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Research & Reviews: A Journal of Dairy Science and Technology
Volume 1, Issue 2, August 2012, Pages
__________________________________________________________________________________________
ISSN: 2319-3409© STM Journals 2012. All Rights Reserved
Page 1
Isolation and Biochemical Characterization of Lactobacillus species
Isolated from Dahi
Aarti Bhardwaj1, Monica Puniya
2, K. P. S. Sangu
1, Sanjay Kumar
3, Tejpal Dhewa
4*
1Depatment of Dairy Science and Technology, Janta Vedic College, Baraut-250611,
Uttar Pradesh, India 2Dairy Cattle Nutrition Division,
3Dairy Microbiology Division, National Dairy Research Institute,
Karnal-132001, Haryana, India 4Bhaskaracharya College of Applied Sciences (University of Delhi), New Delhi-110075, India
*Author for Correspondence Email: tejpal_dhewa07@rediffmail.com, Tel: + 91-8836325454
1. INTRODUCTION
Generally, the healthiness of food has been
linked to a nutritionally rich diet
recommended by specialists and the role of it
in totality has been emphasized instead of
emphasizing on individual components.
During the past few decades, the lifestyle in
the developed and developing countries has
been changing fast with regard to living
standards, diet, hygiene and usage of
antibiotics. The prevalence of chronic diseases
like allergies and gut-associated disorders
(e.g., Crohn’s disease, ulcerative colitis, and
inflammatory bowel disease) are of rising
importance in the world nowadays. Hence, the
balance in microbiota of gut is considered to
provide the colonization resistance against
infectious agents and promote anti-allergic
processes, stimulate immune system and
reduce hypersensitivity [1–4].
Milk, very first food of mammals including
humans, is surrounded with emotional and
cultural importance in the society. Men have
been habituated to think of milk as nature’s
most perfect food for them. Therefore, milk
and dairy products have long been recognized
as an important constituent of balanced diet for
human beings as these products provide a wide
range of essential nutrients.
ABSTRACT
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 organisms (probiotics). Hence, the present study is
directed towards the study of prevalence of lactobacillus species in Dahi. A total of 40 samples of dahi
were collected for the isolation of Lactobacilli using Lactobacillus selection MRS agar. Thirty-eight
colonies were randomly picked based on colonial morphology. All the isolates were subjected to cell
morphology, physiology and an array of biochemical characterization. The isolates showed different
growth patterns at different temperatures (15 ° C and 45 ° C), oxygen and at different concentrations of
NaCl (2.0, 4.0 and 6.5%). On the basis of physiological tests and sugar utilization pattern, all the
seventy-eight isolates were confirmed to the different species of Lactobacillus: Lactobacillus casei
(24.35%), Lactobacillus brevis (3.84%), Lactobacillus fermentum (6.41%), Lactobacillus plantarum
(7.69%), Lactobacillus helveticus (5.12%), Lactobacillus rhamnosus (6.41%), Lactobacillus viridiscence
(5.12%), Lactobacillus lactis (3.84%), Lactobacillus acidophilus (37.17%). Among isolates, L.
acidophilus was found to be prevalent in dahi.
Keywords: Dahi, probiotics, biochemical characterization, MRS agar
Research & Reviews: A Journal of Dairy Science and Technology
Volume 2, Issue 2, August 2012, Pages
__________________________________________________________________________________________
ISSN: 2319-3409© STM Journals 2012. All Rights Reserved
Page 2
In addition, the evidence of health benefits of
milk associated with the presence of specific
components or beneficial bacteria is gaining
scientific credibility at a rapid pace. Therefore,
the best known examples of functional foods
(i.e., that benefit the consumers beyond
nutrition) are fermented milks containing
friendly bacteria and are called probiotics
[5, 6]. Milk itself is much more than the total
sum of its nutrients as it is also a natural
source of biologically active compounds that
exert potential impact on human health.
Probiotic group of microorganisms that are
present in fermented milk products (i.e., dahi,
yoghurt, cheese, etc.) beneficially affect the
host by improving the intestinal microbial
balance. The effect of probiotics includes
alleviation of intestinal disorders (i.e., lactose
intolerance, acute gastroenteritis due to enteric
pathogens, constipation, and inflammatory
bowel disease) and a number of food allergies.
