Isolation and biochemical characterization of δ-aminolevulinic acid dehydratase from Streptomyces...

Research & Reviews: A Journal of Dairy Science and Technology

Volume 1, Issue 2, August 2012, Pages

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ISSN: 2319-3409© STM Journals 2012. All Rights Reserved

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: [email protected], 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



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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



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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



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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.



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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



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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



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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



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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



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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



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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



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(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



__________________________________________________________________________________________


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.



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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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