World J Microbiol Biotechnol

DOI 10.1007/s11274-014-1625-0

SHORT COMMUNICATION

Antibacterial, anti-inflammatory and probiotic potential

of Enterococcus hirae isolated from the rumen of Bos primigenius
Selvaraj Arokiyaraj • Villianur Ibrahim Hairul Islam • R. Bharanidharan •
Sebastian Raveendar • Jinwook Lee • Do Hyung Kim • Young Kyoon Oh •
Eun-Kyung Kim • Kyoung Hoon Kim

Received: 14 November 2013 / Accepted: 17 February 2014

Ó Springer Science+Business Media Dordrecht 2014

Abstract In the present study bacterial strains were iso-
lated from the rumen fluids of Bos primigenius and inves-
tigated their in vitro probiotic properties with potent
antibacterial activity and anti-inflammatory effects. 9 g
positive bacterial isolates were obtained and three isolates
could able to tolerate gastric conditions, high bile salt
concentrations and exhibited significant bactericidal effect
against the enteric pathogens Vibrio cholera, Enterococcus
faecalis, Enterobacter aerogens, Pseudomonas aeruginosa,
Escherichia coli and Salmonella typhi. Moreover it showed
above 70 % cell surface hydrophobicity, significant low-
invasion ability and potential adherence capacity in Caco-2
cells when compared with the control. The proinflammatory
cytokines (TNF-a) was greatly reduced in rumen bacteria
treatment and ARBS-1 modulate the immune response by
activating the IL-4 secretion in parallel to TNF-a suppres-
sion. The 16s rRNA gene sequence of the active isolates
were identified as Enterococcus hirae (ARBS-1), Pedio-
coccus acidilactici (ARBS-4) and Bacillus licheniformis
(ARBS-7). This study revealed the probiotic bactericidal
properties of E. hirae obtained from the rumen of B.
primigenius with potential antibacterial and anti-inflam-
matory effects. Future studies with the strains may yield
some novel probiotic product for livestock’s.
Keywords Antibacterial activity  Anti-inflammatory 
Enterococcus hirae  Hemolytic assay  Probiotic  Rumen 
TNF-a
Introduction
The rumen constitutes an effective animal-microbe inter-
dependency system from that each partner derives benefit.
In recent years several rumen microbes have been isolated,
characterized and recommended as feed additive for
improving the overall growth or production of animals
(Mamen et al. 2010). Probiotics are live microorganisms
that favor health to the hosts by maintaining and improving
the intestinal microbial balance and its immune response;

S. Arokiyaraj  K. H. Kim (&) S. Raveendar

College of Agriculture and Life Sciences, Seoul National
University, Seoul, Republic of Korea
e-mail: khhkim@snu.ac.kr
S. Arokiyaraj  J. Lee  D. H. Kim  Y. K. Oh
Department of Animal Nutrition and Physiology, National
Institute of Animal Science, RDA, Suwon, Republic of Korea
V. I. Hairul Islam
Division of Infection and Immunity, Pondicherry Centre for
Biological Science, Pondicherry, India
Animal Genomics and Bioinformatics Division, National
Institute of Animal Science, RDA, Suwon, Republic of Korea
E.-K. Kim
Division of Food Bio Science, Konkuk University, Seoul,
Republic of Korea
E.-K. Kim
Korea Nokyong Research Center, Konkuk University, Seoul,
Republic of Korea

R. Bharanidharan
Department of Biotechnology, Vel Tech High Tech Dr. RR Dr.
SR Engineering College, Avadi, Chennai, India

