Bharadwaj et al., 2012

Research & Reviews: A Journal of Dairy Science and Technology Volume 1, Issue 2, August 2012, Pages __________________________________________________________________________________________

Isolation and Biochemical Characterization of Lactobacillus species Isolated from Dahi Aarti Bhardwaj1, Monica Puniya2, K. P. S. Sangu1, Sanjay Kumar3, Tejpal Dhewa4* 1 Depatment of Dairy Science and Technology, Janta Vedic College, Baraut-250611, Uttar Pradesh, India 2 Dairy Cattle Nutrition Division, 3Dairy Microbiology Division, National Dairy Research Institute, Karnal-132001, Haryana, India 4 Bhaskaracharya College of Applied Sciences (University of Delhi), New Delhi-110075, India 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

*Author for Correspondence Email: [email protected], Tel: + 91-8836325454 balance in microbiota of gut is considered to

1. INTRODUCTION

provide the colonization resistance against Generally, the healthiness of food has been

infectious agents and promote anti-allergic

linked

processes, stimulate immune system and

to

a

nutritionally

rich

diet

recommended by specialists and the role of it

reduce hypersensitivity [1–4].

in totality has been emphasized instead of emphasizing

on

components.

Milk, very first food of mammals including

During the past few decades, the lifestyle in

humans, is surrounded with emotional and

the developed and developing countries has

cultural importance in the society. Men have

been changing fast with regard to living

been habituated to think of milk as nature’s

standards,

of

most perfect food for them. Therefore, milk

antibiotics. The prevalence of chronic diseases

and dairy products have long been recognized

like allergies and gut-associated disorders

as an important constituent of balanced diet for

(e.g., Crohn’s disease, ulcerative colitis, and

human beings as these products provide a wide

inflammatory bowel disease) are of rising

range of essential nutrients.

diet,

individual

hygiene

and

usage

importance in the world nowadays. Hence, the

ISSN: 2319-3409© STM Journals 2012. All Rights Reserved Page 1


In addition, the evidence of health benefits of

functional foods has been developed for the

milk associated with the presence of specific

consumers.

components or beneficial bacteria is gaining scientific credibility at a rapid pace. Therefore,

The microbial ecology in the gastrointestinal

the best known examples of functional foods

tract influences many functions in our body

(i.e., that benefit the consumers beyond

(i.e.,

nutrition) are fermented milks containing

detoxification and ultimately the functioning

friendly bacteria and are called probiotics

of immune system). All these aspects make the

[5, 6]. Milk itself is much more than the total

gut a target organ for development of

sum of its nutrients as it is also a natural

functional foods that can help in maintaining

source of biologically active compounds that

the relative balance of microorganisms in the

exert potential impact on human health.

gastrointestinal tract. The establishment of

Probiotic group of microorganisms that are

microbial balance by shifting it towards a

present in fermented milk products (i.e., dahi,

beneficial one with the help of specific dietary

yoghurt, cheese, etc.) beneficially affect the

components (i.e., probiotics and prebiotics)

host by improving the intestinal microbial

has opened the gateway for the development

balance. The effect of probiotics includes

of functional foods ensuring more benefits to

alleviation of intestinal disorders (i.e., lactose

the host’s health [9].

digestion,

absorption

of

nutrients,

intolerance, acute gastroenteritis due to enteric pathogens, constipation, and inflammatory

Probiotics are defined as live microbes which

bowel disease) and a number of food allergies.

transit the gastro-intestinal tract and in doing

The therapeutic value of fermented dairy

so, benefit the health of the consumer [10, 11].

products has been utilized, dating back to

Probiotic microorganisms are found in many

Biblical days. These foods confer the added

food products, especially in the fermented

health

disease-prevention

foods. Therefore, the probiotic lactic acid

characteristics beyond the basic nutrition of

bacteria can be isolated from the fermented

the food [7]. About 65% of the total functional

milk products like acidophilus milk, yoghurt

foods market is covered with dairy probiotic

and dahi. Dahi, naturally fermented milk by

products [8]. Although the primary aim of

lactic cultures, is widely consumed throughout

food is to provide enough nutrients to fulfil the

South

body requirements, yet various functions of

subcontinent. The micro-flora of dahi varies

the host body are modulated by the diet

from one geographical region to the other and

consumed. Hence, in order to compensate for

also with seasonal variations of the year. Apart

deficiency of certain nutrients in the diet due

from the starter lactic cultures, dahi can also

to

benefits

changes

industrialized

in

or

nutritional

nations,

the

Asia,

especially

in

the

Indian

habits

of

have some probiotics like Lactobacillus

concept

of

acidophilus, Lactobacillus casei, Lactobacillus



bulgaricus, Streptococcus thermophilus, etc.

