Food Safety and Health

eISSN 2617-0094

Volume 1 Issue 1 February 2021 Pages 1–7

Bangladesh Society for Safe Food

doi: 10.5455/fsh.2021.25 www.bssf-bd.org

Original Research

Isolation and biochemical characterization of probiotic bacteria obtained from

selective regional Yoghurts in Bangladesh
Khondoker Moazzem Hossain1, Md. Shamim Gazi1, Pallob Barai1, Md. Faysal Al-Mazid1, Mohammad Abdul Jalil1 ,
Mohammod Abdul Hamid2, and Mahmudur Rahman3

 iotechnology and Genetic Engineering Discipline,

B
Abstract
1

Khulna University, Khulna, 9208, Bangladesh.

2
School of Agriculture and Rural Development, Bangla- Probiotics are live microorganisms which show several health benefits. The aim of the study
desh Open University, Gazipur, 1705, Bangladesh.
was to analyze probiotic properties of isolated probiotic bacteria from selective regional
3
Department of Medical Biotechnology, Bangladesh
University of Health Sciences, Mirpur, 1216, yoghurt samples in Bangladesh. Ten lactobacillus bacteria were isolated from yoghurt sam-
Bangladesh. ples of Sylhet and Mymensingh regions in Bangladesh. Yoghurt samples were cultured on
Man, Rogosa, and Sharpe Agar media with appropriate dilution for the isolation of potential
Article History
Received: November 07, 2019
probiotic bacteria and pure cultures were obtained by continuous sub-culturing. Isolated
Accepted: January 21, 2021 bacteria were well characterized by biochemical tests. Probiotic properties study of those
Published Online: XX isolates were carried out by pH resistance test, bile salt resistance test, NaCl tolerance test,
phenol tolerance test, and sugar fermentation tests. All the isolates were resistant at pH 3,
0.3% bile, and 0.1%, 0.2%, 0.3%, and 0.4% phenol. In case of NaCl tolerance test, the iso-
lates survived well at 2% and 4% NaCl and a lesser survival rate were observed at 6% and
8% concentrations. All the isolates showed antimicrobial activity against eight human and
animal enteric pathogens. Selected isolates from yoghurt samples fulfilled the most criteria
of probiotic bacteria. Numerous probiotics containing food and feed products can be devel-
oped and also be used as a biotherapeutic agent for different metabolic disorders and they
also may be a good focus for further research regarding the development of probiotic food
products for human.
Keywords: Native probiotics; Lactobacillus, Yoghurt