123

used to minimize the necessity to use antibiotics, and also
increase the economic productivity and public health
(Asahara et al. 2004; Doron et al. 2008). Probiotic has been
recognized as an antimicrobial agent against various
pathogens and several probiotic strains are reported to
enhance cell mediated immune responses and tumoricidal
activity of natural killer cells (Mera et al. 2012; Gill et al.
2001a; Matsumoto et al. 2005). The most important prop-
erty of a probiotic includes tolerance in highly acidic and
bile salt conditions present in the stomach and small
intestine (Pereira et al. 2003); have high adhesion proper-
ties into the epithelial cells (Kravtsov et al. 2008; Espeche
et al. 2012) and has the capability to inhibit the pathogen
adhesion by specific competition (Reid 2006). For the past
few years, researchers have investigated and studied
extensively for the isolation of probiotics from various
sources (Hairul Islam et al. 2011; Vidhyasagar and Jeeva-
ratnam 2013). But the thorough review of literature
revealed that, only few studies have been carried in
obtaining the probiotics from the rumen of mammals. In
our present study, we aim to isolate the probiotic microbial
strains from the bovine rumen and to evaluate the potential
strains that are tolerant to upper and lower gastric juices
present in the rumen of Bos primigenius and its biotic and
abiotic functional. The potential microbe isolates were
identified and facilitate the brief information of rumen
bacteria and its functional role was evaluated in our
research study to be used as probiotic feed.
Materials and methods
Collection of sample and isolation of bacteria
The B. primigenius rumen were collected by the method
described by Khampa et al. (2006) and stored in an aseptic
condition at 4 °C. The total bacteria present in the rumen
were cultured and isolated using MRS media (Man, Ragosa
and Sharpe Media, Hi media, India) and 0.5 % rumen fluid
media. Pure isolates were grown under limited aeration at
37 °C and Gram stained.
Resistance to simulated gastric and intestinal fluids
Simulated gastric and small intestinal juices were prepared
by following the method described by Hairul Islam et al.
(2011). The visible colony counts were recorded after 24 h in
simulated gastric and intestinal fluid. The percentage of
bacterial survival was calculated as: CFUassay/CFUcontrol 9
100, where CFUassay represents CFU after incubation in
simulated gastric or simulated intestinal fluids and CFUcontrol
represents the CFU after incubation in PBS as a control.
In vitro hemolytic activity.
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World J Microbiol Biotechnol
Freshly collected human red blood cells were taken and
washed thrice with 150 mM NaCl. The serum was
removed and the cells were suspended in 100 mM sodium
phosphate buffer solution before the treatment. The isolates
were then treated in a multiple of infection [MOI] ratio
1:50 and later mixed with 200 lL of RBC solutions. The
final reaction mixture volume was made up to 1 ml by
adding sodium phosphate buffer solution. This reaction
mixture was then placed in the incubator for 1 h at 37 °C.
After the incubation period the reaction mixture was col-
lected and the optical density was measured at 541 nm
wavelength absorbance. The sterile water was used as
positive hemolytic activity for the sample reaction mixture
chosen.
Cell surface hydrophobicity
The in vitro cell surface hydrophobicity was determined by
the bacterial adherence to hydrocarbon assay, modified
from the methods of Hairul Islam et al. (2013) and the
value of cell surface hydrophobicity (H%) was calculated
according to Nostro et al. (2004) method, which considers
the percentage hydrophobicity index is greater than 70 %.
The percentage of cell surface hydrophobicity (H%) of the
strain adhering to hexadecane was calculated using the
equation: H% = ([A0 - A]/A0 9 100).
Cell lines and intestinal cells maintenance
RAW 264.7 murine macrophage cell lines and Human
colon adenocarcinoma Caco-2 cell line were obtained from
National Center for Cell Science (NCCS, Pune, India). The
cells were grown and maintained in DMEM (Dulbecco’s
modified Eagle medium, Invitrogen, Carlsbad, CA, USA)
supplemented with 10 % fetal bovine serum (FBS). Cell
cultures were performed at 5 % CO2 in a humidified
atmosphere at 37 °C grown to 85–95 % confluency and
passaged every 2–4 days.
Adhesion to Caco-2 cells
Caco-2 cells were seeded with 1 ml culture medium con-
taining 106 cells/well in 24-well tissue culture plates. Test
bacteria from 18 h culture in MRS broth were harvested
and washed twice with PBS solution. These bacteria iso-
lates were resuspended in non-supplemented DMEM to
consecrate 108 cfu/ml in the tubes. Caco-2 epithelial cells
totaling 1 9 105 were infected with selected strains for the
assessment of adhesion and invasion abilities for 30 min.
Yersinia enterocolitica was used as standard for assessing
invasion and L. acidophilus was used as a standard for
