European Journal of Biotechnology and Bioscience
Volume: 3, Issue: 8, 9-15
Aug 2015
www.biosciencejournals.com
ISSN: 2321-9122
Impact Factor: 3.742
Anupama Gupta
Department of Basic
Science, Dr. Y S Parmar
University of Horticulture
and Forestry, Nauni, Solan,
Himachal Pradesh 173 230,
India.
Nivedita Sharma
Department of Basic
Science, Dr. Y S Parmar
University of Horticulture
and Forestry, Nauni, Solan,
Himachal Pradesh 173 230,
India.
Correspondence
Anupama Gupta
Department of Basic
Science, Dr. Y S Parmar
University of Horticulture
and Forestry, Nauni, Solan,
Himachal Pradesh 173 230,
India.
Assessment of cell surface properties and adhesion
potential of lactic acid bacteria isolated from Lasoda
bari - A rare fermented food of Himachal Pradesh
(India)
Anupama Gupta, Nivedita Sharma
Abstract
One of the most significant groups of probiotic organisms are the lactic acid bacteria, with
fermentative, biopreservative and various health benefits. Most important criterion during probiotic
selection is the adhesion to the luminal epithelium to colonize the gastrointestinal tract of a human and
to provide health benefits and safety against GI disorders after being consumed. Present study was
aimed to evaluate in vitro adhesion of lactic acid bacteria isolated from Lasoda bari- a rare fomented
food product of Himachal Pradesh (India) to gut mucosa and epithelial cell lines. Pediococcus
pentosaceus LB-CC and Pediococcus pentosaceus LB-WC were found to exhibit 87.0 and 88.0 %
hydrophobicity, produced exopolysaccharides, showed 68.56 and 49.62% adherence to gastric mucin
and efficiently adhered to human epithelial cell lines. Overall, both isolates fulfilled the essential
criteria for their selection and could be recommend for the nutraceutical product formulations to
provide health benefits to human beings.
Keywords: Probiotic, Pediococcus, Lasoda bari, Mucin, Adherence, Himachal Pradesh
1. Introduction
Microbial cultures have been exploited for thousands of years in fermentation industry and
have undergone scientific inspection for their therapeutic as well as biopreservative potential
[1]
. Probiotics are live microorganisms thought to be beneficial to the host organisms upon
ingestion in an adequate amount [2]. Many probiotics are lactic acid bacteria which are
reported to provide health benefits by balancing the intestinal flora, preventing the growth of
food borne as well as spoilage causing microorganisms, improving digestion and immune
responses. The major well established genera of probiotics are Lactobacilli and
Bifidobacteria. But, some minor genera Pediococci, Streptococci, Bacillus, Brevibacillus and
Saccharomyces have also been reported and recommended as potential probiotics in food
products [3]. Among them, Pediococci are used as beneficial microorganisms in the context of
food industry and can be used as a probiotics and starter culture in various vegetable
fermentation processes [4]. In order to carry out beneficial effects, probiotic strains must
survive passage through the upper gastrointestinal tract (GIT) by tolerating gastric acidity
and bile salt concentration, and colonizing the GIT by adhering to mucin or intestinal derived
epithelial cells [5].
Proposed mechanisms using which the ingested probiotic microorganisms may later benefit
their host includes the production of antimicrobial substances, competition for nutrition,
degradation of toxins and immune modulation [6]. However, adherence to intestinal epithelia
is thought to be paramount among the main criteria for selecting probiotic strains [7]. The
ability to adhere to epithelial cells and mucosal surfaces has been suggested as being an
important property of many probiotic bacteria strains. Several workers have suggested that
the ability of beneficial microbes to aggregate and adhere aids in colonization of the gut and
in the establishment a barrier which prevents enteropathogens from establishing an infection
[8]
. Also, as adhesion to host tissues is essential for many gastro-intestinal pathogens, the
paradigm of competitive exclusion through competition for binding sites has evolved.
Therefore, assessment of adhesion potential of probiotic isolates is of major importance
when developing functional foods and nutraceuticals based on the administration of
probiotics to promote gut health. In the present study, therefore, the adhesion ability of
probiotic bacteria to gastric mucin as well as to the epithelial cell line has been evaluated for
their efficient colonization to the human gut.