© 2021 The authors. This is an Open Access article distributed under
the terms of the Creative Commons Attribution 4.0 Licence (CC-BY)
Correspondence:
Khondoker Moazzem Hossain ( kmhossainbt@yahoo.com.au)
 Biotechnology and Genetic Engineering Discipline, Khulna
University, Khulna, 9208, Bangladesh.
How to cite: Hossain KM, Gazi MS, Barai P, Al-Mazid MF, Jalil MA,
Hamid MA, Rahman M. Isolation and biochemical characterization
of probiotic bacteria obtained from selective regional yoghurts in
Bangladesh. J Food Saf Health. XXXX;XX(XX):XX–XX. doi:XXXXX
Introduction
Dairy foods have established health benefits. Dahi is a Bangladeshi
homemade variant of yoghurt. Dahi has been considered as a functional
food due to its several health benefits, i.e., antidiabetic, antidiarrheal,
anticarcinogenic, cholesterol-lowering, and antiatherogenic properties [1,2].
Yoghurt is a potential source of probiotic lactobacilli. The term probiotic comes
from the Greek language meaning “for life”, but the meaning of probiotics has
evolved over time concurrently with the expanding interest in the use of live
bacterial supplements and in relation to the progress made in understanding
their mechanisms of action. Here, the term was used to describe the stimulation
of the growth of other microorganisms by substances produced by one
microorganism and was later used to describe tissue extracts that stimulated
microbial growth and animal feed supplements applying a beneficial effect on
animals by contributing to their intestinal flora balance [3]. The most updated
definition of probiotics is “Live strains of strictly selected microorganisms
which, when administered in adequate amounts, confer a health benefit on the
host” [4]. According to the FAO, “probiotics is defined as ‘Live microorganisms’,
Food Saf Health, 1(1):1–7, February 2021
which when administered in adequate amounts confer a
health benefit on the host” [5].
The effect of probiotic benefits has been
investigated for improving immune function, lowering
blood pressure, and improving lipids. There are
different strains of lactic acid bacteria (LAB), such as
Lactobacillus and Bifidobacterium, which are considered
as important probiotics regimens. Fermented dairy
products, such as yoghurt, contain adequate probiotic
LAB and are well established for several health benefits
[6]. Probiotics is well characterized by their capability
to survive for an unspecified time period in the upper
digestive tract and to colonize in the intestinal lumen
and colon. It is reported that functional foods containing
probiotics are safe for consumers and no hazardous
reports have been found or produced by these
strains of any particular toxin [7]. It is also reported
that antimicrobial substances like bacteriocins
are produced by some probiotics. Temporarily, the
rate of mitosis in enterocytes is increased by some
probiotic strains and they can reduce intestinal transit
time and improve the quality of migrating motor
complexes [8]. Lactobacillus and Bifidobacterium are
the most common probiotics and in general most of
the probiotics are Gram-positive, usually catalase-
negative, rods with rounded ends, occur in pairs, short,
or long chains, non-flagellated, non-motile, and non-
spore-forming, and are intolerant to salt; optimum
growth temperature is 37°C but some strains such as
Lactobacillus casei prefer 30°C and optimum pH for
initial growth is 6.5–7.0 [7]. Lactobacillus acidophilus
is microaerophilic and anaerobic and they have
capability of aerobic growth. Bifidobacterium are
also anaerobic, but some species are aero-tolerant.
According to probiotics fermentation capacity, they
are either obligate homofermentative (L. acidophilus,
Lactobacillus helvelicas), obligate heterofermentative
(Lactobacillus brevis, Lactobacillus reuteri), or
facultative heterofermentative (L. casei, Lactobacillus
plantarum) [9]. Various beneficial compounds are
secreted by probiotics such as antimicrobials, lactic
acid, hydrogen peroxide, and a variety of bacteriocins
[10,11] and it is very necessary for a healthy gut
environment. Probiotics also interact with the host
microflora and competitor for microbial pathogens
[11]. In accordance of the World Health Organization,
Food and Agriculture Organization, and the European
Food Safety Authority, probiotic strains must fulfill
both safety and functionality criteria and these criteria
should also fulfill their technological usefulness [12].
Literature reveals so far that no research works have
been conducted regarding isolation, identification,
and biochemical characterization of native probiotic
2
bacteria from regional yoghurt samples in Bangladesh.
Therefore, the objectives of the present study were to
isolate, identify, and biochemically study the native
probiotic isolates from selective yoghurt samples from
Sylhet and Mymensingh regions in Bangladesh.
Materials and Methods
Collection of samples
Ten yoghurt samples were collected from Sylhet
and Mymensingh regions from different dairy product
shops. All the samples were properly collected in
plastic containers labeled with the name of the shops,
locations, and dates. After collection, the samples
were stored aseptically at 4°C and at −20°C to protect
from deterioration and contamination in order to
immediate use and long-term storage for subsequent
use, respectively.
Isolation of probiotics from yoghurt
Man, Rogosa, and Sharpe (MRS) media was used to
isolate of probiotic bacteria. At first, the stock sample of
yoghurt was thawed. A total of six test tubes were used
to carry out serial dilution. Peptone water (53.8 ml)
was prepared for this procedure. Here, 9.8 ml peptone
water was transferred to the first test tube and 9 ml for
another five test tubes carefully. Then, 2 mg yoghurt
sample was taken into the first test tube containing 9.8
ml peptone water and then vortexed properly. Then, 1
ml of the vortexed solution was pipetted to the second