The therapeutic value of fermented dairy
products has been utilized, dating back to
Biblical days. These foods confer the added
health benefits or disease-prevention
characteristics beyond the basic nutrition of
the food [7]. About 65% of the total functional
foods market is covered with dairy probiotic
products [8]. Although the primary aim of
food is to provide enough nutrients to fulfil the
body requirements, yet various functions of
the host body are modulated by the diet
consumed. Hence, in order to compensate for
deficiency of certain nutrients in the diet due
to changes in nutritional habits of
industrialized nations, the concept of
functional foods has been developed for the
consumers.
The microbial ecology in the gastrointestinal
tract influences many functions in our body
(i.e., digestion, absorption of nutrients,
detoxification and ultimately the functioning
of immune system). All these aspects make the
gut a target organ for development of
functional foods that can help in maintaining
the relative balance of microorganisms in the
gastrointestinal tract. The establishment of
microbial balance by shifting it towards a
beneficial one with the help of specific dietary
components (i.e., probiotics and prebiotics)
has opened the gateway for the development
of functional foods ensuring more benefits to
the host’s health [9].
Probiotics are defined as live microbes which
transit the gastro-intestinal tract and in doing
so, benefit the health of the consumer [10, 11].
Probiotic microorganisms are found in many
food products, especially in the fermented
foods. Therefore, the probiotic lactic acid
bacteria can be isolated from the fermented
milk products like acidophilus milk, yoghurt
and dahi. Dahi, naturally fermented milk by
lactic cultures, is widely consumed throughout
South Asia, especially in the Indian
subcontinent. The micro-flora of dahi varies
from one geographical region to the other and
also with seasonal variations of the year. Apart
from the starter lactic cultures, dahi can also
have some probiotics like Lactobacillus
acidophilus, Lactobacillus casei, Lactobacillus
Research & Reviews: A Journal of Dairy Science and Technology
Volume 2, Issue 2, August 2012, Pages
__________________________________________________________________________________________
ISSN: 2319-3409© STM Journals 2012. All Rights Reserved
Page 3
bulgaricus, Streptococcus thermophilus, etc.
Therefore, dahi can be used as a source for
isolation of probiotic bacteria [5, 6] and also a
product of immense importance for human
consumption.
In view of the above facts, the present study
has been designed to isolate and identify a
Lactobacillus species from dahi samples
collected from the local market.
2. MATERIALS AND METHODS
2.1. Isolation of Lactic Acid Bacteria
Dahi samples (40) for the isolation of lactic
acid bacteria were collected from different
rural and urban locations in order to get a
wider diversity of lactic acid bacterial strains.
Each sample was taken in a sterile container
separately and placed in a polyethylene bag
during transportation to the laboratory
employing standard conditions for sample
collection. One gram of curd sample was
immediately processed under aseptic
conditions by suspending in 9 mL of normal
saline (0.85%) and was vortexed for proper
mixing. One milliliter of each well-mixed dahi
sample was enriched in 9 mL of sterile
Lactobacillus selection MRS broth for 24 h at
37 °C. Before inoculation of sample, the pH of
MRS broth was adjusted to 6.5 ± 0.2. The
enriched samples were streaked on the Petri
plates containing Lactobacillus selection MRS
agar with the help of calibrated inoculating
loop and incubated aerobically at 37 °C for
48 h and observed for the growth of colonies.
2.2. Identification of Selected Lactobacilli
The identification and further characterization
of Lactobacilli isolates grown on MRS agar
was done mainly with the help of the
following tests: microscopic examination
(Gram staining), catalase test, growth at
different temperatures 10 + 1 ºC and
42 + 1 ºC), growth under aerobic and
anaerobic conditions, growth at different NaCl
concentration, fermentation of different
carbohydrates, etc.
a) Microscopic Examination: The purity
morphological identification of the isolates
as Lactobacilli was confirmed
microscopically by performing Gram
staining, for which single colony of each
isolate was picked up and stained as per
the standard protocol and viewed under oil
immersion for similar type of cells.
b) Micrometry: Each isolate after Gram
staining was subjected to microscopic
measurements employing ocular and stage
micrometer. To determine the size of
Lactobacilli isolates, prepared slides were
observed under oil-immersion objective
and number of ocular divisions occupied
by each bacillus was recorded and
interpreted as per the above formula.
