Viability of Lactobacillus bulgaricus as Yoghurt Culture Under Different Preservation Methods

INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
1560–8530/2003/05–1–46–48
http://www.ijab.org
Viability of Lactobacillus bulgaricus as Yoghurt Culture Under
Different Preservation Methods

ZAHOOR, T., S.U. RAHMAN† AND UMAR FAROOQ
Departments of Food Technology and †Veterinary Microbiology, University of Agriculture, Faisalabad–38040, Pakistan

ABSTRACT

In present study, Lactobacillus bulgaricus (yoghurt starter culture) was isolated from indigenous sources and preserved by
three different methods namely on agar slopes, under oil and in liquid form conditions using MRS medium. Best method of
preservation was suggested on the basis of viability, morphology and Gram’s staining ability of culture during storage of two
months. Viability checks were made at 0, 15, 30, 45 and 60 days of storage. Under oil preservation method was found to be
the best method for maintenance and preservation of starter culture.

Key Words: Lactobacillus bulgaricus; Yoghurt; Culture; Preservation

INTRODUCTION
Morphological examination of culture. Morphological

and cultural examination was carried out by using Gram’s

Yoghurt is a fermented milk product and is most
staining method as described by Awan and Rahman (2002).
popular in South Asia. It is more nutritive as compared to
Purification of isolates. All the colonies from Milk agar,
milk in terms of vitamins content, digestibility and as a
showing frequent rods, pair or chain forming pattern and
source of calcium and phosphorus (Foissy, 1983). Tamime
Gram positive character were separately studied and later on
and Robinson (1985) suggested that yoghurt starter culture
purified on Acetate agar (AA) and Rogosa agar (RA) by
is consisted of two symbiotically growing bacteria
applying streak plate techniques as described by Awan and
Lactobacillus bulgaricus and Streptococcus thermophillus
Rahman (2002). Growth obtained after 24 h at 37oC was
in the ratio of 1:1. In Pakistan, starter cultures for the
then examined for morphological and cultural
production of yoghurt are normally imported from other
characteristics. Pure colonies of these cultural isolates were
countries which are preserved in the form of liquid, spray
finally transferred onto, de Man, Rogosa and Sharpe agar
dried, freeze dried and in frozen form. These imported
(MRS agar) plates and broth. Incubation was made at 37oC
cultures result in added costs to the end product. Keeping in
for 24 h and growth thus obtained was again examined for
view the existing situation, the project was planned to
morphological and cultural characteristics following the
isolate mono starter culture from local sources and
procedure mentioned by Harrigan and McCance (1976) and
subsequently to preserve it for further use in yoghurt
Cappuccino and Sherman (1996).
production under available facilities at laboratory scale.
Identification of pure culture. Pure culture thus isolated in

the above process was identified up to species level on the
MATERIALS AND METHODS
basis of their growth at different temperatures and sugar

fermentation tests as recommended by Harrigan and
Procurement of samples. Six samples (10 mL each) of
McCance (1976). Biochemical tests like indole test, methyl
commercial curd were collected in sterilized screw-capped
red test, voges proskauer test, citrate utilization test and
bottles (sterilized at 171oC for 30 min in hot-air-oven) from
catalase test were also performed for identification of
different areas of Faisalabad. The samples were brought to
culture isolates according to the method described by Awan
the Department of Food Technology, University of
and Rahman (2002).
Agriculture Faisalabad and stored immediately under
Preservation of pure culture. Pure culture of Lactobacillus
refrigeration conditions for further processing.
bulgaricus was preserved by three different methods as
Isolation of Lactobacillus spp. Isolation of the particular
described by Harrigan and McCance (1976). Briefly, (A)
microorganisms was done according to the method given by
On Agar Slopes Methods: Pure culture of Lactobacillus
Harrigan and McCance (1976) and Holt et al. (1994). Curd
spp. was preserved on MRS agar slopes after taking a
samples were diluted to 1:10 in sterilized normal saline
growth of 48 h at 37oC at ordinary refrigeration temperature,
solution and 1mL volume from each dilution was inoculated
(B) Under Oil Methods: Pure culture was grown on agar
onto the surface of Milk agar (MA) and Nutrient agar (NA)
slants and then completely covered with sterile liquid
and incubated at 37oC for 24 h. The growth thus obtained
paraffin sterilized at 160oC for 1-2 h in hot-air-oven and
was checked for morphological and cultural characteristics.
placed under refrigeration conditions, and (C) In Liquid
Form Conditions: Growth of pure culture was taken in MRS

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VIABILITY OF Lactobacillus bulgaricus AS YOGHURT CULTURE / Int. J. Agri. Biol., Vol. 5, No. 1, 2003
broth and then stored for two months at refrigeration
showing the suitability of Acetate agar for isolation of
temperature i.e. 4oC.
Lactobacillus spp. as also recommended by Harrigan and
Viability if culture. Viability, morphology and Gram’s
McCance (1976) and Cappuccino and Sherman (1996).
staining ability of the cultures was estimated as described by
Purification of isolates. The colonies on Acetate agar for S1
Awan and Rahman (2002) at 0, 15, 30, 45 and 60 days of
showing G +ve chain forming long rods were further
storage.
purified onto the surface of MRS agar plates and in MRS
Statistical analysis. The data obtained were statistically
broth. Single types of colonies were obtained after 24 h
analyzed as described by Steel et al. (1996).
incubation at 37oC. Colonies found on MRS agar plates

were white in color, convex in shape, small in size, pinpoint,
RESULTS AND DISCUSSION
smooth and 1- 2.2 mm in diameter; whereas, there was

