Screening and Analysis of Probiotic Properties
of Lactobacillus Spp. From Famous Commercial
Brand of Yogurt Found in Kathmandu Valley
Asmita Khanal1,2 and Niranjan Koirala1*
1Dr. Koirala Research Institute for Biotechnology and Biodiversity, Department of Natural Products Research, Nepal
2Research Center for Applied Science and Technology, Tribhuvan University, Nepal
Submission: February 20, 2019; Published: March 18, 2019
*Corresponding author: Dr. Niranjan Koirala PhD, Dr. Koirala Research Institute for Biotechnology and Biodiversity, Department of Natural Products Research, Kathmandu, Nepal
How to cite this article: Asmita K, Niranjan K. Screening and Analysis of Probiotic Properties of Lactobacillus Spp. From Famous Commercial Brand
of Yogurt Found in Kathmandu Valley. Curr Trends Biomedical Eng & Biosci. 2019; 18(4): 555992.
Probiotics are live microbial strains that have a positive impact on human health by maintaining the gut microflora. Probiotics are present in fermented food products. Out of many probiotics, most belongs to Lactobacillus genera which are also used in commercial food products. Yoghurt is a potential source of probiotic lactobacilli. The purpose of this study is to identify and analyse the probiotic properties of isolated Lactobacillus spp. from different five commercial brand of yoghurt that are famous in Kathmandu valley.
All the isolate was identified on the basis of their morphological and biochemical characteristics. These identified isolates were further examined to analyze the probiotic properties which includes tolerance to inhibitory substances like phenol(0.4%), Nacl (1-9%) and bile acid (0.1-1%); the ability to grow in acidic (pH 2.5) and alkaline (pH8.5) condition and their antimicrobial activities along with susceptibility to selected nine antibiotics which was determined by disk diffusion method. The result of this present study indicates that the isolates from commercial yoghurts sample fulfill the most common criteria of probiotic bacteria with slight variation among the sample in different properties.
Keywords: Probiotics; Lactobacillus spp; Antimicrobial; Yoghurts; Kathmandu Valley
Probiotics are living microorganism that stimulates the health of the host by maintaining the intestinal microflora. Probiotic bacteria mainly consists a genera of Lactic Acid-Producing Bacteria (LAB), including Lactobacillus and Bifidobacterium, streptococcus and some enterrococcus spp. . Traditionally fermented food product including dairy product such as yoghurt is the major source of probiotic .
Yoghurt is a cultured dairy product that has been fermented with LAB. Fermentation of lactose by these bacteria produces lactic acid, whose action on milk proteins results the texture and characteristics flavour of yogurt. The properties like lactic acid production, antimicrobial properties, antibiotic tolerance, bile tolerance, pH tolerance and gastric juice resistance are some criteria for the in vitro selection of lactobacilli to be used as probiotic . It is very important that probiotic strain should survives in the area where it is supposed to be active.
The strain should able to proliferate and colonize at the specific location to boost maximum activity. Probiotics for human consumption should have ‘generally regarded as safe’. It should be tolerated by immune system and should not be pathogenic,
allergic, or mutagenic . Probiotic assist in the prevention and treatment of Gastrointestinal infections and diseases . The benefits of Lactobacillus spp ranges from reducing allergies, preventing antibiotic-associated diarrhea , removing cholesterol , prevent urinary tract infection, stimulation and development of immune system of the host .
Yoghurt has been a part of a human daily diet, with the increasing awareness about the health benefits of yoghurts consumption, the interest and market of yoghurt has increased globally. In Nepal, yoghurt is locally called ‘dahi’ and consumed as a part of a daily diet. Here, yoghurt has been produced in a household level but with the increasing number of population and changing lifestyle, the demand of commercial yoghurt is rising. Respectable evidence continues to emerge that shows that the members of the genus Lactobacillus are widely used in the manufacturing of yoghurt .
Despite growing use and consumption of Lactobacillus as probiotics, which can enhance the health status of large segment of communities, there is a lack of scientific research on local commercial yoghurts in Kathmandu. Therefore, this study is undertaken to find potential probiotics properties of Lactobacillus spp. from a famous commercial brand of yoghurt in Kathmandu Valley.
Total five yoghurt samples from different commercial dairy
in Kathmandu were collected from market as freshly as prepared.
The samples were stored aseptically in low temperature (4°C) refrigerator
to protect from contamination and deterioration immediately
The bacteria Lactobacillus spp. was isolated from each sample
by using DeMann, Rogosa and Sharpe (MRS) media both MRS
broth and MRS agar media . In order to improve the specificity
of the medium for isolation of Lactobacillus 0.05% cysteine was
added . The final pH of the media was adjusted to 6.5.
All the required instrument and media were autoclaved for 15
min at 120°C. One gram of each sample was suspended in 100ml
respective MRS broth and after dissolving they were shaken homogeneously.