2.2. Identification of Selected Lactobacilli

Therefore, dahi can be used as a source for

The identification and further characterization

isolation of probiotic bacteria [5, 6] and also a

of Lactobacilli isolates grown on MRS agar

product of immense importance for human

was done mainly with the help of the

consumption.

following

tests:

microscopic

examination

(Gram staining), catalase test, growth at In view of the above facts, the present study

different

temperatures

has been designed to isolate and identify a

42 + 1 ºC),

Lactobacillus species from dahi samples

anaerobic conditions, growth at different NaCl

collected from the local market.

concentration,

growth

10 + 1 ºC

under

fermentation

and

aerobic

of

and

different

carbohydrates, etc. a) Microscopic Examination: The purity

2. MATERIALS AND METHODS

morphological identification of the isolates 2.1. Isolation of Lactic Acid Bacteria

as

Dahi samples (40) for the isolation of lactic

microscopically

acid bacteria were collected from different

staining, for which single colony of each

rural and urban locations in order to get a

isolate was picked up and stained as per

wider diversity of lactic acid bacterial strains.

the standard protocol and viewed under oil

Each sample was taken in a sterile container

immersion for similar type of cells.

separately and placed in a polyethylene bag during

transportation

to

was

by

confirmed

performing

Gram

b) Micrometry: Each isolate after Gram

laboratory

staining was subjected to microscopic

employing standard conditions for sample

measurements employing ocular and stage

collection. One gram of curd sample was

micrometer. To determine the size of

immediately

aseptic

Lactobacilli isolates, prepared slides were

conditions by suspending in 9 mL of normal

observed under oil-immersion objective

saline (0.85%) and was vortexed for proper

and number of ocular divisions occupied

mixing. One milliliter of each well-mixed dahi

by each bacillus was recorded and

sample was enriched in 9 mL of sterile

interpreted as per the above formula.

processed

the

Lactobacilli

under

Lactobacillus selection MRS broth for 24 h at 37 °C. Before inoculation of sample, the pH of

2.2.1.

MRS broth was adjusted to 6.5 ± 0.2. The

Isolates

enriched samples were streaked on the Petri

After confirming the purity of culture, each

plates containing Lactobacillus selection MRS

isolate was further assessed for growth at two

agar with the help of calibrated inoculating

different temperatures.

loop and incubated aerobically at 37 °C for

a) Growth of isolates at (10 °C and 42 °C):

48 h and observed for the growth of colonies.

Physiological

Characterization

of

The isolates were tested for their ability to



grow in MRS broth at 10 + 1 °C for 7 days

For this, culture from a typical colony was

and

for

placed onto a clean grease-free glass slide

24–48 h. For this, 10 mL of MRS broth

and drop of 3% hydrogen peroxide

tubes

of

solution was added onto the culture and

Lactobacilli cultures. The development of

closely observed for the evolution of

turbidity in culture tubes was recorded as

bubbles.

the ability of isolates to grow at 10 °C and

indicated positive catalase reaction and

42 °C and results were noted as positive or

was recorded accordingly for the presence

negative.

or absence of enzyme.

42 °C

were

by

incubating

inoculated

@

1%

The

production

of

bubbles

b) Oxygen requirement of the isolates: All

b) Gas from Glucose: Sterile test tubes of

the isolates were inoculated in MRS broth

10 mL glucose broth containing Durham’s

and

tube

were

kept

differently

under

(inverted

and

dipped),

were

oxygenated condition; in dessicator with

inoculated with Lactobacilli cultures at the

burned

micro-aerophilic

@1% and incubated at 37 °C for 24–48 h.

condition) and in anaerobic jar with gas

Gas production that appeared in the form

pack at 37 °C for 24–48 h to determine the

of a hollow space in Durham’s tube was

impact of oxygen on the growth of the

recorded as a positive result.

candle

(for

Lactobacilli isolates and results were noted as positive or negative.

c) Arginine Hydrolysis: Autoclaved arginine hydrolysis broth tubes were inoculated with the isolated cultures (1%) and

2.2.2. Effect of NaCl Concentrations on

incubated at

Growth of Isolates

incubation, 3–5 drops of the Nessler's

The isolates were inoculated in MRS broth

reagent were added to each test tube and

having different NaCl concentration (2.0%,

observed for the change in color (yellow

4.0% and 6.5%) and incubated at 37 °C for

to orange color), indicating a positive

24–48 h. The culture tubes were observed for

result for arginine hydrolysis.

the presence or absence of growth.