assessing adhesion.
World J Microbiol Biotechnol
Quantification of inflammatory cytokines
RAW 264.7 cells adjusted to a density of 5 9 105 cells/ml
were exposed with 2 lg/ml LPS from Escherichia coli
(Sigma-Aldrich, Oakville, Canada) for 60 min and super-
natant media were aspirated. The probiotics strain in the
ratio of 1:50 MOI were co-cultured for 16 h. The positive
control consisted of LPS stimulated RAW 264.7 macro-
phage cells and the negative control consisted of RAW
264.7 macrophage cells exposed to DMEM supplemented
with 10 % FBS. After co-culture incubation, the culture
supernatant was collected and stored at -20 °C until
cytokines analysis. The induction of TNF-a and IL-4
cytokines was assayed using commercial mouse ELISA
kits (Tocris, Peprotech). The units are expressed in pg/ml.
Antimicrobial activity by soft agar overlay method
Antimicrobial activity was evaluated by the soft agar
overlay method. The test isolates were inoculated by
placing in the middle of the media and these plates were
incubated for 24 h at 37 °C. Soft agar (0.8 %) was pre-
pared and the test pathogens of 1 9 106/ml obtained by
serial dilution were inoculated as an overlay on the surface
of 24 h pre-incubated media plates. The pathogens used for
evaluating the bactericidal activity are E. coli ATCC
25922, Klebsiella pneumonia ATCC 15380, Salmonella
typhi MTCC 1254, Vibrio cholera MTCC 3906, Pseudo-
monas aeruginosa ATCC 27853, Enterococcus faecalis
ATCC 29212 and Enterobacter aerogenes MTCC 111. All
these entero pathogens were obtained from clinical isolate
repository of Madras Medical College (MMC), Chennai,
India.
Identification of active bacterial isolates
The genomic DNA was extracted from the active probiotic
isolates by the genomic purification kit (Promega, Madi-
son, WI, USA). For PCR analysis the nucleotide sequence
of the forward primer was 12f universal 50 -GTTTGATCC
TGGCTCAG-30 and the reverse primer 1492r universal
50 -ACGGGCGGTGTGTAC-30 were used. The primers
were tested in PCR amplification with E. coli genomic
DNA. Cycling parameters were: (1) 95 °C for 2 min; (2)
35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for
30 s, and (3) 72 °C for 7 min. The 16S rDNA sequence
similarity was analyzed in the Ribosomal database project
(RDP) (http://rdp.cme.msu.edu/seqmatch/seqmatch_intro.
jsp) and the National Center for Biotechnology Information
(NCBI) (http://blast.ncbi.nlm.nih.gov/). The phylogenetic
tree was constructed by following the method of Fitch and
Margoliash (1967). Bootstrap values derived from the
neighbor-joining method are given in parentheses.
Statistical analysis
All experiments were performed in triplicates and the
results were expressed as mean ± SD. The level of sig-
nificance was analyzed by ANOVA (p \ 0.05) using SPSS
17.0 for Windows (SPSS Inc.).
Results and discussion
This research study was aimed to understand the underly-
ing mechanism of action of a novel microbe isolates from
rumen of bovine against in vitro challenging parameters
like bactericidal activity besides being supplementing the
growth of an individual or a mammal.
Culture of bacterial isolates from B. primigenius rumen
region
In total, nine microbe isolates were isolated and segregated
into individual strains from the rumen sample by repeated
tubing method (serial dilution). These colonies showed
catalase positive and predominantly gram positive cell
surface in nature were selected for storage and further
characterization studies.
Effect of simulated gastric juice and small intestinal
transit on the viability
The ability of microorganisms to survive in the gastroin-
testinal tract condition is one of the main important char-
acteristics of any probiotic bacteria. The survival ability of
the microbe isolates in the presence of the MRS at pH 3.0
and 0.45 % bile salts is presented in Fig. 1. In this study,
ARBS-1, ARBS-4, ARBS-6 and ARBS-7 were selected, as
they were tolerable to acidic conditions and bile juices.
These strains survived efficiently at pH 3.0 up to 3 h
without much loss in the cell viability. Whereas the earlier
studies by Succi et al. (2005) shown that the survival
properties and tolerance of Lactobacillus rhamnosus at pH
3.0 is limited only up to 2 h with loss in cell viability. The
ability to survive in the conditions that are prevailing in the
rumen of mammals will facilitate the knowledge about the
possibility of probiotics to acclimatize and colonize the
host unfavorable environment system. The acidic pH also
helps the host against the pathogenic action and its colo-
nization in the upper intestine. Some studies revealed that
Pediococcus strains survived in the digestive tract and its
traits will facilitate the functional food metabolism and
digestion (Mikulski et al. 2012). Thus, the survival strains
must have the ability to adhere and make a biofilm in the
rumen of host environment.
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Gastric juice tolerance