2.2.1. Physiological Characterization of
Isolates
After confirming the purity of culture, each
isolate was further assessed for growth at two
different temperatures.
a) Growth of isolates at (10 °C and 42 °C):
The isolates were tested for their ability to
Research & Reviews: A Journal of Dairy Science and Technology
Volume 2, Issue 2, August 2012, Pages
__________________________________________________________________________________________
ISSN: 2319-3409© STM Journals 2012. All Rights Reserved
Page 4
grow in MRS broth at 10 + 1 °C for 7 days
and 42 °C by incubating for
24–48 h. For this, 10 mL of MRS broth
tubes were inoculated @ 1% of
Lactobacilli cultures. The development of
turbidity in culture tubes was recorded as
the ability of isolates to grow at 10 °C and
42 °C and results were noted as positive or
negative.
b) Oxygen requirement of the isolates: All
the isolates were inoculated in MRS broth
and were kept differently under
oxygenated condition; in dessicator with
burned candle (for micro-aerophilic
condition) and in anaerobic jar with gas
pack at 37 °C for 24–48 h to determine the
impact of oxygen on the growth of the
Lactobacilli isolates and results were
noted as positive or negative.
2.2.2. Effect of NaCl Concentrations on
Growth of Isolates
The isolates were inoculated in MRS broth
having different NaCl concentration (2.0%,
4.0% and 6.5%) and incubated at 37 °C for
24–48 h. The culture tubes were observed for
the presence or absence of growth.
2.2.3. Biochemical Characterization of
Isolates
a) Catalase Test: The test was performed in
order to determine the ability of the
isolated cultures to degrade the hydrogen
peroxide by producing the enzyme
catalase. The test was carried out as the
slide method, using an inoculating needle.
For this, culture from a typical colony was
placed onto a clean grease-free glass slide
and drop of 3% hydrogen peroxide
solution was added onto the culture and
closely observed for the evolution of
bubbles. The production of bubbles
indicated positive catalase reaction and
was recorded accordingly for the presence
or absence of enzyme.
b) Gas from Glucose: Sterile test tubes of
10 mL glucose broth containing Durham’s
tube (inverted and dipped), were
inoculated with Lactobacilli cultures at the
@1% and incubated at 37 °C for 24–48 h.
Gas production that appeared in the form
of a hollow space in Durham’s tube was
recorded as a positive result.
c) Arginine Hydrolysis: Autoclaved arginine
hydrolysis broth tubes were inoculated
with the isolated cultures (1%) and
incubated at 37 °C for 48 h. After
incubation, 3–5 drops of the Nessler's
reagent were added to each test tube and
observed for the change in color (yellow
to orange color), indicating a positive
result for arginine hydrolysis.
d) Aesculin Hydrolysis: The isolates were
also assessed for their ability to hydrolyze
glycoside aesculin to aesculetin and
glucose. For this, bile aesculin agar plates
were streaked with the isolated cultures
and incubated at 37 °C for 24–48 h. After
incubation, the plates were examined for
the presence of a dark brown to black halo
around the bacterial growth, showing a
positive result for aesculin hydrolysis.
Research & Reviews: A Journal of Dairy Science and Technology
Volume 2, Issue 2, August 2012, Pages
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ISSN: 2319-3409© STM Journals 2012. All Rights Reserved
Page 5
e) Nitrate Reduction Test: Nitrate reduction
is an important criterion for differentiating
and characterizing different types of
bacteria. Therefore, the isolates were
incubated at 37 °C for 24 h in trypticase
nitrate broth. After incubation, 0.5 mL
each of sulphanilic acid (0.8%, in 5N
Acetic acid) and -naphthylamine (0.5%,
in 5N Acetic acid) were added into the
tubes. The appearance of red or pink color
indicated the positive test for nitrate
reduction and was recorded accordingly
for the isolates tested in the present study.
f) Citrate Utilization Test : The isolates were
inoculated in Simmons citrate agar
incubated at 37 °C for 24 h. After
incubation, the appearance of blue
coloration indicated the positive test for
citrate utilization and was recorded
accordingly for the isolates tested.
