EVALUATION OF THE LACTIC ACID BACTERIA IN DIFFERENT TYPES OF YOGURT CONSUMED IN PAKISTAN

Abstract

Lactic acid bacteria are industrially essential components to produce milk products such as yogrut, cheese, buttermilk, and kefir. Different gram-positive species are used for this purpose, including Streptococcus thermophilus, and Lactobacillus delbrueckii spp. bulgaricus bacteria were assayed by a viable count method. Different available commercial yogurts were purchased from the local market; among these, six were selected for assay. Samples were named as Nestle Podina Raita (NPR), Nestle Smooth Yogrut (NSY), Nestle Karachi Khase (NKK), Nestle Zeera Podina (NZR), Nestle Fruit Yogrut (NFY), and National Original Yogrut (NOY). From results, it was observed that NOY has the highest mean count i.e. 12.08 ± 0.25 log10 CFU/ml and 11.02 ± 0.13 log10 CFU/ml for S.thermophilus and L.bulgaricus, respectively. Titratable acidity was highest in NOY (0.93 %) with mean viable LAB counts were found to be 11.26 ± 0.24 and 10.61 ± 0.12 for lactobacillus bulgaricus and streptococcus thermophilus, respectively.

Full text

Pak. J. Biotechnol. Vol. 17 (3) 149-153 (2020) pISSN: 1812-1837 eISSN 2312-7791 www.pjbt.org
http://doi.org/10.34016/pjbt.2020.17.3.149
EVALUATION OF THE LACTIC ACID BACTERIA IN DIFFERENT TYPES OF
YOGURT CONSUMED IN PAKISTAN
Abdul Sami Dahri1, Asim Patrick1, Nasirudin Shaikh1, Jamaluddin Mangi2, Asif Ali
Bhatti1 and Altaf Ahmed Simair3,*
1Government College University, Hyderabad, 71000 Sindh, Pakistan.
Email: sameedahri@gmail.com, asim.patrick@gmail.com, shaikhdrnasir@gmail.com
and asifali00083@gmail.com. 2Institute of Plant Sciences, University of Sindh,
Jamshoro, 76080 Sindh, Pakistan. Email: jamal.mangi@usindh.edu.pk. 3College of
Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai
20160, China. Email: *altafsimair@dhu.edu.cn.
Article received 28.7.2020, Revised 8.9.2020, Accepted 18.9.2020
ABSTRACT
Lactic acid bacteria are industrially essential components to produce milk products such as
yogrut, cheese, buttermilk, and kefir. Different gram-positive species are used for this purpose,
including Streptococcus thermophilus, and Lactobacillus delbrueckii spp. bulgaricus bacteria were
assayed by a viable count method. Different available commercial yogurts were purchased from
the local market; among these, six were selected for assay. Samples were named as Nestle Podina
Raita (NPR), Nestle Smooth Yogrut (NSY), Nestle Karachi Khase (NKK), Nestle Zeera Podina (NZR),
Nestle Fruit Yogrut (NFY), and National Original Yogrut (NOY). From results, it was observed
that NOY has the highest mean count i.e. 12.08 ± 0.25 log10 CFU/ml and 11.02 ± 0.13 log10
CFU/ml for S.thermophilus and L.bulgaricus, respectively. Titratable acidity was highest in NOY
(0.93 %) with mean viable LAB counts were found to be 11.26 ± 0.24 and 10.61 ± 0.12 for
lactobacillus bulgaricus and streptococcus thermophilus, respectively.
Keywords: Lactic acid Bacteria (LAB) Viable counts, Streptococcus thermophilus and
Lactobacillus bulgaricus.
