Probiotic potential of lactic acid bacteria isolated from chicken gastrointestinal digestive tract pot

ORIGINAL PAPER
Probiotic potential of lactic acid bacteria isolated from chicken
gastrointestinal digestive tract
H. Musikasang Æ A. Tani Æ A. H-kittikun Æ
S. Maneerat
Received: 28 October 2008 / Accepted: 16 March 2009 / Published online: 31 March 2009
Ó Springer Science+Business Media B.V. 2009
Abstract This study was conducted in order to evaluate
the probiotic properties of lactic acid bacteria (LAB) iso-
lated from intestinal tract of broilers and Thai indigenous
chickens. The major properties, including the gastric juice
and bile salts tolerance, starch, protein and lipid digesting
capabilities, and the inhibition on certain pathogenic bac-
teria were investigated. Three-hundred and twenty-two and
226 LAB strains were isolated from ten broilers and eight
Thai indigenous chickens, respectively. The gastrointesti-
nal transit tolerance of these 548 isolates was determined
by exposing washed cell suspension at 41°C to simulated
gastric juice (pH 2.5) containing pepsin (3 mg ml
-1
), and
to simulated small intestinal juice (pH 8.0) in the presence
of pancreatin (1 mg ml
-1
) and 7% fresh chicken bile,
mimicking the gastrointestinal environment. The survival
of 20 isolates was found after passing through the gastroin-
testinal conditions. The survival rates of six strains; KT3L20,
KT2CR5, KT10L22, KT5S19, KT4S13 and PM1L12 from
the sequential study were 43.68, 37.56, 33.84, 32.89, 31.37
and 27.19%, respectively. Twelve isolates exhibited protein

digestion on agar plate but no isolates showed the ability to
digest starch and lipid. All 20 LAB showed the antimicrobial
activity against Salmonella sp., Staphylococcus aureus and
Escherichia coli except one strain which did not show the
inhibitory activity toward E. coli. Accordingly, five isolates
of selected LAB (KT2L24, KT3L20, KT4S13, KT3CE27
and KT8S16) can be classified as the best probiotics and were
identified as Enterococcus faecalis, Enterococcus durans,
Enterococcus faecium, Pediococcus pentosaceus, and
Enterococcus faecium, respectively. The survival rate of
microencapsulation of E. durans KT3L20 under simulated
small intestine juice after sequential of simulated gastric
juice was also investigated. An extrusion technique exhib-
ited a higher survival rate than emulsion technique and free
cell, respectively.
Keywords Probiotic Á Lactic acid bacteria Á
Chicken intestinal tract Á Broiler Á Thai indigenous
chicken
Introduction
In recent years considerable interest has been shown in
using some probiotic microorganisms and organic acids as
an alternative to the use of antibiotics in feeds (Guerra et al.
2007). Probiotics are a live microbial feed supplements
which positively affects the health of the host animal by
improving its intestinal balance (Fuller 1989). LAB is one
of the probiotic groups which make up a large group of
microorganism in gastrointestinal tract of all human and
animals. The basic requirements for an LAB strain which is
to be used as probiotic have been described as follows.
They should be tolerant to acid and bile and be able to:

adhere to the intestinal epithelium of the hosts; show an
antagonistic activity against pathogenic bacteria and; keep
their viability during processing and storage (Lin et al.
2007). The most probiotic microorganisms used are: Lac-
tobacillus (e.g. L. bulgaricus, L. acidophilus, L. casei,
L. helveticus, L. lactis, L. salivarius, L. plantarum); Bifi-
dobacterium; Bacillus; Streptococcus; Pediococcus;
H. Musikasang Á A. H-kittikun Á S. Maneerat (&)
Department of Industrial Biotechnology, Faculty of
Agro-Industry, Prince of Songkla University, Hat Yai 90112,
Thailand
e-mail:
A. Tani
Research Institute for Bioresources, Okayama University,
Okayama, Japan
123
World J Microbiol Biotechnol (2009) 25:1337–1345
DOI 10.1007/s11274-009-0020-8
Enterococcus; and yeasts such as Saccharomyces cerevi-
siae and S. boulardii (Fuller 1989; Hyronimus et al. 2000).
The use of probiotic bacteria and their metabolites has
many beneficial effects on cattle, pigs and chickens. These
include the improvement of: general health; feed conver-
sion ratios; growth rates; resistance to diseases; promoting
body weight and increase in milk yield; quality and egg
production (Ahmad 2006; Guerra et al. 2007; Hyronimus
et al. 2000). They have been used as a substitute of anti-
biotics in considerable amounts and as growth promoters in
broilers production (Ahmad 2006).
When selecting LAB for use as dietary adjuncts, a