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European Journal of Biotechnology and Bioscience
2. Materials and Methods
2.1 Isolation and identification of Lactic acid bacteria
LAB strains were isolated from Lasoda bari - a rare and
traditional fermented food product of Himachal Pradesh (Fig.
1) using De Man Rogosa and Sharpe (MRS) broth [9] by
serial dilutions method and incubated at 35°C for 24-48 h
anaerobically. Lasoda bari is traditionally prepared from
Lasoda (Cordia dichotoma) and urd dal (Phaseolus radiata).
Lasoda fruits have been reported to have many medicinal
uses including anti-inflammatory, diuretic, anti-ulcer and as
antidiabetic drug. The isolates obtained on MRS were
identified according to their morphological, cultural,
physiological and biochemical characteristics. Molecular
identification of the isolates selected after screening (on the
basis of biochemical and antagonistic potential) was carried
out on the basis of 16S rRNA gene sequence.
Fig 1: Lasoda bari-A traditional fermented food of Himachal
Pradesh
Tolerance to acid and bile conditions
The ability to tolerate low pH had been evaluate by
following the method of [10] with slight modifications. Effect
of bile salts on the growth of selected isolates was studied by
the method given by [11].
Hydrophobicity
The test of adhesion to hydrocarbons (xylene) was adopted to
screen isolates for their cell surface hydrophobicity.
Microbial adhesion to hydrocarbons (MATH) in terms of the
cell surface hydrophobicity (%H), was determined according
to the method of [12] with slight modifications. The decrease
in the absorbance of the aqueous phase was taken as the
measure of the cell surface hydrophobicity (H%) which was
calculated with the following formula:
%H = [{A0- At}/A0)] × 100
Where At represents the absorbance at time t=2h and A0 the
absorbance at t =0h.
Production of exopolysaccharides
Qualitative estimation of exopolysaccharide (EPS) was
evaluated as reported by [13] while the quantitative estimation
was done by using the method described by [14] with minor
modifications. Total EPS (expressed as µg/ml) were
estimated in each sample by Phenol-Sulphuric acid method
of [15] using glucose as standard.
β-galactosidase production
Quantitative β-galactosidase estimation has been done by o-
nitrophenyl-β-D-galactopyranoside (ONPG) assay [16] with
minor modifications. An aliquot of 1ml of the sonicated cell
suspension was taken in a tube and 4ml of o-nitrophenyl-β-
~ 10 ~
D-galactopyranoside (ONPG, HiMedia Laboratories, Pvt.,
Ltd., Mumbai, India) (4 mg/ml) was added. Tubes were
placed in a water bath at 35oC for 15 min. 0.5 ml of 1M
sodium carbonate (Na2CO3) was added to each tube to stop
the reaction. Absorbance values at both 420 and 560 nm
were recorded for each tube.
β -Galactosidase activity was calculated (Miller Units) as
follows:
MU= [(A420/ (15minx1mlxA560)] x 1000
Mucin binding assay
Ability of P. pentosaceus LB-CC and P. pentosaceus LB-
WC to adhere mucin type III form porcine stomach (Sigma
Aldrich) was investigated by following the method given by
[17]
. Gastric mucin (0.5mg/ml in PBS) was immobilized
passively into microtiter plate wells (Maxisorp; Nunc,
Denmark) by overnight incubation at 4 oC. Bacterial cells
were added as a volume of 150µl into microtiter plate wells
already coated with mucin and allowed to adhere at 37 oC for
1 h. Adherent bacteria were fixed at 65oC for 45 min and
stained with crystal violet (150µl/well; 0.1% solution. The
stain bound to bacterial cells was released by adding 150µl
of Citrate buffer (50mM, pH 4.3). The absorbance values at
620nm were determined using Microtiter plate reader.
Stained mucus without added cells was used as negative
control and absorbance values of this control were subtracted
from absorbance values of the sample.