37 °C for 48 h. After

d) Aesculin Hydrolysis: The isolates were also assessed for their ability to hydrolyze

2.2.3.

Biochemical

Characterization

of

glycoside aesculin to aesculetin and

Isolates

glucose. For this, bile aesculin agar plates

a) Catalase Test: The test was performed in

were streaked with the isolated cultures

order to determine the ability of the

and incubated at 37 °C for 24–48 h. After

isolated cultures to degrade the hydrogen

incubation, the plates were examined for

peroxide

enzyme

the presence of a dark brown to black halo

catalase. The test was carried out as the

around the bacterial growth, showing a

slide method, using an inoculating needle.

positive result for aesculin hydrolysis.

by

producing

the



e) Nitrate Reduction Test: Nitrate reduction

autoclaving. One sugar disc was aseptically

is an important criterion for differentiating

added to each tube. Each tube was inoculated

and characterizing different types of

with 0.1 mL of inoculum, incubated at 37 °C

bacteria. Therefore, the isolates were

for 24–48 h and the results of color change

incubated at 37 °C for 24 h in trypticase

were recorded as positive or negative. A

nitrate broth. After incubation, 0.5 mL

control using 0.1 mL sterile water as inoculum

each of sulphanilic acid (0.8%, in 5N

was used to compare the color change. Sugars

Acetic acid) and -naphthylamine (0.5%,

used to determine the fermentation profile of

in 5N Acetic acid) were added into the

Lactobacilli

tubes. The appearance of red or pink color

cellobiose,

indicated the positive test for nitrate

maltose,

reduction and was recorded accordingly

raffinose,

for the isolates tested in the present study.

sorbitol, sucrose and xylose. The cultures were

f) Citrate Utilization Test : The isolates were inoculated

in

incubated at incubation,

Simmons

citrate

agar

isolates fructose,

mannitol,

were galactose, mannose,

rhamnose,

trehalose,

arabinose, lactose, melibiose, salicin,

identified based on the pattern of sugar utilization [12].

37 °C for 24 h. After

the

appearance

of

blue

2.3.

Maintenance

and

Propagation

of

coloration indicated the positive test for

Cultures

citrate

For their further assessment of probiotic

utilization

and

was

recorded

attribute and other analysis all the isolated

accordingly for the isolates tested.

Lactobacilli cultures were maintained in chalk 2.2.4. Carbohydrate Fermentation Pattern by

litmus milk at refrigeration temperature after

the Isolates

their growth at 37 °C overnight. The cultures

Most microorganisms obtain their energy

were sub-cultured at regular intervals in chalk

through a series of orderly and integrated

litmus milk and stored under refrigeration

enzymatic reactions leading to the bio-

conditions. Before use, the cultures were

oxidation

a

activated in MRS broth. All the isolates were

carbohydrate. Thus, different sugars were used

also maintained at −70 °C in glycerol stock in

for determining the fermentation profile and

triplicates for use in experiment at different

further

stages.

of

a

substrate,

characterization

of

frequently

Lactobacilli

isolates. For this, Lactobacilli cultures were subjected to sugar fermentation reactions using

2.4. Purity of Cultures

CHL medium for knowing their fermentation

The cultures of Lactobacilli species and

pattern. CHL medium was used as basal

indicator strains were regularly tested for their

medium. Four milliliter of the medium was

purity by microscopic examination and/or

taken

in

each

tube

and

sterilized

by



catalase test for confirmation and presence of

(Table I) were picked up after plating 1 mL of

contamination, if any.

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

3. RESULTS AND DISCUSSION

done 3–4 times in MRS agar by incubating at 3.1, Isolation of Lactobacilli from Dahi from

37 °C for 24–48 h. Finally, the well-isolated

Nearby Vicinity

colonies were streaked on the solidified Petri

Out of several colonies developed on agar

plates of Lactobacillus selection MRS agar

plates,

colonial

with inoculating loop incubated aerobically at

morphology (i.e., color, size, margin and shape

37 °C for 48 h for further identification and

of the colony) such as white, greyish white or

characterization

cream color, size varying from 0.5–2.3 mm in

physiological and biochemical tests used

diameters, with entire or undulate margins

routinely in microbial taxonomy.