Bile juice tolerance
125

100
% of survival CFU
75
* * *
**
50
** ** **
** ** **
25 **

s
1

ilu
S-

S-

S-

S-

S-

S-

S-

S-

S-
B

ph
R

o
A

id
ac
L.
Fig. 1 Screening of gastric and bile juice tolerance values represent mean ± SEM for triplicate. *Values decreased significantly from positive
control (L. acidophlus). **Values decreased in highly significant manner from positive control

Table 1 Screening of isolates for hydrophobic cell surface capacity PBS

a Adherence Lactobacillus acidophilus
and hemolytic properties
100 Yersinia enterocolitica
% CFU adherence

Isolates Hydrophobic percentage Haemolytic activity ARBS-1

* *
80 ARBS-2
*
ARBS-3
ARBS-1 70.32 ± 4.16** 27.1 ± 0.22* 60 ARBS-4
ARBS-2 81.333 ± 2.51 85.8 ± 0.19** ARBS-5
40
ARBS-3 39.333 ± 2.08** 32.6 ± 0.16** ARBS-6
20 ARBS-7
ARBS-4 80.21 ± 7.21* 23 ± 0.14** ARBS-8
ARBS-5 21.33 ± 8.08** 86.1 ± 0.16 0 ARBS-9

ARBS-6 37.666 ± 2.56** 104.9 ± 0.19* Isolates

ARBS-7 74.666 ± 4.25* 14.5 ± 0.22**
PBS
ARBS-8 38.133 ± 8.02** 65 ± 0.25**
b Invasion L. acidophilus
ARBS-9 73.012 ± 4.35** 94.3 ± 0.28* Yersinia enterocolitica
150 ARBS-1
L. acidophilus 82.828 ± 2.93 17.8 ± 0.31
% CFU invasion

ARBS-2
ARBS-3
Values represent mean ± SEM for triplicate; * values decreased 100
ARBS-4
significantly from positive control (L. acidophilus); ** values ARBS-5
decreased in a highly significant manner from positive control (L. ARBS-6
acidophilus) 50
* ARBS-7
* * * ARBS-8
ARBS-9
0
Screening of hydrophobic and hemolytic activity
Isolates

The screening of hydrophobicity depends upon the cell
surface biochemical nature and its properties. If the
microbe isolates cell has more than 70 % of lipophylic in
nature, they were considered as hydrophobic isolates (Ki-
ely and Olson 2000). The isolates ARBS-1, ARBS-4,
ARBS-7 and ARBS-9 showed significant results in our
study as described in Table 1. Cell hydrophobicity is one of
the properties of bacterial cells that have auto-aggregate
and co-aggregation in host tissues (Ram and Chander
2003). These properties indicate that there is an advantage
and importance for the bacterial maintenance and survival
in the human gastrointestinal tract (Vidhyasagar and
Fig. 2 Adhesion and invasion assay. a Adhesion and b invasion abilities
of the selected bacterial strains. Values represent mean ± SEM for
triplicate; *values deviate significantly from negative control (Y.
entrocolitica); **values deviate in highly significant manner from
negative control (Y. entrocolitica); #values deviate significantly from
positive control (L. acidophilus); ##values deviate in highly significant
manner from positive control (L. acidophilus)
Jeevaratnam 2013). The isolates ARBS-1, ARBS-3,
ARBS-4 and ARBS-7 showed less hemolytic activity
compared to other tested isolates as described in Table 1.
Yang et al. (2005) reported, hemolytic study resembles the