Inhibition/Exclusion of pathogen adhesion to intestinal
mucus
The ability of selected isolates to inhibit the adhesion of
pathogens viz. Listeria monocytogenes MTCC 839,
Clostridium perfringenes MTCC 1739 and Bacillus cereus
CRI was assessed by using the same procedure for bacterial
adhesion to gastric mucin with minor modifications i.e.
probiotic bacteria were adhered first followed by pathogen
adhesion. The inhibition of pathogens was calculated as the
difference between the adhesions of the pathogen in the
absence and presence of probiotic bacteria [18].
Displacement of pathogen adhered to intestinal mucus
The ability of probiotic isolates to displace already adhered
pathogens was assessed by following the method used for
microbial adhesion to mucin with minor modifications.
Displacement of pathogens was calculated as the difference
between the adhesion after the addition of the probiotic
strains [18].
Competence between pathogen and probiotic isolates to
adhere to intestinal mucus
Competitive exclusion of pathogens by probiotic isolates was
determined by following the same procedure for microbial
adhesion to gastric mucin with minor modifications i.e.
probiotic bacteria and pathogens were immobilized
simultaneously. Competitive exclusion was calculated as the
percentage of pathogens bound after the combination with
probiotic bacteria relative to pathogens bound in the absence
of probiotic bacteria [18].
Adherence to mammalian cells
The Hep-2C cell line was procured from Department of
Biotechnology, Himachal Pradesh University, Shimla
(India). Cells were maintained in Dulbecco’s modified Eagle
medium (DMEM, HiMedia), supplemented with 10% Fetal
 

European Journal of Biotechnology and Bioscience
Calf serum (FCS, HiMedia) and 100µg/mL Pen strep. For
adhesion assay, monolayer of Hep-2C cells was prepared on
22mm2 glass coverslip which were placed in Leighton tubes
at a concentration of 104cell/ml. The Hep-2C monolayer was
washed twice with PBS, inoculated with 1ml bacterial
suspension (containing approx. 108 CFU/ml) and incubated
for 120 min at 37oC in a humified atmosphere containing 5%
CO2. After incubation the monolayer was washed five times
with sterile PBS, fixed with methanol for 10 min, Gram
stained and examined microscopically under oil immersion.
For each glass cover slip monolayer, the number of adherent
bacteria per 20 epithelial cells was counted in 10 microscopic
fields [7].
3. Results and discussion
A total of 4 isolates were obtained out of which 2 were
confirmed as rods while 2 were confirmed as coccus (tetrad)
as revealed by microscopic examination, were non
sporulating, catalase negative, not able to utilize citrate, no
casein hydrolysis, no urease production and no indole
production were observed. On the basis of microscopic
examination isolates were tentatively identified as
Lactobacillus and Pediococcus sp. Out of four isolates, LB-
CC and LB- WC gave clear halos around the indicator
pathogenic strains using bit/disc method with widest
antimicrobial spectrum and were selected for further study.
The largest diameter of inhibition upto 23.6 mm was
obtained against serious food borne and spoilage pathogens
viz. L. monocytogenes, S. aureus and C. perfringens,
revealing their antagonistic potential and use as safe
Where LB-CC, LB-WC and LB-4 are isolates obtained
from Lasoda bari
Fig.2. Antagonistic potential of isolates by bit/disc method
The adherence of probiotics to the intestinal epithelial tissues
is an important prerequisite, which depends on the
hydrophobicity of the bacterial cell surface [20, 21]. It is the
interaction of the bacterial cell with the organic compounds.
Adhesion to hydrocarbon like xylene, toluene and n-
hexadecane is considered as a biochemical marker for
adherence to the epithelial cell in gut. The adhesion to xylene
(apolar solvent) demonstrates the hydrophobic surface
characteristic of bacteria. The affinities to chloroform (polar
acidic solvent) and ethyl acetate (polar basic solvent)
describe the electron donor and electron acceptor properties
of the bacterial cell surface, respectively. Hence the cell
surface hydrophobicity of P. pentosaceus LB-CC and P.
pentosaceus LB-WC was measured with organic solvent
xylene after 2 hours and were observed to have strong
~ 11 ~
biopreservative (Fig. 2).