All this homogenized sample were subjected to
fivefold dilution, these dilutions were than incubated for 24 hours
for 37°C under anaerobic condition in the presence of 10% CO2 to
remove unwanted bacteria. From each broth, a loopful of culture
was streaked on to the MRS agar media which were incubated for
24 hours at 37°C . Finally, the single colony of bacteria was
isolated and identified by observing their morphological characteristics
and some biochemical tests like Gram staining, catalase,
endospore test, motility test and milk coagulation activities. The
cultures were maintained in the MRS broth.
Determination of optimal growth and pH: For the determination
of optimal pH and growth, the overnight culture of the Lactobacillus
in the MRS broth media with a varying pH ranging from
2.5 to 8.5, using Hcl or NaOH were inoculated with 1% (v/v) and
incubated in anaerobic condition for 24h at 37°C in the presence
of 10% CO2. Bacterial growth was monitored by the determination
of optical density at 560nm using a spectrophotometer against
the uninoculated broth .
Assay for NaCl tolerance: In order to determine the NaCl tolerance,
isolates from each sample were incubated with 1% (v/v)
overnight culture of Lactobacillus in the test tubes containing MRS
broth adjusted with different concentrations (1-10%) of NaCl. The
inoculated medium was incubated in anaerobic condition for 24h
at 37°C. The bacterial growth was determined using spectrophotometer
reading the optimal density at 560nm .
Determination of bile salt tolerance: Bile salt tolerance of
the isolated bacteria was examined by inoculating the freshly cultured
isolates at 1% (v/v) into MRS broth containing Bile salt at
different concentrations (0, 0.1, o.3, o.5 and 1% (w/v). The medium
was then incubated at 37°C for 24 hours in an anaerobic condition.
The growth of the isolates was monitored at 0, 3, 5 and 24 h
by measuring the absorbance of the culture broth at 620nm using
Antimicrobial activity: The antimicrobial activity of the isolated
Lactobacillus was identified by well diffusion method ,
with slight modification. The overnight culture of Lactobacillus
was centrifuged (10,000 rpm for 20min. at 4°C) and the supernatant
was adjusted to pH 7.0 with NaOH to exclude the antimicrobial
effect of H+. Supernatant of each sample of Lactobacillus were
monitored for antibacterial activity against indicator bacteria
(Escherichia Coli, Staphylococcus aureus, Salmonella typhi, Bacillus
subtilis) inoculated on Nutrient agar. Wells of 5mm in diameter
were punctured into the agar plate and aliquots of 50μl from each
solution samples were placed into the wells. The plates were incubated
for bacterial growth and examined for the presence of clear
zones of inhibition around the wells
Antibiotics sensitivity: For the observation of antibiotics
sensitivity test Disk diffusion method was performed . Different
nine antibiotics were tested over the isolates. The appropriate
antimicrobial-impregnated disks were placed on the surface of
the MH agar plate inoculated with each isolate. The zones of inhibition
around the disks were observed and the diameters were
The bacteria were isolated from different five samples of yoghurt
and were identified as Lactobacillus spp. by observing their
colony morphology, physiological as well as some biochemical
characteristic. Microscopic examination showed the isolates were
Gram-positive rod shaped (Figure. 1), non-motile, catalase negative
and absence of Endospore. The isolates were able to coagulate
milk and have the abilities to tolerate inhibitory substances such
as 0.4% bacteriostatic phenol and showed growth in MRS broth
containing 1-9% NaCl. The results are shown in (Table 1).
Maximum growth of isolated Lactobacillus of different sample
was observed at different PH Maximum growth of isolated Lactobacillus
from sample 1(ND’s) yoghurt was observed at pH 5.5
(OD=1.067) similarly, maximum growth of sample 2(KDC) was
also observed at PH 5.5 (OD=1.042), sample 3(DDC plain) at PH
6.5 (OD=1.039), sample 4(DDC probiotic) at PH4.5 (OD=1.025)
and sample 5(ju –ju dhau ) was observed at PH 6.5 (OD=1.048),
after 24 h of incubation in an anaerobic condition. The results are
shown in Table 2.
The isolates from sample were able tolerate 1-9% NaCl. But
these organisms grow best at the broth containing 1% NaCl. The
growth rate decreases with increase in NaCl concentration. The
result are shown in Table 3.
Isolated Lactobacillus spp. were able to survive and multiply
in 0.1, 0.3, 0.5 and 1% bile acid. In Table 4 the optical density values
against incubation time are given. The growth of Lactobacillus
in MRS broth without bile was used as control. All the isolates
showed different growth rate. With increase in the concentration
of bile salt there the growth is decreased. The growth rate is also
highly affected the increased in incubation period.
The isolates showed susceptibility and resistance pattern to
all the nine antibiotics tested. The results are shown in Table 5.
The isolates were more commonly sensitive to ampicillin, penicillin,
erythromycin, nalidicxic acid, chloramphenicol, tetracycline,
gentamycin and amoxicillin which presented diameter of inhibition
zones between 10 and 35mm. The diameter of inhibition
zones varied for different samples (Figure 2). The Antibiotics ciprofloxacin
only showed resistance to all the five isolates.
The isolated spp. was examined to their antimicrobial activity.