78

isolates

based

on

based

on

different

Table I: Colonial Morphology of the Isolated Lactobacilli Cultures from Dahi. Colonial Morphology Isolate(s)

Color

Shape

Size (mm)

Margin

Pin point; circular; 1, 11, 44, 47, 57, 61

Cream

smooth; compact and

0.8

Entire

1.2

Entire

2.1

Undulate

convex 2, 7, 9, 15, 17, 19,23, 25, 30, 32, 34,

White

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,

White

60, 65, 68, 69, 75, 78

Circular

Circular; large; rough and irregular

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

0.8

Entire

20, 28, 29, 66

White

Circular and compact

0.6

Entire

33, 37, 70

Creamish white

Circular

2.3

Entire

Pin point; circular; compact and convex

3.2. Identification and Characterization of

using standard staining procedure. The isolates

Lactobacillus Isolates

were found to be purple colored Gram-positive

The

Gram

reaction

property

and

cell

rods (i.e., straight rods, irregular rods, rods

morphology of all the isolates were examined

with rounded ends) of varying sizes and



arrangements such as rods in single, or in

micrometry.

chains

oil-immersion

indicated that the isolates could belong to the

microscope (Table II). After confirming the

Lactobacilli group and paved the way for

Gram positive character each isolate was

further characterization through physiological

further characterized for cell dimensions using

and biochemical means for confirmation.

(2–5

cells),

under

These

preliminary

results

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,

G +ve

Rods in chains

0.7 × 3.2

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

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

3.3. Physiological Characterization

isolates 02, 07–09, 15–20, 23–25, 28–30,

a) Growth at 10 °C and 42 °C: The isolated

32–34, 37, 39, 42, 43, 46, 48–53, 58, 62–

Lactobacilli cultures were assessed for

64, 66, 67, 70–74, 76 and 77. The other

their growth at two different temperatures

Lactobacilli could not grow at the elevated

(i.e., 10 °C and 42 °C). For this, cultures

temperature of 42 °C. Hence, it can be

were incubated in MRS broth at 10 °C for

concluded form Table III that 37 °C is the

7 days and the turbidity in broth was

optimum temperature for all the isolates

observed as an indication of microbial

and few of these could either survive or

growth.

grow at 10 °C/42 °C or at both the

The

isolates

showed

good

turbidity except that of 30 isolates i.e., 02,

temperatures away from the optimum.

07, 09, 15, 17–20, 23–25, 28–30, 32–34,

b) Oxygen Requirement: After assessing the

37, 50–53, 58, 62–64, 66, 70, 71 and 73,

growth

of

Lactobacilli

where no growth was observed in terms of

temperatures, they were exposed to the

turbidity. On the other hand, isolates were

growth in oxygenic, reduced oxygen and

also incubated at 42 °C for 24 to 48 h and

anoxygenic environments. For this, all the

turbidity was observed in tubes containing

isolates were incubated in MRS broth and


at

different


incubated at 37 °C for 24 h under aerobic,

3.4. Effect of NaCl

micro-aerophilic and in anaerobic gas jars.

The other physiological parameter for growth

In aerobic and micro-aerophilic conditions

of a cell is the requirement of sodium chloride

all the isolates tested showed turbidity in

as the physiological saline prevents the cell

the medium indicating the occurrence of

from osmotic shock. For this, isolated

growth. Under anaerobic condition, the

Lactobacilli cultures were incubated in MRS

turbidity was not observed with isolates

broth at 37 °C for 24 h to assess the effect of

20, 28, 29, 33, 37, 66 and 70; however, all

different NaCl concentrations (i.e., 2.0%,

other isolates showed turbidity and/or

4.0%, and 6.5%) in terms of turbidity, as an

growth. It can be concluded from Table III

indication of microbial growth. At the NaCl

that all the isolates were facultative

concentration of 2.0%, the growth was

anaerobes and can grow in the presence

observed in all the Lactobacilli isolates. With

and absence of oxygen except seven

an increase in NaCl from 2% to 4%, turbidity

isolates (i.e., 20, 28, 29, 33, 37, 66 and 70)

was observed only in thirty-eight isolates 01,

that

anoxygenic

02, 07, 09, 11, 15–19, 23–25, 30, 32, 34, 39,

condition. Hence, it can be stated that the

42–45, 47, 48, 50–53, 57, 58, 61–64, 66, 67,

isolates have both the mechanisms of

71, 73 and 77. The growth pattern at 4% NaCl

oxidative and fermentative processes for

concentration was also reported [13] and

energy generation.