number of factors should be considered. While the func-
tionality of probiotics depends on their ability to survive
and colonize the gastrointestinal tract, the resistance of
cells to bile acids is a property that is necessary (Taranto
et al. 2006). In order to be effective the bacteria must
therefore survive when exposed to the acid in the stomach
and bile in the intestine (Shah 2000). However, many
studies have indicated that probiotic bacteria may not
survive in sufficient numbers when they pass through the
gastrointestinal tract in in vitro test (Lin et al. 2006;
Maragkoudakis et al. 2006). The encapsulation technique is
an approach which is currently receiving significant inter-
est for resisting environmental conditions that are adverse
to probiotics. Entrapment in calcium alginate beads has
frequently been used for the immobilization of LAB.
Alginate has the benefits of being non-toxic to the cells
being immobilized, and is an accepted food additive. The
reversibility of encapsulation, i.e. solubilizing alginate gel
by sequestering calcium ions, and the possible release of
entrapped cells in the human or animal intestine is another
advantage (Chandramouli et al. 2004; Kailasapathy 2002;
Sheu and Marshall 1993).
Therefore, this study was carried out in order to isolate
and screen LAB as probiotics from gastrointestinal diges-
tive tracts of marketable broilers and Thai indigenous
chickens. The enhancement of LAB survival through the
application of microencapsulation to use as feed supple-
ment for broilers was also studied.
Materials and methods
Lactic acid bacteria isolation

The gastrointestinal digestive tracts (crop, small intestine,
large intestine and cecum) of ten marketable broilers and
eight Thai indigenous chickens were used as LAB sources.
Each part of the intestinal tract was washed in 70% ethanol
and washed twice with sterile distilled water. Twenty-five
grams of washed section gut was homogenized in 225 ml
phosphate-buffered saline (PBS : 50 mM potassium
di-hydrgenphosphate, 50 mM di-potassium hydrogen
phosphate trihydrate, 0.85% sodium chloride, pH 7.0) for
5 min using a stomacher (Stomacher
Ò
400 Circulator,
Seward Ltd., UK). Appropriate serial dilutions were plated
onto Man, Rogosa and Sharpe (MRS) agar (HiMedia
Laboratories, Pvt. Ltd., India) supplemented with 0.02%
bromocresol purple (Ajax Finechem, Australia) and incu-
bated anaerobically for 24 h at 41°C. Colonies which
exhibited a clear halo were randomly selected from the
highest dilutions of each MRS agar plate. Bacterial colo-
nies were then purified by re-streaking on MRS agar 2–3
times. The pure cultures were characterized using Gram
stain, cell morphology and catalase reaction tests. Gram-
positive and catalase-negative isolates were stored at -20°C
in MRS broth supplemented with 25% (v/v) glycerol. For
routine analysis, the strains were subcultured twice in MRS
broth for 24 h at 41°C. The selected isolates were further
identified along full length of 16S rRNA sequence based on
the methods of Gonza
´
lez et al. (2007).