test tube containing 9 ml peptone water so that the final
volume was 10 ml and again vortexed for 30 seconds.
Then, 1 ml of vortexed solution was transferred to the
next fresh test tubes from each test tube, respectively.
One milliliter of the last vortexed solution from the
final test tube was poured into each labeled Petri dish
containing MRS agar media and spread on the surface
of the medium properly with a glass rod spreader.
Finally, the plates were incubated at the incubator
(INQUCELL) at 37°C for 24–48 hours. After incubation,
a single colony was obtained by streaking. Well-isolated
bacterial strains were picked up and stored in MRS
broth for further studies.
pH measurement test
The isolated LABs were grown in MRS broth at 37°C
for 24 hours. MRS broth medium was prepared with
pH 3 by using 5N HCl. Then, 20 µl overnight grown
bacterial cultures were inoculated in the 15 ml broth
medium. After that, both media were incubated at 37°C,
absorbance was taken at 0, 4, 8, 12, and 16 hours at
620 nm to determine the pH tolerance and viability of
the bacteria. Uninoculated medium at pH 3 served as
control.
Food Saf Health, 1(1):1–7, February 2021
Bile salt tolerance test
MRS broths with 0.3% of bile were used to determine
the tolerance and growth rate of isolated LABs. For the
bile salt tolerance test, a fresh overnight culture was
prepared. Twenty microliter overnight grown cultures
of LAB was inoculated with 15 ml MRS broth medium
containing 0.3% bile. Then, the tubes were incubated at
37°C and optical density was measured at 620 nm for
every 4–16 hours.
Antimicrobial activity test
Each of the isolates was co-cultured with a particular
pathogen at a time and antimicrobial activity was checked
by cross-line checking in the MRS agar plate. A total of
eight different pathogens were used for the antimicrobial
activity test of each probiotic isolate separately.
NaCl tolerance test
For the NaCl tolerance test, fresh overnight culture
was prepared. Then, individual MRS broth containing
2%, 4%, 6%, and 8% NaCl salts were prepared. After
sterilization, 1% fresh overnight culture of LABs was
inoculated in each test tube containing 10 ml MRS
broth with different concentration of salt. After 0, 4,
8, 12, and 16 hours, the optical density was measured
at 620 nm for determining the tolerance against NaCl.
Maximum growth rate was indicated as double positive
(++), normal growth as (+) positive, and no growth was
indicated with a negative (−) sign.
Phenol tolerance
For phenol tolerance test, fresh overnight culture was
prepared. Individual MRS broth containing 0.1%, 0.2%,
0.3%, and 0.4% phenol was prepared. After sterilization,
20 µl overnight culture of LABs was inoculated in each
test tube containing 10 ml MRS broth with different
concentration of salt and was incubated at 37°C for 24
hours. After 0, 4, 8, 12, and 16 hours, the optical density
was measured at 620 nm for determining the tolerance
against phenol.
Coagulase test
To determine the milk coagulation pattern, 200 µl
of a pure culture containing single isolates of probiotic
bacteria was inoculated into 9.8 ml fresh cow sterilized
milk and was incubated for 24 hours. Clotting of the solid
from liquid was observed for every 2-hour intervals.
Milk coagulation due to the association of lactic acid
forming bacteria was observed. Milk coagulation ability
was expressed by a positive sign (+) and non-coagulation
result was expressed by negative sign (-).
Motility test
At first, broth culture was prepared from the pure
culture and then these broth cultures were placed in an
3
incubator overnight. Using a sterilized needle, a well-
isolated colony was picked up and stabbed the medium
within 1 cm of the bottom of the tube. The needle was
kept in the same line and removed in the same line.
Test tube containing broth was labeled and foiled with
aluminum foil paper. Test tube was incubated at 37°C for
24–48 hours. A positive motility test was indicated by a
diffuse cloud of growth away from the line of inoculation
and a negative motility test was indicated no diffuse
cloud of growth away from the line of inoculation.
Sugar fermentation test
For sugar/carbohydrate fermentation test, fresh
overnight culture was prepared. At first MRS broth
containing media was autoclaved. After cooling, MRS
broth was poured into screw-capped test tubes.
Different concentrations of sugar solutions (5%)
(filtered/sterilized) was added and then phenol red
(0.01 gm/l) was added to the tube as pH indicator. Then,
200 µl overnight liquid cultures were inoculated into
the broth. At last, a Durham tube was placed inversely
at each of the test tube to determine the gas production.
Incubation was carried out anaerobically at 37°C for
24 hours. If fermenting bacteria are grown in a liquid
culture medium containing the carbohydrates, they may
produce organic acids as byproducts of the fermentation.
These acids are released into the medium and lower its
pH. For positive fermentation, the medium was changed
from its original color to yellow. Culture containing no
sugar solution and sugar solution containing no active
cell culture were prepared to be used as negative and
positive controls, respectively.
Results and Discussion
Results
All of the isolated bacteria were Gram-positive,
catalase-negative, rod-shaped, non-motile, and
coagulase positive.
pH tolerance test
All the isolated bacteria were able to survive at pH
3 for a period of 24 hours with 4-hour intervals. The
absorbance of cell concentration of all the isolates were
taken at 620 nm (Fig. 1a). Isolated bacteria from Shaad
and Co. (SC) and Dayamay Mistanno Bhandar (DM)
showed highest tolerance at pH 3 after a 4-hour period
(Table 1).
Bile tolerance test
All the isolated bacteria were able to survive in 0.3%
bile salt. The absorbance of cell concentration of all the
isolates was taken at 620 nm (Fig. 1b). However, isolated
bacteria from Rifat and Co. (RC), Shaad and Co. (SC) and
Food Saf Health, 1(1):1–7, February 2021 4