concluded wide variation in growth of

failed

to

grow

in

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. Physiological Characteristics Growth at Isolate No. (s)

Effect of NaCl

Oxygen Requirement

Temperature 10 °C

42 °C

Aerobic

+

-

+

-

+

+

8, 46, 49

Micro-

(%)

Anaerobic

2

4

6.5

+

+

+

+

-

+

+

+

+

+

-

-

+

+

+

+

-

-

+

+

+

+

+

+

-

-

16, 39, 48, 67, 77

+

+

+

+

+

+

+

-

18, 24,43, 51, 63

-

+

+

+

+

+

+

-

20, 28, 29, 66

-

+

+

+

-

+

-

-

33, 37, 70

-

+

+

+

-

+

-

-

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

aerophilic

Symbols: + = able to grow; - = not able to grow



3.5.

Biochemical

Characterization

of

production of gas. It was observed from

Lactobacilli a) Catalase

Production:

an

Table IV that isolates 03–06, 10, 12–14,

extracellular enzyme secreted by several

18, 21, 22, 24, 26, 27, 31, 33, 35–38, 40,

microorganisms, helps in degradation of

41, 43, 45, 51, 54–56, 59, 60, 63, 65, 68–

hydrogen

during

70, 72, 74–76 and 78 produced gas in

energy

broth containing glucose as the sole source

peroxide

carbohydrates production,

b)

fermentation test in order to see the

Catalase,

produced

utilization thereby

for

its

or

of carbon while other isolates could not

absence in a microbial cell can be used as

produce any gas as shown by a hollow

a significant diagnostic tool. The catalase

space in the inverted Durham’s tubes and,

is involved in catalyzing the breakdown of

therefore, resulted as negative for gas

toxic hydrogen peroxide to produce

production from glucose. Thus, the above

molecular

generates

isolates showed fermentation of glucose in

vigorously while producing effervescence,

the medium for their growth. These

when a microbial culture is mixed with an

variations in results were also reported

equal volume of 3% solution of hydrogen

[13, 15].

oxygen

presence

that

peroxide. Absence of effervescence is

c) Arginine Hydrolysis: After confirming the

taken as indicative as negative for catalase

glucose utilization pattern of the isolates,

enzyme production. In the present study,

these were checked for the ability to

all the isolates were found to be catalase

hydrolyze arginine that results in the

negative

results

production of ammonia, making the pH of

obtained for catalase further support the

the medium alkaline, and hence, changing

identification of isolates as Lactobacilli

the color apparently from yellow to orange.

and

further

In arginine hydrolysis test, 14 isolates

characterization of the isolates and are in

showed a negative reaction and four

agreement with [14, 15].

isolates (20, 28, 29 and 66) showed weak

Gas from Glucose: The microorganisms

hydrolysis, 29 isolates (03–06, 10, 12–14,

use carbohydrates in a different pattern

21, 22, 26, 27, 31, 35, 36, 38, 40, 41, 45,

depending on their enzyme complement.

54–56, 59, 60, 65, 68, 75 and 78) were

In

as

observed for a variable reaction, while the

undergo

rest 31 isolates were observed to be positive

produce

for arginine hydrolysis (Table IV).

(Table

pave

IV).

the

way

fermentation,

carbohydrates anaerobic

These

in

substrates

and

alcohols

dissimilation

and

such

organic acids that may be accompanied by

d) Aesculin

Hydrolysis:

The

medium

the production of gases such as hydrogen

contains esculin and peptone for nutrition

or carbon-dioxide. Therefore, all the

while ferric citrate is added as a color

Lactobacilli were subjected to glucose

indicator. Esculin is a glycoside (a sugar



molecule bonded by an acetyl linkage to

indicating a positive test. The esculin

an alcohol) composed of glucose and

hydrolysis test was used to check the

esculetin.