Resistance to simulated intestinal juice after sequential
incubation in simulated gastric juice of isolated LAB
A simulated gastric juice was prepared by suspending
3mgml
-1
pepsin (Fluka, Biochemika, Japan) in sterile
saline (0.85% NaCl, w/v) and adjusted the pH to 3.0 with
1.0 M HCl. Twenty-four hour 1.0 ml cultures of the strains
were subjected to centrifugation in an Eppendrof centrifuge
(Eppendorf Centrifuge 5415R, Hamberg, Germany) at
10,000 rev min
-1
for 10 min and washed twice with sterile
saline before being re-suspended in simulated gastric juice.
Resistance was assessed in terms of the viable colony count
and enumerated after incubation at 41°C for 2 h. After
120 min of gastric digestion, cells were harvested and
suspended in simulated intestinal fluid which contained
1mgml
-1
pancreatin (Sigma, Germany) and 7% fresh
chicken bile at pH 8.0. The suspension was incubated at
41°C for 6 h and the viable count was determined (modi-
fied from Madureira et al. 2005).
Starch, protein and lipid digesting capabilities
Modified MRS agar containing skimmed milk (HiMedia
Laboratories Pvt. Ltd., India), tributyrin (Fluka, USA) and
soluble starch (Labchem, Ajax Finechem, Australia) was
used for detecting the protein, lipid and starch digesting
capabilities of selected LAB strains, respectively. The

overnight cultures of LAB (10 ll) were dropped on the
modified MRS agar and incubated at 41°C for 24 h. The
diameters of the holo zone on the agar plate were then
measured. The digesting capability of the tested strains was
classified as positive when the diameters of clear zone were
1338 World J Microbiol Biotechnol (2009) 25:1337–1345
123
more than 1 mm. Each assay was performed in triplicate
(Thongsom 2004).
Antibacterial activity
Antibacterial activity was studied using the agar diffusion
method (Makras and Vuyst 2006). The indicator strains
used in this study were gram-negative strains as the main
pathogenic microorganisms in the intestinal tract of chicken
such as Escherichia coli, and Salmonella sp. In addition,
some bacterial species potentially pathogenic to humans,
Staphylococcus aureus, was also used. All strains were
obtained from Songklanagarind Hospital, Prince of Songkla
University, Thailand. Indicator strains were cultivated in
nutrient broth (HiMedia Laboratories Pvt. Ltd., India) at
41°C for 18 h. To measure the antibacterial activity, LAB
were cultivated in MRS broth at 41°C for 18 h. The culture
containing 10 ll of LAB (10
8
cfu ml
-1
) was dropped on
MRS agar and incubated at 41°C under anaerobic condition
for 18 h. The LAB on MRS agar plate were overlaid with
9 ml of soft nutrient agar with 1 ml of culture of indicator

strains activated overnight (10
6
cfu ml
-1
). The agar plates
were incubated at 41°C for 18 h and diameters of inhibition
zone on the agar plate were measured. Each assay was
performed in triplicate. The antibacterial activity was cal-
culated as follows:
The antibacterial activity ðmmÞ
¼ Diameter of inhibition zone
À Diameter of LAB colony
Cell preparation for microencapsulation
The 20% starter cultures of selected LAB were inoculated
in 50 ml MRS broth and incubated at 41°C for 24 h to
obtain a cell density of about 10
9
cfu ml
-1
. Harvesting of
cells was done by centrifugation at 8,500 rev min
-1
for
20 min at 4°C. Cell pellet was washed twice with sterile
saline. Washed cells were then suspended in 1 ml of sterile
saline and stored at 4°C until use.
Microencapsulation and enumeration
of microencapsulated LAB
Washed cells were prepared for encapsulation by extrusion
and emulsion techniques. For the extrusion technique,