INTRODUCTION
Fermented milk products have been con-
sidered to be beneficial for health. Yogurt
is one of the fermented milk products and
the best human diet. Presence of viable
lactic acid bacteria in fermented milk like
yogurt, such as Streptococcus thermophi-
lus and Lactobacillus delbrueckii ssp. Bul-
garicus has been correlated with vari-ous
benefits for human health. It is suggested
that the minimum level for probiotic bact-
eria should be in the range of 10-5 to 10-6
viable cells per mL (Dave and Shah,
1997). France and Spain set a mini-mum
viable lactic acid bacteria number in
yogurt's shelf life of 5×108 CFU/ml (Birollo
et al., 2000). In medical science, it is belie-
ved that yogurt starter microbes are prot-
eolytic and produce bioactive peptides from
milk protein. Fermented milk and yogurt
contain peptides with angiotensin over
protein inhibitory action (Guevarra and
Barraquio, 2015). They have anticarcino-
genic, antimutagenic effects, improvised
of lactose digestion, and enhancement of
the Immune system. The essential probio-
tic organism naturally associated with the
human GIT is Lactic acid Bacteria (LAB)
(Dicks and Botes, 2010). Usually, Lactic
acid bacteria are classified on the bases of
their morphology, mode of fermentation of
glucose, and growth at various tempera-
tures and fermentation of various carbo-
hydrates (Axels-son, 2004; Khalid, 2011).
Nowadays, the production of probiotic on
an industrial scale in food and pharma-
ceutical has a beneficial impact on human
and animal life (Mattila-Sandholm et al.,
2002).
The majority of the population who use
probiotics believe that the use of such
products not only reduces the risk of gett-
ing many GIT related infections but also
helps in many other critical diseases (San-
ders, 2003). To gain a guaranteed medical
advantage to humans, the minimum avai-
lability of probiotic microorganisms in the
fermented milk considers that it would be
around 109-10 colony forming units (CFU)
/g. However, the dosage is not fixed in the
evaluation of probiotic (Ouwehand, 2015).
A vital parameter in evaluating the quality
of the product is by observing suitable for

A.S. Dahri et al., Pak. J. Biotechnol.
150
probiotic microbes selectively. To ensure
that the required number of probiotic
organisms in the end-product is present,
fast and reliable approaches require rout-
ine count.
Moreover, such techniques are addition-
ally essential to screen any physiological
or biochemical changes in the population
of probiotic bacteria amid the product’s
commercial storage (Vinderola et al.,
2002). Besides, a few research studies
have shown that some probiotic items in
the market don't meet the minimal pres-
cribed count of probiotic strain(s), parti-
cularly in those containing bifidobacteria
(Fasoli et al., 2003). The presence of pro-
biotics in yogurt is affected by many other
factors such as strain of bacteria is used,
the interaction between the species in it,
what is the conditions of culture, produc-
tion of hydrogen peroxide due to bacterial
metabolism, and at the end, acrid smell,
and combination of acidic acid and lactic
acid. Moreover, during the fermentation of
milk due to the growth of bacteria, the pH
of the medium is also decreased which
affects the feasible amount of probiotic in
it (Shah and Jelen, 1990). The yogurt
which reaches consumers may raise few
doubts on the actual presence of probio-
tics in it because many sweeteners, pre-
servative, flavoring ingredients are used in
it (Birollo et al., 2000). Till now, no one
has studied the actual presence of probio-
tic in commercially available yogurts in
Pakistan. Now, questions can be raised
here whether such products can give the
said health benefits to the consumers due
to the presence of probiotic in lower range.
The objective of this research is to check
the prescribed amount of probiotic pres-
ent in commercially available products
and Lactic acid Bacteria is available in
such items or not. The information from
this investigation will be valuable to check
the various yogurt makers regarding the
health benefits of live and active culture
present in the products.
MATERIALS AND METHODS
All chemicals used were of reagent grade.
The study was carried out from January
to April 2019 in the Biotechnology lab,
Government College University Hyder-
abad, Pakistan. The commercial yogurt
starter culture containing S. thermophi-
lus and L. delbrueckii ssp. bulgaricus was
selected because its mild acidity protects
bifidobacteria. Both yogurt starter cultu-
res were obtained from local vendors. BS-
MIX-28 Dancing Fairy-Vortexer Smart
was used to mix the samples.
Collection of Samples: Six yogurt samp-
les were bought as fresh as possible from
the local market of Hyderabad namely:
Nestle Podina Raita (NPR) Nestle Smooth
Yogurt (NSY) Nestle Karachi Khas (NKK)
Nestle Zeera Raita (NZR) Nestle Original
Yogurt (NOY) and Nestle Fruit Yogurt (NF-
Y). All the sample results were an average
of 5 tests. All samples were stored in 4 to
6°C temperature to minimize the acidific-
ation. The production and expiry date of
all samples were recorded in the first
instance.