turbidity, sedimentation and small white suspensions in
Isolation of microorganisms. Three different types of
MRS broth (Table III). MRS medium was found to be the
colonies were observed on Nutrient agar and Milk agar
most suitable selective medium for Lactobacillus spp. as
plates (Table I). Most of the colonies were white, irregular,
also confirmed by Harrigan and McCance (1976). Results of
circular and pinpoints. The cultural and morphological
sugar fermentation tests, growth at different temperatures
characteristics were further resolved on the basis of
and that of biochemical tests for culture isolates (from
microscopic examination. Majority of microorganisms were
selected S1) are shown in Tables IV and V. On the basis of
amongst Gram’s positive (G +ve) rods and cocci shaped
these results it was confirmed that the culture isolated (from
bacteria. Similar findings have also been reported by Masud
S1) was a pure culture of Lactobacillus bulgaricus.
et al. (1991) and Amoroso et al. (1992). S
Preservation of pure culture. After isolation and
1 and S2 were
found to have maximum numbers of G +ve indicating
purification, the pure culture (S1) was preserved by three
Lactobacillus spp. The growth from two samples S
different methods in order to find out the best method of
1 and S2
was then specially transferred on to the surface of Rogosa
preservation. For this viable count, morphology and Gram’s
agar and Acetate agar plates for more specifications.
staining ability of culture were considered as basic
Exuberant growth (Table II) of Lactobacillus spp. was
parameters.
obtained on Acetate agar as compared to Rogosa agar plates

Table I. Cultural and morphological characters of microorganisms isolated from yoghurt collected from different
areas of Faisalabad on general and selective media

Yoghurt
NA plates (colonies)
Morphology
M.A. Plates
Morphology
samples
(colonies)
S1
White, irregular,
G+ve, Rods, curved shape, round
White, Pin point
G + ve rod, Chains,
big circular

G+ve cocci, G+ve clusters.
S2
White, irregular
G+ve rods, Spherical, Curved shape
White, circular
G+ve rod, Chains, G+ve cocci
S3
White,
G+ve, G–ve,
White, dark yellow
G+ve rod, Chains, G+ve, G-ve,
circular
Rods, round, coccobacilli
cocci
S4
- -
-
-
S5
White, small
G+ve, G-ve, Round, coccobacilli
Whit big circular
G +ve, G –ve, cocci
S6 -
-
-
-
N.A. = Nutrient agar, M.A. = Milk agar

Table II. Cultural and morphological characteristics observed on selective and differential media with special
reference to G+ve chain forming bacteria isolated form yoghurt samples

Yoghurt
Differential medium rogosa agar Morphology
Selective medium acetate Agar Morphology
sample No.
plates (colonies)
Plates (colonies)
S1
White,sharp, smooth,Irregular,
G+ve, rods, chains
White, sharp, circular,
pinpoints,
G+ve, long rods,

chains
S2
White,irregular, small, circular,
G
+ve,
rods,
White,
irregular,
sharp,
G +ve, short rods,

cocci,Chain
cocci

Table III. Cultural and Morphological characteristics of G +ve chain forming bacteria on MRS agar medium and in
MRS broth

Yoghurt sample
MRS Agar plates
Morphology MRS-broth
(colonies)
Morphology
No.
(colonies)
S1
White,circular,
G+ve rods,
Turbidity, sedimentation, white
G + ve, long rods,
1-2.2 mm
chains,single
suspensions
chains


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ZAHOOR et al. / Int. J. Agri. Biol., Vol. 5, No. 1, 2003
Table IV. Results indicating sugar fermentation tests
Lactobacillus bulgaricus. Although, these all above values
recorded for the culture isolates from yoghurt sample
were reduced after 60 days but the effect of days was found
(S1)
to be non-significant when statistically observed. Mode of

preservation and methods showed significant effect on the
Sugars Acid
production
Gas
production
preservation of the starter culture (Table VII).
Lactose +
-

Sucrose -
-
Table VII. Analysis of variance for viability of culture
Maltose -
-
(s
+ = Acid production, Gas production, – =No acid production, No gas production
1) observed during its storage


Table V. Results indicating biochemical tests recorded
S.O.V. D.F.
S.S. M.S. F.value
for the culture isolates from yoghurt samples (S
Days 4
67.222
16.805
3.3061NS
1)
Methods 2 111.037 55.518 10.9220**

Error 8
40.665
5.083

Experimental procedure
Observation
Results
Total 14
218.924


Indole production
Layer not red
-ve
**= Highly significant
Methyl red test
Bright red color
+ve
LSD test for culture’s viability
Voges prospkauer test
Pink color
+ve
A B C
Citrate utilization
Green Color
- ve
Catalase activity
No bubbling
-ve
5.034 9.220 2.636
Growth at 15oC No
growth
-ve
B A B
Growth at 45oC Growth
observed
+ve


CONCLUSIONS
Viable count. It is evident from the results (Table VI) that

the original figure of viable count 9.9x106 in method A and

It is concluded that preservation under paraffin oil
4.9x106 in method C had decreased to 3.5x105 and 2.5x105,
method was the best method of preservation. Therefore, this
respectively after 60 days of storage. As far as method of
method can be employed for culture preservations at
preservation under oil (B) is concerned, it gave the desired
laboratory scale.
trend and the figure for viable count almost remained same

i.e., 9.5x106 at 0 days and 9.0x106 at 60 days of storage,
REFERENCES
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A
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8.10x106 7.8x106 5.8x106 3.24x106 3.21x106

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also concluded by Greig et al. (1970) who observed that
(Received 01 October 2002; Accepted 11 November 2002)
freeze-drying is much better than the preservation in nutrient
broth. Silva et al. (1983) also worked on the preservation of

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