Int.J.Curr.Microbiol.App.

Sci (2016) 5(4): 1042-1049

International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 5 Number 4 (2016) pp. 1042-1049
Journal homepage: http://www.ijcmas.com

Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.504.119

Isolation and Biochemical Characterization of

Lactobacillus species Isolated from Dahi

P. Kavitha*, D. Sindhuja and M. Banumathi

Department of Microbiology Srimad Andavan College, Tamilnadu, India

*Corresponding author

ABSTRACT

Keywords Dahi (curd) is a fermented milk product, most commonly used by Indian
population. Trials are in process to establish dahi as a source of health beneficial
Dahi, organisms (probiotics). Hence, the present study is directed towards the study of
probiotics, prevalence of Lactobacillus species in dahi .A total of 40 samples of dahi were
Biochemical collected for the isolation of Lactobacilli using Lactobacillus selection MRS agar.
characterization, Thirty-eight colonies were randomly picked based on colonial morphology. All the
MRS agar isolates were subjected to cell morphology, physiology and an array of biochemical
Article Info characterization. The isolates showed different growth patterns at different
temperatures (15°C and 45°C), oxygen and at different concentrations of NaCl (2.0,
Accepted: 4.0 and 6.5%). On the basis of physiological tests and sugar utilization pattern, all
18 February 2016 the three isolates were confirmed to the different species of Lactobacillus:
Available Online:
Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus lactis. Among
10 April 2016
isolates, L. acidophilus was found to be prevalent in dahi.

Introduction

Generally, the healthiness of food has been products provide a wide range of essential
linked to a nutritionally rich diet nutrients.
recommended by specialists and the role of
it in totality has been emphasized instead of The microbial ecology in the gastrointestinal
emphasizing on individual components. tract influences many functions in our body
(i.e., digestion, absorption of nutrients,
Milk, very first food of mammals including detoxification and ultimately the functioning
humans, is surrounded with emotional and of immune system) (2,3). All these aspects
cultural importance in the society. Men have make the gut a target organ for development
been habituated to think of milk as nature’s of functional foods that can help in
most perfect food for them (1). Therefore, maintaining the relative balance of
milk and dairy products have long been microorganisms in the gastrointestinal tract.
recognized as an important constituent of The establishment of microbial balance by
balanced diet for human beings as these shifting it towards a beneficial one with the