2.2.4. Carbohydrate Fermentation Pattern by
the Isolates
Most microorganisms obtain their energy
through a series of orderly and integrated
enzymatic reactions leading to the bio-
oxidation of a substrate, frequently a
carbohydrate. Thus, different sugars were used
for determining the fermentation profile and
further characterization of Lactobacilli
isolates. For this, Lactobacilli cultures were
subjected to sugar fermentation reactions using
CHL medium for knowing their fermentation
pattern. CHL medium was used as basal
medium. Four milliliter of the medium was
taken in each tube and sterilized by
autoclaving. One sugar disc was aseptically
added to each tube. Each tube was inoculated
with 0.1 mL of inoculum, incubated at 37 °C
for 24–48 h and the results of color change
were recorded as positive or negative. A
control using 0.1 mL sterile water as inoculum
was used to compare the color change. Sugars
used to determine the fermentation profile of
Lactobacilli isolates were arabinose,
cellobiose, fructose, galactose, lactose,
maltose, mannitol, mannose, melibiose,
raffinose, rhamnose, trehalose, salicin,
sorbitol, sucrose and xylose. The cultures were
identified based on the pattern of sugar
utilization [12].
2.3. Maintenance and Propagation of
Cultures
For their further assessment of probiotic
attribute and other analysis all the isolated
Lactobacilli cultures were maintained in chalk
litmus milk at refrigeration temperature after
their growth at 37 °C overnight. The cultures
were sub-cultured at regular intervals in chalk
litmus milk and stored under refrigeration
conditions. Before use, the cultures were
activated in MRS broth. All the isolates were
also maintained at −70 °C in glycerol stock in
triplicates for use in experiment at different
stages.
2.4. Purity of Cultures
The cultures of Lactobacilli species and
indicator strains were regularly tested for their
purity by microscopic examination and/or
Research & Reviews: A Journal of Dairy Science and Technology
Volume 2, Issue 2, August 2012, Pages
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ISSN: 2319-3409© STM Journals 2012. All Rights Reserved
Page 6
catalase test for confirmation and presence of
contamination, if any.
3. RESULTS AND DISCUSSION
3.1, Isolation of Lactobacilli from Dahi from
Nearby Vicinity
Out of several colonies developed on agar
plates, 78 isolates based on colonial
morphology (i.e., color, size, margin and shape
of the colony) such as white, greyish white or
cream color, size varying from 0.5–2.3 mm in
diameters, with entire or undulate margins
(Table I) were picked up after plating 1 mL of
sample that was previously enriched in 9 mL
of Lactobacillus selection MRS broth for 24 h
at 37 °C. The sub-culturing of isolates was
done 3–4 times in MRS agar by incubating at
37 °C for 24–48 h. Finally, the well-isolated
colonies were streaked on the solidified Petri
plates of Lactobacillus selection MRS agar
with inoculating loop incubated aerobically at
37 °C for 48 h for further identification and
characterization based on different
physiological and biochemical tests used
routinely in microbial taxonomy.
Table I: Colonial Morphology of the Isolated Lactobacilli Cultures from Dahi.
Isolate(s)
Colonial Morphology
Color Shape Size
(mm) Margin
1, 11, 44, 47, 57, 61 Cream
Pin point; circular;
smooth; compact and
convex
0.8 Entire
2, 7, 9, 15, 17, 19,23, 25, 30, 32, 34,
42, 50, 52, 53, 58, 62, 64, 71, 73 White Circular 1.2 Entire
3–6, 10, 12, 13–14,, 21, 22, 26, 27,
31, 35, 36, 38, 40, 41, 45, 54–56, 59,
60, 65, 68, 69, 75, 78
White Circular; large; rough
and irregular 2.1 Undulate
8, 46, 49 Greenish white Circular 1.7 Entire
16, 39, 48, 67, 77 White Circular and large 2.1 Undulate
18, 24, 43, 51, 63 Grayish white Pin point; circular;
compact and convex 0.8 Entire
20, 28, 29, 66 White Circular and compact 0.6 Entire
33, 37, 70 Creamish white Circular 2.3 Entire
3.2. Identification and Characterization of
Lactobacillus Isolates
The Gram reaction property and cell
morphology of all the isolates were examined
using standard staining procedure. The isolates
were found to be purple colored Gram-positive
rods (i.e., straight rods, irregular rods, rods
with rounded ends) of varying sizes and
Research & Reviews: A Journal of Dairy Science and Technology
Volume 2, Issue 2, August 2012, Pages
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ISSN: 2319-3409© STM Journals 2012. All Rights Reserved
Page 7
arrangements such as rods in single, or in
chains (2–5 cells), under oil-immersion
microscope (Table II). After confirming the
Gram positive character each isolate was
further characterized for cell dimensions using
micrometry. These preliminary results
indicated that the isolates could belong to the
Lactobacilli group and paved the way for
further characterization through physiological
and biochemical means for confirmation.