Isolation of Microorganisms: Pour plate
count (30-300 colonies) method was used
after incubation. All results are shown in
log CFU/mL) (SERT et al., 2011). MRS
and M17 agar were used to identify the S.
thermophilus and L. bulgaricus in yogurt
samples Serial dilution was performed
with peptone diluents. The micropipette
was used to transfer a 1mL blended yog-
urt sample to a test tube of 9ml sterile
diluent, which stands as 10-1 dilution?
Samples were mixed for 1 minute on a
vortex mixer. 1ml was taken from a 10-1
dilution tube and transferred to 9 ml ste-
rile diluent to prepare a 10-2 dilution.
This activity was repeated until the
expected dilution is obtained by utilizing
new and sterile pipettes and diluents. To
calculate the bacterial count, one ml of
the prepared dilution was moved into the
Petri dishes, then MRS agar medium 12 to
15 ml is added at 45°C was filled each
Petri dish with the proper dilution. (Karna
et al., 2007). The substance of the Petri
dish was blended cautiously by rotating
the Petri dish multiple times clockwise
and counter-clockwise. It was left at room
temperature to solidify at a smooth sur-
face. Plates were placed in an anaerobic
jar for incubation at 37o C for 72 hours.
Plates were then incubated anaerobically
for 48 hours at 37oC then 25 to 250 colo-
nies on Petri plates counted by colony-
forming unit method CFU/ml by the
following formula (De Man et al., 1960)

Vol. 17 (3) 2020 Evaluation Assay of lactic acid ….
151
𝑁 = 𝛴𝐶/[(1.0 × 𝑛1)+(0.1 × 𝑛2)𝑑
Where:
N = number of colonies per ml of sample.
∑C = sum of all of the colonies in all
plates counted.
n1 = number of plates in the lower
dilution counted.
n2 = number of plates in the next higher
dilution counted.
d = dilution from which the first counts
were obtained
Characterization on Bases of Morpho-
logy: Morphological characterization of
colonies was carried out by testing their
catalase reaction by observing the colony
growth gram reaction, cell morphology for
verifications of colony count for S. thermo-
philus and L. bulgaricus. Catalase activity
and procedure for gram staining was
performed according to reported methods
(Murray et al., 1994; Smibert, 1994)
Well-isolated colonies were inoculated on
MRS agar for L.bulgaricus and M17 agar
for S. thermophiles, incubated at their
ideal temperature 37°C for 48-72 hours.
The culture was Gram-stained, and a test
for catalase activity was performed. Col-
ony confirmation was accomplished for
the Gram-positive, catalase-negative cha-
ins of cocci or diplococci on account of S.
thermophilus and non-spore-forming,
Gram-positive, and catalase-negative rod
on account of L. bulgaricus (Guevarra and
Barraquio, 2015).
Acidity Test: APHA method was used for
the acidity test of yogurt samples. Acidity
was calculated from following equation:
% Lactic Acid = (Volume of NaOH used x
Normality of NaOH x 90/100)/9 ×100
Statistical analysis: Complete Randomi-
zed Design (CRD) was applied to date so
that each experimental unit has the same
chance of receiving any one treatment. All
experiments were performed in three bat-
ches (n = 3), and the average was taken.
Data were expressed as mean ± standard
error using one-way ANOVA by SPSS®
version 17.0. Means were compared using
Duncan’s multiple range tests, and statis-
tical significance was standard by ANOVA
at p < 0.05.
RESULT AND DISCUSSION
Bacterial count of Yogurt samples:
Table 1 shows the mean viable lactic acid
bacterial counts in log10 CFU/ml. It was
observed the there is no significant differ-
ence in all six commercial yogurt samples
for both bacterial strains. The highest
mean value was overserved in NOY sam-
ple for S. thermophilus and L. delbrueckii
ssp. bulgaricus i.e. 12.08 ± 0.25 and 11.02
± 0.13, respectively. NKK shows least all
yogurt samples with mean viable counts
of 10.01 ± 0.58 and 9.51 ± 0.35 Thermo-
philus and L. delbrueckii ssp. Bulgaricus,
respectively.