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help of specific dietary components (i.e., MRS broth for 24h at 37°C. Before
probiotics and prebiotics) has opened the inoculation of sample, the pH of MRS broth
gateway for the development of functional was adjusted to 6.5±0.2. The enriched
foods ensuring more benefits to the host’s samples were streaked on the Petri plates
health (4, 13, 14). containing Lactobacillus selection MRS
agar with the help of calibrated inoculating
Probiotics are defined as live microbes loop and incubated aerobically at 37°C for
which transit the gastro-intestinal tract and 48h and observed for the growth of colonies.
in doing so, benefit the health of the and dahi. Dahi, naturally fermented milk by
consumer. Probiotic microorganisms are characterization of Lactobacilli isolates
found in many food products, especially in grown on MRS agar was done mainly with
the fermented foods. Therefore, the the help of the following tests microscopic
probiotic lactic acid bacteria can be isolated examination (Gram staining), catalase test,
from the fermented milk products like growth at different temperatures 10+1ºC and
acidophilus milk, yoghurt lactic cultures, is 42+1ºC),growth under aerobic and
widely consumed throughout South Asia, anaerobic conditions, growth at different
especially in the Indian subcontinent. The NaCl concentration, fermentation of
micro-flora of dahi varies from one different carbohydrates,etc (7, 8, 9, 15, 16).
geographical region to the other and also
with seasonal variations of the year. Apart Microscopic Examination
from the starter lactic cultures, dahi can also
have some probiotics like Lactobacillus The purity morphological identification of
acidophilus, Lactobacillus bulgaricus etc. the isolates as Lactobacilli was confirmed
Therefore, dahi can be used as a source for microscopically by performing Gram
isolation of probiotic bacteria and also a staining, for which single colony of each
product of immense importance for human isolate was picked up and stained as per the
consumption (5, 6). standard protocol and viewed under oil
immersion for similar type of cells.
Materials and Methods
Micrometry
Isolation of Lactic Acid Bacteria
Each isolate after Gram staining was
Dahi samples (40) for the isolation of lactic subjected to microscopic measurements
acid bacteria were collected from different employing ocular and stage micrometer. To
rural and urban locations in order to get a determine the size of Lactobacilli isolates,
wider diversity of lactic acid bacterial prepared slides were observed under oil
strains. Each sample was taken in a sterile immersion objective and number of ocular
container separately and placed in a divisions occupied by each bacillus was
polyethylene bag during transportation to recorded and interpreted as per the above
the laboratory employing standard formula.
conditions for sample collection. One gram
of curd sample was immediately processed Physiological Characterization of Isolates
under aseptic conditions by suspending in
9mL of normal saline (0.85%) and was After confirming the purity of culture, each
vortexed for proper mixing. One milliliter of isolate was further assessed for growth at
each well-mixed dahi sample was enriched two different temperatures.
in 9mL of sterile Lactobacillus selection
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Growth of Isolates at (10°C and 42°C) milliliter of the medium was taken in each
tube and sterilized by autoclaving. One
The isolates were tested for their ability to sugar disc was aseptically added to each
grow in MRS broth at 10+1°C for 7 days tube. Each tube was inoculated with 0.1mL
and42°C by incubating for 24–48h. For this, of inoculum, incubated at 37°C for 24–48h
10 mL of MRS broth tubes were inoculated and the results of color change were
at 1% of Lactobacilli cultures. The recorded as positive or negative. A control
using 0.1mL sterile water development of
Identification of Selected Lactobacilli turbidity in culture tubes was recorded as the
ability of isolates to grow at 10°C and 42°C
The identification and further accordingly and results were noted as positive or
for the presence or absence of enzyme. negative.

Gas from Glucose Effect of NaCl Concentrations on Growth

of Isolates
Sterile test tubes of 10mL glucose broth
containing Durham’s tube (inverted and The isolates were inoculated in MRS broth
dipped), were inoculated with Lactobacilli having different NaCl concentration (2.0%,
cultures at the at 1% and incubated at 37°C 4.0 % and 6.5%) and incubated at 37°C for
for 24–48h. Gas production that appeared in 24–48h. The culture tubes were observed for
the form of a hollow space in Durham’s tube the presence or absence of growth.
was recorded as a positive result.
Biochemical Characterization of Isolates
Citrate Utilization Test
Catalase Test
The isolates were inoculated in Simmons
citrate agar incubated at 37°C for 24h. After The test was performed in order to
incubation, the appearance of blue determine the ability of the isolated cultures
coloration indicated the positive test for to degrade the hydrogen peroxide by
citrate utilization and was recorded producing the enzyme catalase. The test was
accordingly for the isolates tested. carried out as the slide method, using an
inoculating needle f or this, culture from a
Carbohydrate Fermentation Pattern by typical colony was placed onto a clean
the Isolates grease-free glass slide and drop of 3%
hydrogen peroxide solution was added onto
Most microorganisms obtain their energy the culture and closely observed for the
through a series of orderly and integrated evolution of bubbles. The production of
enzymatic reactions leading to the bio- bubbles indicated positive catalase reaction
oxidation of a substrate, frequently a and was recorded
carbohydrate. Thus, different sugars were
used for determining the fermentation Identification and Characterization of
profile and further characterization of Lactobacillus Isolates
Lactobacilli isolates. For this, Lactobacilli
cultures were subjected to sugar The Gram reaction property and cell
fermentation reactions using CHL medium morphology of all the isolates were
for knowing their fermentation pattern. CHL examined using standard staining procedure.
medium was used as basal medium. Four The isolates were found to be purple colored