Analysis of the 16S rRNA sequences of selected strains
revealed that lactic acid bacteria isolated from Lasoda bari
displayed 99% homology with Pediococcus pentoseceus
DSM 20336 and Pediococcus pentoseceus ATCC 25745
(Fig.3). The 16S rRNA gene sequences were deposited in
gene bank under accession no. KM251460 and KJ489415 for
Pediococcus pentoseceus LB-CC and Pediococcus
pentoseceus LB-WC, respectively. Pediococcus pentoseceus
has been isolated and reported for the first time from Lasoda
bari. To be a successful probiotic, a bacterial strain must
resist harsh conditions in stomach and gut region and must
be able to colonize intestinal epithelium for its probiotic
action. Selected isolates were able to survive pH 3 upto 3h
and incubation with 2.0% bile salts (Data not shown).
Resistance to stomach pH, bile salts, and pancreatic fluid is
of great importance in predicting the survival and growth of
the potential probiotic strains in the gastrointestinal
conditions. Tolerance to gastric acidity (pH 2.0–2.5) is
considered as a key functional requirement for probiotics,
which enables them to survive during passage through the
gastrointestinal tract. Similarly, Tokatl et al. [19] reported a
40–76% survival of strains Pediococcus ethanolidurans
isolated from traditional pickle at pH 2.5 for 4h and Ribeiro
et al. [4] found 45.9% and 72.4% survival rate of P.
acidilactici B14 at pH 2.5 and 0.3% bile salts, respectively.
Nevertheless, the majority of the probiotic carrier foods
possess pH higher than 3.0. Therefore, the slight reduction in
the viability of the tested strains would not affect their
suitability to be considered as probiotic candidates.
affinity of organic solvent (87.0 and 88.0 %, respectively)
(Table 1).
Hydrophobocity of Lactic acid bacterial strains has been
studied by various workers. Duary et al. [22] reported that
among the tested faecal isolates, L. plantarum Lp91 showed
maximum percentage hydrophobicity (35.73±0.40 for n-
hexadecane and 34.26±0.63 for toluene) closely followed by
L. plantarum Lp9 (35.53±0.29 for n-hexadecane and
33.00±0.57 for toluene). Also Ng et al. [23] found L.
plantarum 0103 as the most adherent (55.14%) to n-
hexadecane among the selected Lactobacillus strains.
Probiotic strains with more than 40% affinity to polar
solvents generally are more hydrophobic [24]. In the present
study, both isolates had a hydrophobic surface. According to
Del Re et al. [25], strains with a hydrophobic surface have a
higher capacity to adhere to intestinal cells and solid
materials.
 
European Journal of Biotechnology and Bioscience
 
 
 

Table 1: Hydrophobicity of P. pentosaceous LB-CC and P. pentosaceous LB-WC

OD600♦ % Hydrophobicity**
S. No. Name of hydrocarbon Indication
LB-CC LB-WC LB-CC LB-WC
1. Xylene 0.13 (±0.01) 0.12 (±0.02) 87.0 88
2. Chloroform 0.29(±0.01) 0.21 (±0.00) 71.0 79 Strong
3. Ethyl acetate 0.20 (±0.01) 0.36 (±0.04) 80.0 64
♦OD: Mean (±SD) of results from three different experiments
**Hydrophobicity % = [(A-A0)/A] x 100

Production of exopolysaccharides is related to the adhesion
ability of probiotic cells as the proteins and carbohydrates
enhance the attachment process. In this study, both isolates
were able to form ropy colonies on Ruthenium red agar
plates as well as to produce 474.31 and 392.73 µg/ml of EPS
during their growth cycle, thereby proving their ability to
adhere efficiently on the cell surface gut epithelia. On
contrary Nikolic et al. [26] reported that exopolysaccharides
produced by Lactobacillus paraplantarum BGCG11 did not
improved the survival of the producing strains and the
presence of EPS layer hindered the adhesion to epithelial cell
lines. Thus, the effect of EPS on adhesion to cell line might
be strain specific.
P. pentosaceus LB-CC and P. pentosaceus LB-WC were
assessed for their potential to produces β-galactosidase and
were found to produce 177.7 and 173.3 MU, respectively.