Table II: Cell Morphology of the Isolated Lactobacilli from Dahi.
Isolate No. (s) Cell Morphology
Gram Reaction Shape and Arrangement Size (µm)
1, 11, 44, 47, 57, 61 G +ve Straight rods with rounded ends 0.9 × 3.0
2, 7, 9, 15, 17, 19, 23, 25, 30, 32, 34, 42, 50,
52, 53, 58, 62, 64, 71, 73
G +ve Rods in chains 0.7 × 3.2
3–6, 10, 12, 13, 14, 21, 22, 26, 27, 31, 35, 36,
38, 40, 41, 45, 54–56, 59, 60, 65, 68, 69, 75,
78
G +ve Rods with rounded ends 0.6 × 3.9
8, 46, 49 G +ve Irregular rods with rounded ends 0.8 × 2.7
16, 39, 48, 67, 77 G +ve Rods 0.9 × 2.4
18, 24, 43, 51, 63 G +ve Rods 0.8 × 4.2
20, 28, 29, 66 G +ve Rods in chains 0.7 × 2.4
33, 37, 70 G +ve Rods 0.9 × 4.8
3.3. Physiological Characterization
a) Growth at 10 °C and 42 °C: The isolated
Lactobacilli cultures were assessed for
their growth at two different temperatures
(i.e., 10 °C and 42 °C). For this, cultures
were incubated in MRS broth at 10 °C for
7 days and the turbidity in broth was
observed as an indication of microbial
growth. The isolates showed good
turbidity except that of 30 isolates i.e., 02,
07, 09, 15, 17–20, 23–25, 28–30, 32–34,
37, 50–53, 58, 62–64, 66, 70, 71 and 73,
where no growth was observed in terms of
turbidity. On the other hand, isolates were
also incubated at 42 °C for 24 to 48 h and
turbidity was observed in tubes containing
isolates 02, 07–09, 15–20, 23–25, 28–30,
32–34, 37, 39, 42, 43, 46, 48–53, 58, 62–
64, 66, 67, 70–74, 76 and 77. The other
Lactobacilli could not grow at the elevated
temperature of 42 °C. Hence, it can be
concluded form Table III that 37 °C is the
optimum temperature for all the isolates
and few of these could either survive or
grow at 10 °C/42 °C or at both the
temperatures away from the optimum.
b) Oxygen Requirement: After assessing the
growth of Lactobacilli at different
temperatures, they were exposed to the
growth in oxygenic, reduced oxygen and
anoxygenic environments. For this, all the
isolates were incubated in MRS broth and
Research & Reviews: A Journal of Dairy Science and Technology
Volume 2, Issue 2, August 2012, Pages
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ISSN: 2319-3409© STM Journals 2012. All Rights Reserved
Page 8
incubated at 37 °C for 24 h under aerobic,
micro-aerophilic and in anaerobic gas jars.
In aerobic and micro-aerophilic conditions
all the isolates tested showed turbidity in
the medium indicating the occurrence of
growth. Under anaerobic condition, the
turbidity was not observed with isolates
20, 28, 29, 33, 37, 66 and 70; however, all
other isolates showed turbidity and/or
growth. It can be concluded from Table III
that all the isolates were facultative
anaerobes and can grow in the presence
and absence of oxygen except seven
isolates (i.e., 20, 28, 29, 33, 37, 66 and 70)
that failed to grow in anoxygenic
condition. Hence, it can be stated that the
isolates have both the mechanisms of
oxidative and fermentative processes for
energy generation.