Table 1: S. thermophilus and L. bulgaricus
Mean viable count
Yogurt
samples
Mean viable LAB counts
(log10 CFU/ml) n = 5
S.
thermophilus
L.
bulgaricus
Nestle Podina
Raita (NPR)
11.36 ± 0.41
10.39 ±
0.44
Nestle Smooth
Yogrut (NSY)
10.25 ± 0.36
9.98 ± 0.22
Nestle Karachi
Khas (NKK)
10.01 ± 0.58
9.51 ± 0.35
Nestle Zeera
Raita (NZR)
10.68 ± 0.49
10.22 ±
0.62
Nestle Original
Yogrut (NOY)
12.08 ± 0.25
11.02 ±
0.13
Nestle Fruit
Yogrut (NFY)
10.57 ± 0.67
10.14 ±
0.38
n = number of samples examined per yogurt
brand
Properties of lactic acid bacterial colo-
nies: Properties of S.thermophilus and L.
bulgaricus colonies were observed by a
well-isolated colony that appeared on the
Petri plate. A single colony was aseptically
transferred on stab/slat to observe the
growth pattern on tangible media. The
colony of S.thermophilus on M17 agar
plate seemed to be smooth white, around
0.5 to 3.0 mm in circular, diameter, low
convex demonstrated unequal develop-
ment on a slant. While Lactobacillus gene-
rally has little size settlements having 2–5
mm size with the whole margin, convex,
shiny, and cloudy, without pigment (Vos
et al., 2011).
Table 2: Streptococcus thermophilus and
Lactobacillus viable lab counts, and Titratable
acidity in a yogurt samples.

A.S. Dahri et al., Pak. J. Biotechnol.
152
Yogurt
sample
%
Lactic
acid
Mean viable LAB countsa
(log10 CFU/ml)
Streptococcus
thermophilus
Lactobacillus
bulgaricus
NPR
0.71
9.67 ± 0.25
9.55 ± 0.36
NKK
0.85
10.25 ± 0.68
9.63 ± 0.40
NZR
0.83
10.67 ± 0.46
10.21 ± 0.6
NOY
0.93
11.26 ± 0.24
10.61 ± 0.12
NFY
0.69
9.54 ± 0.19
8.84 ± 0.14
NSY
0.79
10.9 ± 0.49
10.32± 0.37
aMean viable counts with the same sup-
erscript are not significantly different (P ≤
0.05)
Morphology of Lactic acid bacteria:
Gram-positive staining of 48-hour old cul-
ture was used to characterize the morp-
hology of LAB samples. A bacterium for S.
thermophiles and gram-positive staining
of 72-hour old culture was used for the
determination of morphology of L. bulga-
ricus. Microscopic visualization showed
that S. thermophilus cells were bowl and
circular, and they usually occur in aggr-
egated form. They were non-motile, Gram-
positive, catalase-negative, and non-spore
forming. Lactobacillus delbrueckii ssp.
Bulgaricus is a rod-shaped cell; someti-
mes, they appear almost coccoid and in a
short chain. L. bulgaricus facultatively
anaerobic, Gram-positive, and non-spor-
ing. Their ideal temperature is 30–40°C
and their digestion is fermentative and
saccharolytic, in any event, half of the
finished result of carbon is lactate (Holt,
1977).
Comparison of acidity and viable cou-
nts: Table 2 shows lactic acid acidity in
different yogurt samples. Acidity was obs-
erved highest in NOY sample, which
correlated with viable counts of the same
sample. No official standards have been
set on minimum titratable acidity for fro-
zen yogurts yet. So it is suggested that
food industries should ensure the mini-
mum base titratable acidity value of 0.25
% lactic acid (Holt, 1977).