The production of β-galactosidase reveals the ability of
probiotic bacteria to metabolize lactose and the prevention of
lactose intolerance in the host after their consumption.
Mustapha et al. [27] studied the influence of bile sensitivity,
lactose transport, and acid tolerance of Lactobacillus
acidophilus on in vivo digestion of lactose and found the
isolate to secrete β-galactosidase and to improve lactose
digestion and tolerance.
In the strains with probiotic functions, adhesion is an
important feature that favors the colonization and
establishment of beneficial microbiota in the intestinal tract
[28]
. Generally the substances responsible for adherence are
adhesins. In the present investigation, the ability of P.
pentosaceus LB-CC and P. pentosaceus LB-WC to adhere to
gastrointestinal mucus, which mimics the GI conditions, was
evaluated. The cells of P. pentosaceus LB-CC and P.
pentosaceus LB-WC adhered significantly to the gastric
mucin with adherence percentage of 68.56 and 49.62%,
respectively. The mucin binding ability exhibited by isolates
P. pentosaceus LB-CC and P. pentosaceus LB-WC
contributes to its adhesion property and provides resistance
to peristaltic elimination by providing competitive advantage
in gut ecosystem. These results suggested the strains being
potential probiotic for their use as good probiotic candidates
in food as well as in pharmaceutical industry.
The ability to adhere to the gastrointestinal mucosa as well as
competitive exclusion of pathogens are most frequent
mechanism tools for the search of new probiotics as the most
important criteria for selection of probiotics. However, few
studies are available on the adhesion interactions of
Pediococcus spp. in the gastrointestinal mucus. Adhesion of
P. pentosaceus LB-CC and P. pentosaceus LB-WC
influenced the pathogens adhesion to the gastrointestinal
mucus, either by enhancing or decreasing the adhesion. The
competition, inhibition and displacement abilities of
probiotics against pathogenic bacteria are strain dependent.
P. pentosaceus LB-CC and P. pentosaceus LB-WC inhibited
the adhesion of the tested pathogens viz. L. monocytogenes,
C. perfringenes and B. cereus with 46.11 and 54.85; 57.45
and 78.47; 71.72 and 80.63%, respectively. P. pentosaceus
LB-CC and P. pentosaceus LB-WC were able to displace C.
perfringens and B. cereus (68.8, 73.67% and 66.84, 69.63%,
respectively).
P. pentosaceus LB-CC displaced L. monocytogenes adhered
to gastric mucin (39.3%) while L. monocytogenes remained
adhered to the wells coated with mucin (-2.91%) in case of
wells treated with P. pentosaceus LB-WC. Competition for
adhesive site between P. pentosaceus LB-CC and P.
pentosaceus LB-WC and pathogens was studied and it was
found that the strains were able to compete for mucus site
with C. perfringens and B. cereus (75.0, 80.46% and 62.47,
72.77%, respectively) while L. monocytogenes exhibited
more competition for mucus sites (-7.28 and -3.39%) (Table
2 & 3). In this study, we are able to demonstrate that P.
pentosaceus LB-CC and P. pentosaceus LB-WC exhibited
good inhibition property against serious food borne and
spoilage causing pathogens thus can be considered as
excellent candidates to be used in fermented food products.
The isolates could increase the beneficial effects in the health
regarding their pathogen adhesion inhibition properties and
its influence in their colonization.
Lee et al. [29] reported that L. rhamnosus GG and L. casei
Shirota were able to exclude and compete with a limited
range of GI bacteria. Similarly, Collado et al. [30] observed
the ability of commercial Lactobacillus strains in
combination with Bacillus spp. to inhibit, displace and
compete with model pathogens viz. E. coli, S. typhimurium,
C. difficile, and S. aureus and found that the combination
was efficient to inhibit, displace and compete with model
pathogens.