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Gram-positive rods (i.e., straight rods, by several microorganisms, helps in

irregular rods, rods with rounded ends) of degradation of hydrogen peroxide produced
varying sizes and arrangements such as rods during carbohydrates utilization for energy
in single, or in chains (2–5 cells), under oil production, thereby its presence or absence
immersion microscope After confirming the in a microbial cell can be used as a
Gram positive character each isolate was significant indicated that the isolates could
further characterized for cell dimensions belong to the Lactobacilli group and paved
using micrometry. These preliminary results the way for further characterization through
as inoculum was used to compare the color physiological and biochemical means for
change. Sugars used to determine the confirmation.
fermentation profile of Lactobacilli isolates
were arabinose, cellobiose, fructose, Physiological Characterization
galactose, lactose, maltose, mannitol,
mannose, melibiose, raffinose, rhamnose, Growth at 10°C and 42°C
trehalose, salicin, sorbitol, sucrose and
The isolated Lactobacilli cultures were
xylose. The cultures were identified based
assessed for their growth at two different
on the pattern of sugar utilization.
temperatures (i.e., 10°C and 42°C). For this,
Results and Discussion cultures were incubated in MRS broth at
10°C for 7days and the turbidity in broth
Isolation of Lactobacilli from Dahi from was observed as an indication of microbial
Nearby Vicinity growth. The isolates showed good turbidity
except that of 30 isolates i.e., Lactobacillus
Out of several colonies developed on agar acidophilus, Lactobacillus bulgaricus,
plates, based on colonial morphology (i.e., Lactobacillus lactis where no growth was
color, size, margin and shape of the colony) observed in terms of turbidity. On the other
such as white, greyish white or cream color, hand, isolates were also incubated at 42°C
size varying from 0.5–2.3mm in diameters, for 24 to 48h and turbidity was observed in
with entire or undulate margins were picked tubes containing isolates Lactobacillus
up after plating 1mL of sample that was acidophilus, Lactobacillus bulgaricus,
previously enriched in 9mL of Lactobacillus Lactobacillus lactis.
selection MRS broth for24h at 37°C. The
sub-culturing of isolates was done 3–4 times The other Lactobacilli could not grow at the
in MRS agar by incubating at 3°C for 24– elevated temperature of 42°C. Hence, it can
48h. Finally, the well isolated colonies were be concluded form that 37°C is the optimum
streaked on the solidified Petri plates of temperature for all the isolates and few of
Lactobacillus selection MRS agar with these could either survive or grow at
inoculating loop incubated aerobically at 10°C/42°C or at both the temperatures away
37°Cfor 48h for further identification and from the optimum.
characterization based on different
physiological and biochemical tests used Effect of NaCl
routinely in microbial taxonomy.
The other physiological parameter for
Biochemical Characterization of growth of a cell is the requirement of
Lactobacilli sodium chloride as the physiological saline
Catalase Production prevents the cell from osmotic shock. For
this, isolated Lactobacilli cultures were
Catalase, an extracellular enzyme secreted incubated in MRS broth at 37°C for 24h to
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assess the effect of different NaCl the medium for their growth. These
concentrations (i.e., 2.0%, 4.0%, and 6.5%) variations in results were also reported (10,
in terms of turbidity, as an indication of 11, 12).
microbial growth. At the NaCl concentration
of 2.0%, the growth was observed in all the Citrate Utilization Test
Lactobacilli isolates. With an increase in
NaCl from 2 % to 4 %, turbidity diagnostic For further characterization and
tool. identification, all the isolates were subjected
for their potential to utilize citrate as the sole
The catalase is involved in catalyzing the carbon source. Further, it also attributes to
breakdown of toxic hydrogen peroxide to the better technological property of the was
produce molecular oxygen that generates observed only in three isolates Lactobacillus
vigorously while producing effervescence, acidophilus, Lactobacillus bulgaricus,
when a microbial culture is mixed with an Lactobacillus lactis. The growth pattern at
equal volume of 3% solution of hydrogen 4% NaCl concentration was also reported
peroxide. Absence of effervescence is taken and concluded wide variation in growth of
as indicative as negative for catalase enzyme Lactobacilli cultures. At 6.5% NaCl
production. In the present study, all the concentration, not a single isolate could
isolates were found to be catalase negative. show growth as indicated in basal media
These results obtained for catalase further with different sugar discs. In total, 16 sugars
support the identification of isolates as were used for conformational identification
Lactobacilli and pave the way in further of Lactobacilli and these were: arbinose,
characterization of the isolates and are in cellobiose, fructose, galactose, lactose,
agreement with. maltose, manitol, mannose, melibiose,
raffinose, rhamnose, salicin, sorbitol,
Gas from Glucose sucrose, trehalose and xylose. Different
isolates showed different types of sugar
The microorganisms use carbohydrates in a utilization patterns where some tubes
different pattern depending on their enzyme containing isolates and sugar turned yellow
complement. In fermentation, substrates where as the other remained brown colored,
such as carbohydrates and alcohols undergo indicating the positive and negative tests,
anaerobic dissimilation and produce organic respectively for sugar fermentation. When
acids that may be accompanied by the these sugar utilization patterns were
production of gases such as hydrogen or compared with those given for Lactobacillus
carbon-dioxide. Therefore, all the species in the Bergey’s Manual of
Lactobacilli were subjected to glucose Determinative Bacteriology, the isolates
fermentation test in order to see the were tentatively identified as L.acidophilus,
production of gas. L.bulgaricus, L. lactis. Hence, nine different
species within Lactobacillus genera in the
It was observed from produced gas in broth dahi sample obtained from rural and urban
containing glucose as the sole source of locations were confirmed in the present
carbon while other isolates could not results.
produce any gas as shown by a hollow space
in the inverted Durham’s tubes and, Carbohydrate Fermentation Pattern of
therefore, resulted as negative for gas Isolated Lactobacilli Cultures
production from glucose. Thus, the above
isolates showed fermentation of glucose in For accounting the sugar utilization pattern