Conclusion
In this study, we have assayed commer-
cially available yogurts in Pakistan to con-
form to the presence of S. thermo- philus
and L. bulgaricus. Six samples were taken
and each sample was tested for mean via-
ble count LAB of (S. Thermophilus and L.
bulgaricus). Among all samples, the mean
viable count was significantly lower (P ≤
0.05) in NKK. The lower mean of LAB
count in NKK is due to the freezing of the
sample. There was no significant differe-
nce between the two bacterial strains for
acidity and mean viable counts, tested in
this study. The Viable count of S. thermo-
philus and L. bulgaricus yogurts assayed
and found under prescribed value for the
proposed therapeutic impacts and wellbe-
ing benefits for the consumers
REFERENCES
Axelsson, L., Lactic acid bacteria: classifi-
cation and physiology. Food Science
and Technology-New York-Marcel
Dekker- 139: 1-66 (2004).
Birollo, G., Reinheimer, J.A. and C.G.
Vinderola, Viability of lactic acid mic-
roflora in different types of yoghurt.
Food Research International 33: 799-
805 (2000).
Dave, R.I. and N.P. Shah, Viability of yog-
hurt and probiotic bacteria in yoghu-
rts made from commercial starter
cultures. International Dairy Journal
7: 31-41 (1997).
De Man, J., Rogosa, D.M. and M.E.
Sharpe, A medium for the cultivation
of lactobacilli. Journal of applied Bac-
teriology 23: 130-135 (1960).
Dicks, L. and M. Botes, Probiotic lactic
acid bacteria in the gastro-intestinal
tract: health benefits, safety and
mode of action. Beneficial microbes 1:
11-29 (2010).
Fasoli, S., Marzotto, M., Rizzotti, L., Rossi,
F., Dellaglio, F. and S. Torriani, Bact-
erial composition of commercial pro-
biotic products as evaluated by PCR-
DGGE analysis. International journal
of food microbiology 82: 59-70 (2003).
Guevarra, R. and V.L. Barraquio, Viable
Counts of Lactic Acid Bacteria In Phi-
lippine Commercial Yogurts. Int J
Dairy Sci Process 2: 24-28 (2015).
Holt, J.G., The shorter Bergey's manual of
determinative bacteriology. The shor-
ter Bergey's manual of determinative
bacteriology. 8th edition (1977).
Karna, B., Emata, O. and V. Barraquio,
Lactic acid and probiotic bacteria
from fermented and probiotic dairy

Vol. 17 (3) 2020 Evaluation Assay of lactic acid ….
153
products. Science Diliman 19: 23-24
(2007).
Khalid, K., An overview of lactic acid bact-
eria. International journal of Biosci-
ences 1: 1-13 (2011).
Mattila-Sandholm, T., Myllärinen, P., Cri-
ttenden, R., Mogensen, G., Fondén, R.
and M. Saarela, Technological chall-
enges for future probiotic foods. Inter-
national Dairy Journal 12: 173-182
(2002).
Murray, R., Doetsch, R.N. and C. Robinow,
Determinative and Ctological Light
Microscopy (1994).
Ouwehand, A.C., The role of probiotics in
digestive health. Nutrition and Diet-
ary Supplements 7: 103-109 (2015).
Sanders, M.E., Probiotics: considerations
for human health. Nutrition reviews
61: 91-99 (2003).
SERT, D., Akin, N. and E. Dertli, Effects
of sunflower honey on the physico-
chemical, microbiological and sens-
ory characteristics in set type yoghurt
during refrigerated storage. Internat-
ional journal of dairy technology 64:
99-107 (2011).
Shah, N. and P. Jelen, P., Survival of lactic
acid bacteria and their lactases under
acidic conditions. Journal of Food
Science 55: 506-509 (1990).
Smibert, R., Phenotypic characterization.
Methods for general and molecular
bacteriology (1994).
Vinderola, C.G., Mocchiutti, P. and J.A.
Reinheimer, Interactions among lac-
tic acid starter and probiotic bacteria
used for fermented dairy products.
Journal of Dairy science 85: 721-729
(2002).
Vos, P., Garrity, G., Jones, D., Krieg, N.R.,
Ludwig, W., Rainey, F.A., Schleifer,
K.-H. and W.B. Whitman, Bergey's
manual of systematic bacteriology:
Volume 3: The Firmicutes. Springer
Science & Business Media (2011).