Table 2: Inhibition of adherence of pathogens to gastric mucin by P. pentosaceus LB-CC

Competition Displacement Exclusion
Pathogens
OD620nm % inhibition OD620nm % inhibition OD620nm % inhibition
L. monocytogenes 0.221 (±0.00) -7.28 0.125 (±0.00) 39.3 0.111 (±0.00) 46.11
C. perfringens 0.151 (±0.00) 75.0 0.188 (±0.01) 68.8 0.257 (±0.02) 57.45
B. cereus 0.215 (±0.01) 62.47 0.190 (±0.01) 66.84 0.162 (±0.01) 71.72

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European Journal of Biotechnology and Bioscience
Table 3: Inhibition of adherence of pathogens to gastric mucin by P. pentosaceus LB-WC
Competition
Pathogens
OD620nm % inhibition
L. monocytogenes 0.213 (±0.00) -3.39
C. perfringens 0.118 (±0.00) 80.46
B. cereus 0.156 (±0.01) 72.77
One of the important properties of probiotic bacteria
including lactobacilli is their ability to adhere to the target
sites for their colonization in the gut for expressing optimal
functionality. The use of human epithelial cell lines in
culture for evaluating bacterial adhesion provides useful
advantage in cellular interactions studies since they mimic
the in vivo conditions. Caco2 and HT-29 cell lines have been
extensively used as in vitro models to screen adherence of
probiotic cultures [31, 32]. The level of adhesion of bacterial
strains positively correlates with the number of probiotic
bacteria added at certain point the saturation of potential
binding sites on cell lines probably occurs.
In order to investigate the adhesion capacity of probiotic
Fig 3: In vitro characterization of the adhesion potential of probiotic isolates to Hep-2C cell line (a) Control (Hep-2C cell line without
probiotic isolates (b) Hep-2C cell line with P. pentosaceus LB-CC c) Hep-2C cell line with P. pentosaceus LB-WC
Similarly Argyri et al. [33] observed the variable efficiency of
lactic acid bacteria isolated from table olives to adhere to
epithelial cell lines viz. Caco-2 cells. Seven lactic acid
bacteria isolated from ethnic fermented vegetables of the
Himalayas strains showing more than 70% hydrophobicity
were evaluated for in vitro adhesion and were found to
adhere to the mucus secreting HT29 MTXcells [34]. The
ability to adhere to the mucosal surfaces of the intestine and
the subsequent long or short-term colonization has long been
one of the most commonly encountered criteria for the
selection of probiotic strains [35-37].
4. Conclusions
In the present study, putting all the results together, it has
been concluded that P. pentosaceus LB-CC and P.
pentosaceus LB-WC reported from Lasoda bari for the first
time, were able to survive to the gastrointestinal transit and
efficiently adhere to the gastric mucin as well as the
epithelial cell lines. The results obtained in in vitro studies,
although cannot be directly extrapolated, indicate that the
isolates analyzed in this study could have probiotic potential
specifically the adherence ability towards mucin and cell
lines. Still, in vivo studies to highlight the safety and health
promoting efficiency of such potential probiotic strains needs
to be demonstrated along with human trials.
5. Acknowledgments
We express our sincere gratitude to Dr. S S Kanwar,
Chairmen, Department of Biotechnology, HPU, Shimla (HP)
for providing HEp-2C epithelial cell lines.
Displacement Exclusion
OD620nm % inhibition OD620nm % inhibition
0.212 (±0.00) -2.91 0.093 (±0.00) 54.85
0.159 (±0.01) 73.67 0.130 (±0.02) 78.47
0.174 (±0.01) 69.63 0.111 (±0.01) 80.62
isolates to mammalian epithelial cells, Hep-2C cell lines has
been used for the assay. The adhesion capacity was
expressed as the number of bacteria adhered to 20 Hep-2C
cells counted in 10 microscopic fields. The adhesion
potential of the strain in the present study was assessed on
the following scale: non-adhesive (less than 5 LAB to one
epithelial cell), moderately adhesive (5 to 40) and strongly
adhesive (more than 40). As shown in Figure 3, P.
pentosaceus LB-CC and P. pentosaceus LB-WC have been
able to efficiently adhere on the epithelial cells. The high
degree of hydrophobicity and ability to adhere to cell lines of
these LAB strains probably indicates the potential of
adhesion thus, advocating their probiotic character.
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