Figure 1. (a) pH resistance test. (b) Bile salt tolerance test. (c) NaCl (2%) tolerance test. (d) NaCl (4%) tolerance test. (e) NaCl (6%) tolerance test.
(f) NaCl (8%) tolerance test. (g) Phenol (0.1%) tolerance test. (h) Phenol (0.2%) tolerance test. (i) Phenol (0.3%) tolerance test. (j) Phenol (0.4%)
tolerance test.
Food Saf Health, 1(1):1–7, February 2021 5

Mamoni Sweets (MS) showed highest tolerance against
0.3% bile after a 4-hour period (Table 1).
NaCl tolerance test
All the isolated bacteria were able to well survive
in 2% and 4% NaCl salt, but a lesser survival rate
was observed at 6% and 8%Nacl salt concentrations
(Table 1). The absorbance of cell concentration of all the
isolates was taken at 620 nm (Fig. 1c–f).
Phenol tolerance test
All the isolated bacteria were able to survive well
in 0.1%, 0.2%, 0.3%, and 0.4% phenol (Table 1). The
absorbance of cell concentration of all the isolates was
taken at 620 nm (Fig. 1g–j).
Carbohydrate fermentation test
All the isolated bacteria showed positive
fermentation results, except sorbitol and mannitol.
Positive fermentation result was found to have a color-
changing pattern (Table 2).
Antimicrobial activity test
All the isolated bacteria were tested for antimicrobial
activity against eight different pathogenic bacteria. The
pathogenic bacteria were Vibrio cholera, Streptococcus
aureus, Micrococcus, Salmonella paratyphi, Bacillus
megaterium, Pseudomonas aeruginosa, Salmonella
typhi, and Escherichia coli. Most of the isolates had
antimicrobial activity. However, isolated bacteria from
Rifat and Co. (RC), Bonoful and Co. (BC), and Anil Ghosh

Table 1. Biochemical and physiological characteristics of the isolated bacteria from selective regional yoghurt samples in Bangladesh.

