O
2
2 H
2
O + O
2
The freshly grown liquid cultures were also used for
catalase activity by dropping 3% hydrogen peroxide
solution onto 1 ml of overnight cultures and their cata-
lase activity was observed.
(Thakkar et al., 2015). MRS broth supplemented with
different Sugars (glucose, lactose and maltose) and phe-
nol red as pH indicator was inoculated with active cul-
tures at 1%, incubated at 37°C for 24 hours. The cul-
tures were identi ed based on acid and gas production
in Durham’s tube after the incubation period. To check
the growth of isolates at various pH, MRS broth sup-
plemented with different pH 2.0, 3.0, 7.0, 8.5 was pre-
pared, 1% of fresh culture was inoculated and then incu-
bated at 37ºC for 28 hours. During incubation, extent of
growth was recorded objectively based on visible turbid-
ity marked as double positive sign (++) for maximum
growth, single positive sign (+) for normal growth and
negative sign (-) for no growth. Turbidity also measured
at 620nm.Overnight active cultures were inoculated at
1% in MRS broth tubes and incubated up to 7 days at 15,
37, 45 and 55ºC. Extent of growth was visually recorded
based on intensity of turbidity. Overnight grown active
cultures were inoculated at 1% in MRS broth tubes
adjusted to various concentrations of Na Cl viz. 3.5, 6.5
and 18% (w/v) along with their respective controls. The
cultures were incubated at 37°C. After 24 hours of incu-
bation, extent of growth was recorded objectively based
on visible turbidity marked as double positive sign (++)
for maximum growth, single positive sign (+) for normal
growth and negative sign (-) for no growth.
Blood hemolysis test was carried out as per the
method of Mabrouk et al.,( 2014). As the strains were
isolated from food material, blood haemolysis test was
performed, to eradicate any chance that our isolates
may be pathogenic. It is also one of the criteria for
assessing the safety of use of probiotics as food supple-
ments. Pathogens produce highly toxic substance which
lyse the RBC and forms a clear zone around them. The
haemolytic activities of isolated strains were determined
according to (Marakoudakis et al., 2009) as follows: all
examined strains were grown in MRS broth at 37°C for
24 hours and then streaked onto Columbia agar base
plates supplemented with 5 % (v/v) whole human blood.
The plates were incubated at 37 ºC for 48 hours. Then
the clear zones and the color of haemolysis around the
growth colonies were observed. Antibiotic susceptibility
test was done using the method of Singh et al., (2014).
Probiotic strains must be sensitive to wards the anti-
biotics. There is a light risk that antibiotic resistance
probiotic strain may transfer the antibiotic resistance
genes to the pathogens via transformation in the gut.
Due to any chance resistant pathogens get introduced
into the human via food chain and cause serious prob-
lems. Sensitivity of probiotics strains towards the antibi-
otics being tested by using Kirby - Bauer disc diffusion
technique. Tetracycline, Penicillin, Vancomycin, Strep-
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS SCREENING OF INDIGENOUS ACTIVE LACTIC ACID BACTERIA ISOLATED 427