ability of the isolated microorganisms to

These

hydrolyzed

linkages

under

Microorganisms

are

acidic split

easily

conditions.

the

hydrolyze

the

glycoside

esculin

to

esculin

esculetin and glucose in the presence of

molecules and use the liberated glucose to

10%–40% bile. In this test, only 11.5%

supply energy needs to release the

isolates were found to be positive, 20.5%

esculetin into the medium. The free

showed variable reaction and rest 67.9%

esculetin reacts with ferric citrate in the

showed

medium to form a phenolic iron complex

accordance with [13, 15].

negative

reaction

and

in

that turns the dark brown media to black

Table IV: The Biochemical Characterization of the Isolated Lactobacilli Cultures from Dahi. Catalase

Isolate No. (s)

Test

1, 11, 44, 47, 57, 61

Gas from Glucose

Arginine

Aesculin

Nitrate

Citrate

Hydrolysis

Hydrolysis

Reduction

Utilization

-

-

-

+

-

+

-

-

+

-

-

+

-

+

V

V

-

+

8, 46, 49

-

-

+

-

-

-

16, 39, 48, 67, 77

-

-

-

+

-

-

18, 24, 43, 51, 63

-

+

+

-

-

-

20, 28, 29, 66

-

-

W

+

-

-

33, 37, 70

-

+

-

+

-

+

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

Symbols: + = able to ferment; - = not able to ferment; v = variable fermentation; w = weak fermentation.

e) Nitrate Reduction Test: In microbial taxonomy,

nitrate

reduction

is

The formation of ammonia changes the pH

an

of media to alkaline thus, changing the

important criterion for characterization

color of media from yellow to cherry red.

and identification of different types of

In the nitrate reduction test, all the isolates

bacteria. This is due to the fact that certain

tested showed negative reactions, as there

bacteria have the capability to reduce

was no formation of red/pink color after

nitrate to nitrite while others are capable

incubation of isolates in the nitrate broth

of further reducing nitrite to ammonia.



(Table

IV),

a

characteristic

of

Lactobacillus group of organism [12, 15]. f) Citrate

Utilization

Test:

For

further

Lactobacilli cellobiose, maltose,

and

these

fructose, manitol,

were:

arbinose,

galactose, mannose,

lactose, melibiose,

characterization and identification, all the

raffinose, rhamnose, salicin, sorbitol, sucrose,

isolates were subjected for their potential

trehalose

to utilize citrate as the sole carbon source.

showed different types of sugar utilization

Further, it also attributes to the better

patterns where some tubes containing isolates

technological property of the product as

and sugar turned yellow whereas the other

citrate utilization leads to a better flavor

remained

production in fermented milk production.

positive and negative tests, respectively for

Certain bacteria utilize citrate with the

sugar fermentation (Table V).

and

xylose.

brown

Different

colored,

isolates

indicating

the

help of enzyme citrate permease and citrase-producing diacetyl, a flavoring

When these sugar utilization patterns were

compound as end product. Following

compared with those given for Lactobacillus

incubation on Simmon’s citrate agar,

species

citrate-positive cultures were identified by

Determinative Bacteriology [12], the isolates

the presence of growth on the surface of

were tentatively identified as L. casei, L.

slant, accompanied by blue coloration

brevis, L. plantarum, L. fermentum, L.

whereas negative cultures did not show

rhamnosus, L. acidophilus, L. helveticus, L.

any growth and medium remained green.

viridescense, L. lactis. Hence, nine different

From Table IV, citrate utilization was

species within Lactobacillus genera in the dahi

confirmed in 74.35% of isolates whereas

sample

25.64% isolates (08, 16, 18, 20, 24, 28, 29,

locations were confirmed in the present

39, 43, 46, 48, 49, 51, 63, 66, 67, 72, 74,

results. Out of 78 Lactobacillus isolates 3.84%

76 and 77) were found to be citrate

were characterized as L. brevis, 24.35% were

negative and are in agreement with [13].

L. casei, 37.17% were L. acidophilus, 5.12%

in

the

obtained

Bergey’s

from

Manual

rural

and

of

urban

as L. viridescence, whereas 6.41% were 3.5. Carbohydrate Fermentation Pattern of

categorized as L. rhamnosus, 5.12% as L.

Isolated Lactobacilli Cultures

helveticus, 7.69% were identified as L.