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Fig. 3 Effect of probiotics adherence in Caco-2 cell lines. a Normal Caco-2 cell lines b L. acidophilus adhered Caco-2 cells, c E. hirae
(KF511962) adhered Caco-2 cells d P. acidilactici e B. licheniformis adhered Caco-2 cells f Yersinia enteroclotica adhered Caco-2 cells
biocompatibility of isolates with in vivo models. Further,
these four bacterial isolates were further tested for patho-
genicity assay against entero pathogens.
Adhesion and invasion assay
Adhesion to the epithelial cells is considered as the prom-
inent characteristics of the probiotic selection experiment.
The adhesion and invasion assay results shown that the
ARBS-1, ARBS-4 and ARBS-7 were in adhesion to Caco-2
cell lines. Figure 2 showed that the invasion activity of the
Y. enterocolitica as positive invasion strain compared with
the L. acidophilus was negative invasive strain. The selec-
ted probiotics showed adherence on the cell surface of
epithelial cell lines whereas, the Y. enterocolitica altered
morphology and viability during adherence (Fig. 3). The
results suggested that the low invasion and significantly
higher adhesive characteristics of probiotics may help to
colonize in the host gut. The L. acidophilus showed sig-
nificantly differed results of invasion. The ARBS-1, ARBS-
4 and ARBS-7 was significantly similar in response to
invasion activity. ARBS-2 showed less invasion and adhe-
sion whereas the ARBS-6 and ARBS-9 showed above 45 %
of adherence and significantly higher invasion properties.
The mechanism behind the adherence properties are charge
based specific binding and hydrophobic interactions in cell
membrane (Roos and Jonsson 2002). The isolates ARBS-1,
ARBS-4 and ARBS-7 showed high adherent properties and
less invasive properties. Isolate ARBS6 and ARBS9 were
selected on all the screening parameters but these two were
showed less adhesion and high invasion in adherence assay.
Effect on cytokine release
To investigate the anti-inflammatory effects of rumen
potential bacteria using LPS stimulated RAW 264.7 mac-
rophages, the TNF-a secretion was significantly increased
in LPS control compared with the negative control
(DMEM supplemented with 10 % FBS) (p \ 0.05). It was
significantly inversed in probiotics treatment group. The
ARBS-1 showed drastic reduction in TNF-levels
(305.3 pg/ml) compared with LPS control (782.33 pg/ml),
the IL-4 was increased significantly and reached up to
80 % of expression compared to control (Fig. 4) and
ARBS-4 and ARBS-7 were mirrored each other, the TNF-
levels were reduced in both treatment up to 428.68 and
500.68 pg/ml compared with LPS induced conditions.
Whereas the IL-4 was not raised significantly, this has
capacity to activate the secretion only 30 % of anti-
inflammatory cytokines. ARBS-1 regulates the immune
response and acts as an anti-inflammatory strain and bal-
ance the cytokine secretion, whereas other two active
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Cytokine quantification
1000

800
TNF-α
IL-4
pg ml -1 600

400

200

0
Control LPS Induced ARBS-1 ARBS-4 ARBS-7

LPS + Probiotic isolates

Fig. 4 Effects of probiotics on the concentration of (A) TNF-a, (B) IL-4
inflammatory cytokines secreted in RAW 264.7 macrophage cells.
ARBS-1, ARBS-4 and ARBS-7 strains were co-cultured with LPS
stimulated RAW 264.7 macrophage cells. Positive and negative controls
consisted of RAW 264.7 cells exposed to, respectively, 2 lg/ml LPS and
DMEM supplemented with 10 % FBS. Concentrations of TNF-a and IL-
4 cytokines were measured (pg/ml). All values are reported as the
mean ± SD (n = 3). Statistical analysis was analyzed using one-way
ANOVA followed by tukey’s post hoc tests to determine the effect of
treatments (p \ 0.05)