3.4. Effect of NaCl
The other physiological parameter for growth
of a cell is the requirement of sodium chloride
as the physiological saline prevents the cell
from osmotic shock. For this, isolated
Lactobacilli cultures were incubated in MRS
broth at 37 °C for 24 h to assess the effect of
different NaCl concentrations (i.e., 2.0%,
4.0%, and 6.5%) in terms of turbidity, as an
indication of microbial growth. At the NaCl
concentration of 2.0%, the growth was
observed in all the Lactobacilli isolates. With
an increase in NaCl from 2% to 4%, turbidity
was observed only in thirty-eight isolates 01,
02, 07, 09, 11, 15–19, 23–25, 30, 32, 34, 39,
42–45, 47, 48, 50–53, 57, 58, 61–64, 66, 67,
71, 73 and 77. The growth pattern at 4% NaCl
concentration was also reported [13]
and
concluded wide variation in growth of
Lactobacilli cultures. At 6.5% NaCl
concentration, not a single isolate could show
growth as indicated in Table III.
Table III: Physiological Characterization of the Lactobacilli Isolates from Dahi.
Isolate No. (s)
Physiological Characteristics
Growth at
Temperature Oxygen Requirement
Effect of NaCl
(%)
10 °C 42 °C Aerobic Micro-
aerophilic Anaerobic 2 4 6.5
1, 11, 44, 47, 57, 61 + - + + + + + -
2, 7, 9, 15, 17, 19, 23, 25, 30, 32, 34, 42, 50, 52,
53, 58, 62, 64, 71, 73 - + + + + + + -
3–6, 10, 12, 13, 14, 21, 22, 26, 27, 31, 35, 36,
38, 40, 41, 45, 54–56, 59, 60, 65, 68, 69, 75, 78 + - + + + + - -
8, 46, 49 + + + + + + - -
16, 39, 48, 67, 77 + + + + + + + -
18, 24,43, 51, 63 - + + + + + + -
20, 28, 29, 66 - + + + - + - -
33, 37, 70 - + + + - + - -
Symbols: + = able to grow; - = not able to grow
Research & Reviews: A Journal of Dairy Science and Technology
Volume 1, Issue 2, August 2012, Pages
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3.5. Biochemical Characterization of
Lactobacilli
a) Catalase Production: Catalase, an
extracellular enzyme secreted by several
microorganisms, helps in degradation of
hydrogen peroxide produced during
carbohydrates utilization for energy
production, thereby its presence or
absence in a microbial cell can be used as
a significant diagnostic tool. The catalase
is involved in catalyzing the breakdown of
toxic hydrogen peroxide to produce
molecular oxygen that generates
vigorously while producing effervescence,
when a microbial culture is mixed with an
equal volume of 3% solution of hydrogen
peroxide. Absence of effervescence is
taken as indicative as negative for catalase
enzyme production. In the present study,
all the isolates were found to be catalase
negative (Table IV). These results
obtained for catalase further support the
identification of isolates as Lactobacilli
and pave the way in further
characterization of the isolates and are in
agreement with [14, 15].
b) Gas from Glucose: The microorganisms
use carbohydrates in a different pattern
depending on their enzyme complement.
In fermentation, substrates such as
carbohydrates and alcohols undergo
anaerobic dissimilation and produce
organic acids that may be accompanied by
the production of gases such as hydrogen
or carbon-dioxide. Therefore, all the
Lactobacilli were subjected to glucose
fermentation test in order to see the
production of gas. It was observed from
Table IV that isolates 03–06, 10, 12–14,
18, 21, 22, 24, 26, 27, 31, 33, 35–38, 40,
41, 43, 45, 51, 54–56, 59, 60, 63, 65, 68–
70, 72, 74–76 and 78 produced gas in
broth containing glucose as the sole source
of carbon while other isolates could not
produce any gas as shown by a hollow
space in the inverted Durham’s tubes and,
therefore, resulted as negative for gas
production from glucose. Thus, the above
isolates showed fermentation of glucose in
the medium for their growth. These
variations in results were also reported
[13, 15].
c) Arginine Hydrolysis: After confirming the
glucose utilization pattern of the isolates,
these were checked for the ability to
hydrolyze arginine that results in the
production of ammonia, making the pH of
the medium alkaline, and hence, changing
the color apparently from yellow to orange.