For accounting the sugar utilization pattern of

plantarum, 6.41% were L. fermentum and

the isolates suspected to be the Lactobacilli

3.84% were tentatively characterized as L.

after their growth on Lactobacillus selection

lactis (Figure 1). The data obtained for genus

MRS agar, the sugar utilization tests were

and

performed using CHL basal media with

number

different sugar discs. In total, 16 sugars were

biochemical and sugar utilization pattern tests

used for conformational identification of

were also subjected to a software called

species of

identification morphological,


comprising

a

physiological,


PIBWIN [16] and the tentative identification

[17]. It was further confirmed that the isolates

was done so as to confirm the obtained results

with both the methods were found to be of

while matching with Bergey’s Manual of

similar species.

Determinative Bacteriology Holt et al., (1994)

Table V: Sugar Fermentation Pattern of the Lactobacilli Isolates from Dahi. Fermentable Sugars Isolate No. (s)

A C

F G L M

V +

+ + +

-

+

-

8, 46, 49

Organism

Mn

Mo

Ml

Rf

Rh

S

Sr

Su

T

X

+

+

+

+

+

-

+

+

+

+

V

L. plantarum

+ + +

+

+

+

-

-

-

+

+

+

+

-

L. casei

+

+ W +

-

+

+

-

+

+

+

-

+

-

+

L. acidophilus

-

-

-

-

+

-

-

-

-

-

-

-

-

-

-

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

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 -

+ +

L. viridescense

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.



Fig. 1: Prevalence (%) of Lactobacillus Species in Dahi.

3. E. Isolauri, P. V. Kirjavainen and S.

4. CONCLUSIONS

Salminen. Gut. 2002. 50. 54–59p. From dahi, a total of 78 Lactobacillus isolates were distributed among nine species and

4. L. M. T. Dicks and M. Botes. Beneficial Microbes. 2010. 1. 11–29p.

identified as L. casei, L. brevis, L. fermentum,

5. T. Dhewa, V. Bajpai, R. K. Saxena, et al.

L. plantarum, L. helveticus, L. rhamnosus, L.

International Journal of Probiotics and

viridiscence, L. lactis, L. acidophilus, where L.

Prebiotics. 2010. 5(1). 45–52p.

acidophilus, was the most prevalent. It can be

6. T. Dhewa, S. Pant and V. Mishra. Journal

stated that L. acidophilus isolated from

of Food Science and Technology. 2011.

traditional Indian dairy product (dahi) could be

DOI 10.1007/s13197-011-0457-2.

exploited as a probiotic after investigating its beneficial characteristics.

7. M. B. Roberfroid. American Journal of Clinical Nutrition. 1998. 73. 406–409p. 8. C. Stanton, G. Gardiner, H. Meehan, et al. American Journal of Clinical Nutrition.

REFERENCES

2001. 73. 4765–4835p. 1. M. Puniya, K. P. S. Sangu, A. Bhardwaj, et

al.

Journal

of

Research

in

Antimicrobials. 2012. 1, 001-011p. 2. T. Dhewa. Lactic Acid Bacteria as Probiotics: Applications and Safety Issues. Germany.

LAMBERT

Publishing. 2011.

Academic

9. T. Dhewa and N. Goyal. The IUP Journal of Life Sciences. 2009. 3. 29–34p. 10. G. W. Tannock, K. Munro, H. J. M. Harmsen, et al. Applied Environmental Microbiology. 2000. 66. 2578–2588p. 11. F. Chaucheyras-Durand and H. Durand. Beneficial Microbes. 2010.1. 3–9p.



12. J. G. Holt, N. R. Kreig, P. H. A. Sneath, et al. Bergey’s Manual of Determinative Bacteriology. 2004. 13. J. Wang, X. Chen, W. Liu, et al. European Food

Research

Technology.

2008.

1555–1561p. 14. J. K. Kaushik, A. Kumar, R. K. Duary, et al. PLoS one. 2009. 4(12). e8099 1–11p. 15. A. K. Puniya, S. Chaitanya, A. K. Tyagi, et al. Journal of Industrial Microbiology and

Biotechnology.

2008.

35.

1223–1228p. 16. T.

N.

Bryant.

Journal

of

Applied

Microbiology. 2004. 97(6). 1326–1327p. 17. J.G. Holt, N.R. Krieg, P.H.A. Sneath, et al., Bergeys Manual of Determinative Bacteriology. 1994. USA. 9th Edn.