Table 2 Screening of isolates Isolates V. cholera E. faecalis K. pneumoniae E. aerogens P. aeruginosa E. coli S. typii
against different intestinal
pathogens ARBS-1 12.2 11.6 8.2 10.3 16.7 12 8.2
ARBS-2 – – – – – – –
ARBS-3 – – 9.3 – – – 9.3
ARBS-4 13.5 14.2 10.2 – 12.2 13.5 10.2
ARBS-5 – – – – – – –
ARBS-6 – 12.2 – – 9.4 – –
Results are shown as
mean ± SD Three independent ARBS-7 16 12.1 – – 15.1 16 –
assays were conducted. Values ARBS-8 16.2 – – 17.5 – 16.2 –
are expressed in zone of ARBS-9 – – – – – – –
inhibition in mm diameter

strains were particular inhibition on anti-inflammatory
cytokines. Perdigón et al. (2002) studied the lactobacillus
sps and some rumen bacteria stimulate low levels of pro-
inflammatory cytokines (TNF-a, IL-6 and IL-8), while
activate high levels of IFN-c and IL-12p70, these two
active cytokines to promote cellular immunity and enhance
the clearance of pathogens. Probiotic strains might boost
host immune status through activation of particular and
nonspecific immune pathway. This also can involve mod-
ulate of humoral, cellular, and nonspecific immunity (Gill
et al. 2001b).
Antimicrobial activity and pathogenicity assay
E. coli as shown in the Table 2. However, the maximum
bactericidal activity was observed with ARBS-1, ARBS-4,
ARBS-6 and ARBS-7 isolates. This bactericidal activity is
may be due to the metabolic products of the isolates that
contains a large number of organic acids and could reduce
the pH of the gastro intestinal tract juices Millette et al.
(2007). Some common soil bacteria and actinomycetes
showed anti-infective activity against intestinal pathogens
(Hairul Islam et al. 2013). Several in vivo and in vitro
studies have established that probiotics can control the
growth of Shigella dysenteriae Moorthy et al. (2007),
Salmonella (Alcaine et al. 2007) and Clostridium difficile
(Sarinena et al. 2012).

The isolates, ARBS-1, ARBS-2, ARBS-4, ARBS-6, Identification of active isolates

ARBS-7 and ARBS-9 showed inhibitory activity against
the tested intestinal entero pathogens V. cholera, E. fae- The bile salt tolerant, hydrophobic, less pathogenic and
calis, K. pneumoniae, E. aerogens, P. aeruginosa and antimicrobial active strains from the rumen isolates were

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Fig. 5 16sRNA gene
phylogeny of ARBS-1 E. hirae
(KF511962)
chosen for sequencing, and the PCR product obtained in
the 16S rDNA was 1400 bp in size. Nucleotide sequences
were submitted to Genbank and obtained accession number
for ARBS-1—Enterococcus hirae (KF511962), ARBS-4—
Pediococcus acidilactici (KF511963), ARBS-7—Bacillus
licheniformis (KF511964). The phylogenetic tree shown
that the E. hirae was 0.001 bootstrap closer to Entero-
coccus species (Fig. 5). P. acidilactici and B. licheniformis
species were 0.005 nucleotide substitution among the
cluster of respective phylogenetic trees. The scale bar
indicates 0.005 substitutions per nucleotide position. In the
present study, in vitro result revealed that the rumen iso-
lates such as E. hirae, P. acidilactici and B. licheniformis
shown to be good candidates for use as probiotics. How-
ever, to our knowledge no studies have been investigated
on E. hirae from the rumen of B. primigenius.
Conclusion
This study indicated that the strain ARBS-1—E. hirae
(KF511962) could able to tolerate gastric and high bile
concentration, exhibited good adhesion to CaCo-2 cells and
also demonstrated anti-microbial and anti-inflammatory
property. From this study, we conclude that the isolated
microbe strains might be used as probiotic feed for control
the intestinal infections, digestion, immunological toler-
ance of the host.
Acknowledgments The authors would like to thank the Seoul
National University and National Institute of Animal Science (NIAS),
Republic of Korea, for its unstinted support and Pondicherry Centre
for Biological Science, Pondicherry, India for carrying out the anti-
microbial assay.
Conflict of interest The authors declare that there are no conflicts
of interest in the work.
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