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of the isolates suspected to be the Lactobacillus selection MRS agar, the sugar
Lactobacilli after their growth on utilization tests were performed using CHL.

Table.1 Morphological, Physiological and Biochemical characterization of Lactobacillus spp

Lactobacillus
acidophilus
Characteristics Strain I Strain II
Characteristics
(Bergy’s Manual)
Morphological
Characterization
Colour Cream-white Cream-white Cream-white
Shape circular circular Circular
Size 0.6µm 0.5µm 0.5-0.8µm
Motility Non-motile Non-motile Non-motile
Gram reaction Positive Positive Positive
Shape rod Rod Rod
Physiological
characterization
Growth at different
temperature
15-20°C - - -
30°C + + +
35°C + + +
37°C + + +
Oxygen requirement
Aerobic + + +
Anaerobic - - -
Microaerophilic + + +
Biochemical
characterization
Indole - - -
Methyl red - - -
Voges proskauer - - -
Citrate utilization - - -
Urease - - -
Triple sugar iron - - -
Nitrate reduction - - -
Catalase - - -
Oxidase - - -

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Table.2 Carbohydrate Fermentation of Lactobacillus spp

Lactobacillus acidophilus
Sugars Strain I Strain II
Characteristics (Bergy’s manual)
Dextrose + + +
Fructose + + +
Galactose + + +
Glucose + + +
Lactose + + +
Maltose + + +
Mannitol + + +
Sucrose + + +
Xylose - - -

In conclusion, from dahi, a total of 3 could be exploited as a probiotic after

Lactobacillus isolates were distributed investigating its beneficial characteristics.
among nine species and identified as, L. product as citrate utilization leads to a better
acidophilus, L. bulgaricus, L. lactis, where flavor production in fermented milk
L. acidophilus, was the most prevalent. It production. Certain bacteria utilize citrate
can be stated that L. acidophilus isolated with the help of enzyme citrate permease
from traditional Indian dairy product (dahi) and citrate-producing diacetyl, a flavoring

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How to cite this article:

Kavitha, P., D. Sindhuja and Banumathi, M. 2016. Isolation and Biochemical Characterization
of Lactobacillus species Isolated from Dahi. Int.J.Curr.Microbiol.App.Sci. 5(4): 1042-1049.
doi: http://dx.doi.org/10.20546/ijcmas.2016.504.119

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