Name of isolates Probiotic properties

PH tolerance Bile salt tolerance NaCl tolerance Phenol tolerance
Sylhet region Short names
pH 3 0.3% 2% 4% 6% 8% 0.1% 0.2% 0.3% 0.4%
Rifat and Co. (Isolate No 1) RC ++ ++ ++ ++ + + ++ ++ ++ +
Fizza and Co. (Isolate No 2) FC ++ ++ ++ ++ + + ++ ++ ++ +
Modhubon (Isolate No 3) MB ++ ++ ++ ++ + + ++ ++ ++ +
Shaad and Co. (Isolate No 4) SC ++ ++ ++ ++ + + ++ ++ ++ +
Bonoful and Co. (Isolate No 5) BC ++ ++ ++ ++ + + ++ ++ ++ +
Mymensingh region
Anil ghosh sweets (Isolate No 6) AG ++ ++ ++ ++ + + ++ ++ ++ +
Dayamay mistanno bhandar DM ++ ++ ++ ++ + + ++ ++ ++ +
(Isolate No 7)
Misti kanon (Isolate No 8) MK ++ ++ ++ ++ + + ++ ++ ++ +
Sudhir ghosh (Isolate No 9) SG ++ ++ ++ ++ + + ++ ++ ++ +
Mamoni sweets (Isolate No 10) MS ++ ++ ++ ++ + + ++ ++ ++ +

Table 2. Sugar fermentation pattern of the isolated bacteria from selective regional yoghurt samples in Bangladesh.

Name of isolates Name of sugar

Sylhet region Short names Glucose Sucrose Lactose Xylose Raffinose Mannitol Glactose Maltose Sorbitol Fructose
Rifat and Co. (Isolate No 1) RC + + + + + − + + − +
Fizza and Co. (Isolate No 2) FC + + + + + − + + − +
Modhubon (Isolate No 3) MB + + + + + − + + − +
Shaad and Co. (Isolate No 4) SC + + + + + − + + − +
Bonoful and Co. (Isolate No 5) BC + + + + + − + + − +
Mymensingh region
Anil ghosh sweets (Isolate No 6) AG + + + + + − + + − +
Dayamay mistanno bhandar DM + + + + + − + + − +
(Isolate No 7)
Misti kanon (Isolate No 8) MK + + + + + − + + − +
Sudhir ghosh (Isolate No 9) SG + + + + + − + + − +
Mamoni sweets (Isolate No 10) MS + + + + + − + + − +
Food Saf Health, 1(1):1–7, February 2021 6

Table 3. Antimicrobial activity of the isolated bacteria obtained from selective regional yoghurt samples in Bangladesh.

Name of isolates Name of pathogens

Sylhet region Short forms V. cholera S. aureus Micrococcus S. paratyphi B. megaterium P. aeruginosa S. typhi E. coli
Rifat and Co. (Isolate No 1) RC − + + + + + + +
Fizza and Co. (Isolate No 2) FC + + + + + + + +
Modhubon (Isolate No 3) MB + + + + + + + +
Shaad and Co. (Isolate No 4) SC + + + − + + + +
Bonoful and Co. (Isolate No 5) BC − + + + + + + +
Mymensingh region
Anil ghosh sweets (Isolate No 6) AG − + + + + + − +
Dayamay mistanno bhandar DM + + + + + + + +
(Isolate No 7)
Misti kanon (Isolate No 8) MK + + + + + + + −
Sudhir ghosh (Isolate No 9) SG − + + + + + − +
Mamoni sweets (Isolate No 10) MS + + − + − − + +