For this, spp. was detected against the indicator microorganisms
Salmonella thyphi, Escherichia coli, Bacillus subtillis and staphylococcus
aureus. These organisms are occasionally found as food
borne microorganisms that might cause gastroenteritis. The results
revealed that the antibacterial activity of the five isolated
Lactobacilli could inhibit the pathogenic bacteria tested, however
at different inhibition levels as shown in Table 6 (Figure 3). Of the
five isolates tested, only isolates from sample 2 can inhibited the
growth of all the pathogens used as indicators. But other samples
1, 3 4, and 5 only inhibit the indicator organism Bacillus and Escherichia
The isolated rod shaped, gram positive, catalase negative, non
-motile with other mention biochemical properties in Table 1, bacteria
were isolated from different yoghurt sample from Kathmandu
valley. These isolates were assigned as Lactobacillus . The
pH factor directly affects the bacterial growth. Probiotic bacteria
are mostly delivered in a food system so these must have to tolerate
acid in order to survive in the human gastrointestinal tract.
In this study we observed the growth of the isolated Lactobacillus
spp. in various pH value ranges from 2.5 to 8.5 to determine
whether the isolated spp. can grow in acidic and alkaline conditions
and also to detect the optimal pH value for good growth. It
was found that the bacteria were able to survive from extreme
acidic (2.5) to basic pH (8.5) and for optimal growth the variation
with the sample is viewed. Probiotic organism have to stand in a
high salt concentration in a human gut . In our study the lactobacillus
isolated from all sample were able to survive in1-9% of
NaCl and the good growth is seen at1% NaCl.
This study also indicate that the growth rate decreases with
the increase in NaCl concentration. The probiotic bacteria must
able to resist the bile salt in order to survive in the human intestine
and 0.3% bile is the maximum concentration present in the
healthy men . So, for the human consumption the probiotics
should be bearable to 0.3% bile concentration . Here, 0.1-1%
of bile salt concentration was used and our isolated Lactobacillus
spp. from all five yoghurts are able to tolerate up to o.3% of bile
In control conditions there were rapid increase in growth
of all three cultures with an increase in incubation period up to
24h and there was rapid decrease observed in all cultures with
increase concentration especially above 0.3% (0.5% and 1%) of
Antibiotic resistance of microorganisms used as probiotic is of
growing concern. Bacteria used as probiotics may serve as host of
antibiotic resistance genes, which can be transferred to pathogenic
bacteria . The antibiotic resistance of pathogenic bacteria is
an increasing medical problem. Therefore, the antibiotic susceptibility
test should be integrated for the safety assessment of the
desired property of the probiotic Lactobacilli.
Our isolates were all sensitive to penicillin, ampicillin, amoxicillin,
tetracycline, erythromycin nalidixic acid and chloramphenicol,
but only resistance to ciprofloxacin. Resistance to ampicillin
ciprofloxacin and vancomycin are commonly found in the genus
Lactobacillus . Since the isolates tested in this work were not
resistant to any of antibiotics, except ciprofloxacin they could be
safe for use by animals and eventually by humans, and their lack
of resistance also indicates that they may not contribute to the
transfer of resistance to other microorganisms.
Resistance of all Lactobacillus spp to ciprofloxacine could be
due to their natural and intrinsic resistance, probably due to the
cell wall structure and membrane impermeability, complemented
in some cases by potential efflux mechanisms . Regarding
antibacterial activities the good probiotics should present their
antimicrobial actions particularly to the pathogens in the gastrointestinal
tract. The inhibitory activity of Lactobacillus spp. varies
within the strains . Lactobacilli are highly competitive due to
their production of several antimicrobial compounds such as organic
acids, hydrogen peroxide, reuterin and bacteriocins .
In this study, Staphylococcus aureus, Salmonella typhi, Bacillus
subtilis and Escherichia coli were used as the test bacteria because
they are occasionally found as food borne microorganisms
that might cause gastroenteritis. The results revealed that the antibacterial activity of the five isolated Lactobacilli could inhibit
all test pathogenic bacteria however at different inhibition levels
as shown in (Table 5). The isolated sample 1, 3, 4 and 5 showed
the most antibacterial potency to E. coli and Bacillus whereas the
isolated sample 2 demonstrated the highest potency to the entire
organism used as indicator.
Those food having the health benefit beyond the basic nutrient
are captivating the consumers. Extensive research is currently
directed toward increasing our understanding of such functional
foods and the food industry is trying to fulfill these demands by
developing products with measurable health benefits. Food with
the probiotics is gaining consumer interest.
The isolated Lactobacillus spp. from different yogurt samples
which most of the people of Kathmandu use to consume fulfills
the required criteria of a probiotics. These isolated strains were
able to survive in a favourable environment of human gastrointestinal
tract such as high salt, low pH and high bile concentration.
Therefore, these Lactobacillus isolates show their potential to be
used as probiotic. This spp. is hence a good candidate for further
investigation under in vitro as well as in vivo conditions to elucidate
their potential beneficial health effects and their possible
application as novel probiotic species in the food industry.
We would like to thank Professor Dr. Rameshwar Adhikari,
Research Center for Applied Science and Technology, Tribhuvan
University, Nepal for his continuous support, guidelines and encouragement
towards this research.