In arginine hydrolysis test, 14 isolates
showed a negative reaction and four
isolates (20, 28, 29 and 66) showed weak
hydrolysis, 29 isolates (03–06, 10, 12–14,
21, 22, 26, 27, 31, 35, 36, 38, 40, 41, 45,
54–56, 59, 60, 65, 68, 75 and 78) were
observed for a variable reaction, while the
rest 31 isolates were observed to be positive
for arginine hydrolysis (Table IV).
d) Aesculin Hydrolysis: The medium
contains esculin and peptone for nutrition
while ferric citrate is added as a color
indicator. Esculin is a glycoside (a sugar
Research & Reviews: A Journal of Dairy Science and Technology
Volume 2, Issue 2, August 2012, Pages
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ISSN: 2319-3409© STM Journals 2012. All Rights Reserved
Page 10
molecule bonded by an acetyl linkage to
an alcohol) composed of glucose and
esculetin. These linkages are easily
hydrolyzed under acidic conditions.
Microorganisms split the esculin
molecules and use the liberated glucose to
supply energy needs to release the
esculetin into the medium. The free
esculetin reacts with ferric citrate in the
medium to form a phenolic iron complex
that turns the dark brown media to black
indicating a positive test. The esculin
hydrolysis test was used to check the
ability of the isolated microorganisms to
hydrolyze the glycoside esculin to
esculetin and glucose in the presence of
10%–40% bile. In this test, only 11.5%
isolates were found to be positive, 20.5%
showed variable reaction and rest 67.9%
showed negative reaction and in
accordance with [13, 15].
Table IV: The Biochemical Characterization of the Isolated Lactobacilli Cultures from Dahi.
Isolate No. (s) Catalase
Test
Gas
from
Glucose
Arginine
Hydrolysis
Aesculin
Hydrolysis
Nitrate
Reduction
Citrate
Utilization
1, 11, 44, 47, 57, 61 - - - + - +
2, 7, 9, 15, 17, 19, 23, 25, 30, 32,
34, 42, 50, 52, 53, 58, 62, 64, 71,
73
- - + - - +
3–6, 10, 12, 13-14, 21, 22, 26, 27,
31, 35, 36, 38, 40, 41, 45, 54–56,
59, 60, 65, 68, 69, 75, 78
- + V V - +
8, 46, 49 - - + - - -
16, 39, 48, 67, 77 - - - + - -
18, 24, 43, 51, 63 - + + - - -
20, 28, 29, 66 - - W + - -
33, 37, 70 - + - + - +
Symbols: + = able to ferment; - = not able to ferment; v = variable fermentation; w = weak fermentation.
e) Nitrate Reduction Test: In microbial
taxonomy, nitrate reduction is an
important criterion for characterization
and identification of different types of
bacteria. This is due to the fact that certain
bacteria have the capability to reduce
nitrate to nitrite while others are capable
of further reducing nitrite to ammonia.
The formation of ammonia changes the pH
of media to alkaline thus, changing the
color of media from yellow to cherry red.
In the nitrate reduction test, all the isolates
tested showed negative reactions, as there
was no formation of red/pink color after
incubation of isolates in the nitrate broth
Research & Reviews: A Journal of Dairy Science and Technology
Volume 2, Issue 2, August 2012, Pages
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ISSN: 2319-3409© STM Journals 2012. All Rights Reserved
Page 11
(Table IV), a characteristic of
Lactobacillus group of organism [12, 15].
f) Citrate Utilization Test: For further
characterization and identification, all the
isolates were subjected for their potential
to utilize citrate as the sole carbon source.
Further, it also attributes to the better
technological property of the product as
citrate utilization leads to a better flavor
production in fermented milk production.
Certain bacteria utilize citrate with the
help of enzyme citrate permease and
citrase-producing diacetyl, a flavoring
compound as end product. Following
incubation on Simmon’s citrate agar,
citrate-positive cultures were identified by
the presence of growth on the surface of
slant, accompanied by blue coloration
whereas negative cultures did not show
any growth and medium remained green.
From Table IV, citrate utilization was
confirmed in 74.35% of isolates whereas
25.64% isolates (08, 16, 18, 20, 24, 28, 29,
39, 43, 46, 48, 49, 51, 63, 66, 67, 72, 74,
76 and 77) were found to be citrate
negative and are in agreement with [13].
3.5. Carbohydrate Fermentation Pattern of
Isolated Lactobacilli Cultures
For accounting the sugar utilization pattern of
the isolates suspected to be the Lactobacilli
after their growth on Lactobacillus selection
MRS agar, the sugar utilization tests were
performed using CHL basal media with
different sugar discs. In total, 16 sugars were
used for conformational identification of
Lactobacilli and these were: arbinose,
cellobiose, fructose, galactose, lactose,
maltose, manitol, mannose, melibiose,
raffinose, rhamnose, salicin, sorbitol, sucrose,
trehalose and xylose. Different isolates
showed different types of sugar utilization
patterns where some tubes containing isolates
and sugar turned yellow whereas the other
remained brown colored, indicating the
positive and negative tests, respectively for
sugar fermentation (Table V).