Sweets (AG) did not show inhibitory effects against V.
cholera. Isolated bacteria from Anil Ghosh Sweets (AG)
and Sudhir Ghosh (SG) did not show inhibitory effect
against S. typhi. Isolated bacteria from Mamoni Sweets
(MS), Shaad & Co. (SC), and Mistikanon (MK) did not
show any antimicrobial activity against Micrococcus,
S. paratyphi, B. megaterium, P. aeruginosa, and E. coli,
respectively (Table 3).
Discussion
The study was designed for biochemical
characterization and determination of probiotic
properties of isolated bacteria from yoghurt samples
collected from Sylhet and Mymensingh regions in
Bangladesh. In the present study, probiotic bacteria
were isolated by a screening process. MRS agar media
was used for culturing these probiotic bacteria. After
growing the isolates on MRS agar media, they were sub-
cultured and colonies were selected for physical and
biochemical characterization. After that 10 isolates were
selected for determination of probiotic properties. The
most important characteristics that probiotic bacteria
have are the ability to resist low pH like gastric juice (pH
1.5–3) while they pass from the stomach [13]. In the
present study, all the isolates showed resistant to low
pH and their multiplication ability was very good. Bile
tolerance is one of the major characteristics of probiotic
bacteria [14]. Bile salt tolerance activity of probiotic
isolates showed more or less nearly equal resistance
against 0.05%, 0.1%, 0.15%, and 0.3% artificial bile acid
concentration after 0, 2, 4, and 24 hours of incubation,
respectively [15]. In the present study, all the isolates
were resistant to 0.3% bile salt and their multiplication
ability was very good. It is reported that Lactobacillus
spp. was isolated from yoghurt and they were tested at
different concentrations of NaCl (1–10%) and 1%–9%
NaCl tolerance was noted [16]. In the present study,
all the isolated bacteria were able to survive well in
2% and 4% NaCl salt, but a lesser survival ability was
observed in 6% and 8% NaCl salt concentrations.
Some authors mentioned that bacteriostatic action in
some microorganisms occurred at 0.4% concentration
of phenol [17]. In the present study, all the isolated
bacteria were able to survive in 0.1%, 0.2%, 0.3% and
0.4% phenol concentrations, respectively. In the present
study, all the isolated bacteria fermented all the 10 sugar
used except sorbitol and mannitol, which was similar
with the study of Ghanbari et al. [18], Karna et al. [19],
and Azizpour et al. [20]. All the isolated bacteria showed
antimicrobial activity and they were resistant to all the
eight human and animal enteric pathogens, which is
similar to the study by Coconnier et al. [21].
Conclusion
Bangladesh is a developing country in which a
significant number of people live below poverty line.
They could not meet their daily nutritional requirements
and a substantial population remains malnourished.
As probiotic bacteria have potential therapeutic or
prophylactic effects, the development of numerous
probiotic products such as fermented milk drinks,
yoghurt, cheese, ice cream, sausages, probiotic juice, and
drinking water with defined culture is severely required.
The antidiabetic, antidiarrheal, and antiallergic effects of
these isolated presumptive probiotic bacteria on animal
model to check the efficacy should also be conducted.
Therefore, future studies should be carried out
on the use of these isolates’ reliability including
molecular techniques like 16S rRNA sequencing for
accurate identification of these presumptive probiotic
Food Saf Health, 1(1):1–7, February 2021
bacteria. It is expected that successful identification,
conservation, and utilization of native probiotic isolates
will help to develop different probiotic food and feed
products for human, domesticated animals, and poultry,
which will subsequently enrich the technological
development regarding health beneficial effects and
poverty alleviation through the establishment of
different commercial safe probiotic food and feed
product manufacturing industries both for human and
domesticated animals for achieving the Government’s
Sustainable Development Goal.
Acknowledgment
The authors are grateful to the authority of National
Agricultural Technology Programs-Phase 2, Project
Implementation Unit, Bangladesh, Agricultural Research
Council (BARC) for providing funding under CRG Sub-
project by the World Bank.
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