When these sugar utilization patterns were
compared with those given for Lactobacillus
species in the Bergey’s Manual of
Determinative Bacteriology [12], the isolates
were tentatively identified as L. casei, L.
brevis, L. plantarum, L. fermentum, L.
rhamnosus, L. acidophilus, L. helveticus, L.
viridescense, L. lactis. Hence, nine different
species within Lactobacillus genera in the dahi
sample obtained from rural and urban
locations were confirmed in the present
results. Out of 78 Lactobacillus isolates 3.84%
were characterized as L. brevis, 24.35% were
L. casei, 37.17% were L. acidophilus, 5.12%
as L. viridescence, whereas 6.41% were
categorized as L. rhamnosus, 5.12% as L.
helveticus, 7.69% were identified as L.
plantarum, 6.41% were L. fermentum and
3.84% were tentatively characterized as L.
lactis (Figure 1). The data obtained for genus
and species identification comprising a
number of morphological, physiological,
biochemical and sugar utilization pattern tests
were also subjected to a software called
Research & Reviews: A Journal of Dairy Science and Technology
Volume 2, Issue 2, August 2012, Pages
__________________________________________________________________________________________
ISSN: 2319-3409© STM Journals 2012. All Rights Reserved
Page 12
PIBWIN [16] and the tentative identification
was done so as to confirm the obtained results
while matching with Bergey’s Manual of
Determinative Bacteriology Holt et al., (1994)
[17]. It was further confirmed that the isolates
with both the methods were found to be of
similar species.
Table V: Sugar Fermentation Pattern of the Lactobacilli Isolates from Dahi.
Isolate No. (s)
Fermentable Sugars
Organism
A C F G L M Mn Mo Ml Rf Rh S Sr Su T X
1, 11, 44, 47, 57, 61 V + + + + + + + + + - + + + + V L. plantarum
2, 7, 9, 15, 17, 19,
23, 25, 30, 32, 34,
42, 50, 52, 53, 58,
62, 64, 71, 73
- + + + + + + + - - - + + + + - L. casei
3–6, 10, 12, 13, 14,
21, 22, 26, 27, 31,
35-36, 38, 40, 41,
45, 54–56, 59, 60,
65, 68, 69, 75, 78
- + + W + - + + - + + + - + - + L. acidophilus
8, 46, 49 - - - - - + - - - - - - - - - - L.
viridescense
16, 39, 48, 67, 77 - - + + + + + + - + + + - + - + L. rhamnosus
18, 24, 43, 51, 63 V V + + + + + - + + - - - + V V L. fermentum
20, 28, 29, 66 - - + + + - - + - - - - - + - - L. helveticus
33, 37, 70 - - - + + + - - + + - + - v + - L. lactis
A = Arabinose; C = Cellobiose; F = Fructose; G = Galactose; L = Lactose; M = Maltose; Mn = Mannitol;
Mo = Mannose, Ml = Melibiose, Rf = Raffinose; Rh = Rhamnose; S = Salicin; Sr = Sorbitol; Su = Sucrose;
T = Trehalose; X = Xylose; (+) = able to ferment sugar; (–) = not able to ferment sugar; v = variable
fermentation; w = weak fermentation.
Research & Reviews: A Journal of Dairy Science and Technology
Volume 2, Issue 2, August 2012, Pages
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ISSN: 2319-3409© STM Journals 2012. All Rights Reserved
Page 13
Fig. 1: Prevalence (%) of Lactobacillus Species in Dahi.
4. CONCLUSIONS
From dahi, a total of 78 Lactobacillus isolates
were distributed among nine species and
identified as L. casei, L. brevis, L. fermentum,
L. plantarum, L. helveticus, L. rhamnosus, L.
viridiscence, L. lactis, L. acidophilus, where L.
acidophilus, was the most prevalent. It can be
stated that L. acidophilus isolated from
traditional Indian dairy product (dahi) could be
exploited as a probiotic after investigating its
beneficial characteristics.
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