probiotic capsules were prepared by mixing the 1 ml of
LAB suspension with 20 ml of 3% sodium alginate (Fluka,
Switzerland). The cell suspension was extruded through
dropping with a 24G syringe needle into 0.1 M calcium
chloride solution (the distance between the syringe and the
calcium chloride collecting solution was 5 cm). The beads
were allowed to stand for 30 min to ensure complete
gelification (Krasaekoopt et al. 2003, 2004; Muthukumar-
asamy and Holley 2007).
For the emulsion technique, 1 ml of washed cell sus-
pension was added to 20 ml of 3% sodium alginate and the
mixture was then emulsified into palm oil (Morakot,
Morakot industry Co. Ltd., Thailand) with the ratio 1:5.
The emulsion was produced by stirring for 20 min at a
constant speed (900 rev min
-1
) until it was creamy.
A solution of 0.1 M calcium chloride was then added
quickly along the side of the beaker. The mixture was
allowed to stand for 30 min. The oil layer was then
removed (Annan et al. 2008; Krasaekoopt et al. 2003;
Sultana et al. 2000).
The beads from the two encapsulation techniques were
harvested by filtration (Whatman No. 4, filter paper, Fisher
Scientific) then rinsed and stored in peptone saline (1 g l
-1
peptone, 8.5 g l
-1
sodium chloride) containing 0.05 M
calcium chloride pending further analysis.

The microencapsulated LAB were enumerated as
described by Kailasapathy (2006) and Annan et al. (2008).
The encapsulated bacteria in the microcapsules were
released by using 1.0 g of a filtered microcapsule and were
re-suspended in 9.0 ml of PBS buffer (pH 7.5) in a plastic
bag. It was homogenized for 10 min to allow complete
release of the bacteria from alginate capsules by using a
stomacher. The homogenized samples were diluted to
appropriate concentrations and drop-plated on MRS agar.
The plates were incubated anaerobically for 24 h at 41°C
and the encapsulated bacteria were enumerated as
cfu ml
-1
.
Survival of encapsulated probiotic in simulated small
intestinal juice after sequential incubation in simulated
gastric juice
One gram of the encapsulated probiotic and 1 ml of non-
encapsulated probiotic samples of individual treatments
were incubated in 9 ml of simulated gastric juice
(3 mg ml
-1
pepsin, pH 2.5) at 41°C for 2 h. Microencap-
sulated beads in simulated gastric juice were then
centrifuged at 8,500 rev min
-1
at 4°C for 20 min and
washed with 0.85% sodium chloride. The obtained cap-
sules were re-suspended in 9 ml of simulated small
intestinal juice (1 mg ml

-1
pancreatin, 7% fresh chicken
bile, pH 8.0) at 41°C for 6 h. The survivals of free cell and
encapsulated probiotic before and after exposure to simu-
lated small intestinal juice for 6 h after sequential
incubation in simulated gastric juice for 2 h were deter-
mined by plating in MRS agar containing 0.02%
bromocresol purple. Plates were incubated anaerobically at
41°C for 24 h using the anaerobic jar (modified from
Madureira et al. 2005).
World J Microbiol Biotechnol (2009) 25:1337–1345 1339
123
Results and discussion
Lactic acid bacteria isolation
Three-hundred and twenty-two and 226 LAB strains were
isolated from 10 marketable broilers and eight marketable
Thai indigenous chickens, respectively (Table 1). The 548
isolates of LAB were isolated from crop, small intestine,
large intestine and caecum of chicken intestinal tracts. The
number of LAB isolated from each organ of broilers or
Thai indigenous chicken was almost similar. In addition,
observation under light microscopic revealed that about
85% of isolated LAB was rod shape and 15% was cocci
(data not shown). The result was in accordance with a
previous study that major LAB in native chicken intestinal
tract was rod shape (Sonplang et al. 2007).
Studies on microbiota of the alimentary tract in animals
show the complex of bacteria. Base on their roles, the
intestinal bacteria may be divided into two groups: LAB
and putrefactive bacteria. LAB are evaluated as beneficial

bacteria by their product of acids (lactic acid), bacteriocin
or bacteriocin-like substances. Putrefactive bacteria are
regarded as harmful bacteria in that they decompose pro-
teins, produce foul-smelling substances and some cause of
diarrhea or produce toxins. For these reasons, LAB are paid
great attention for use as probiotics for animal produce. For
the chicken, the intestinal LAB are mainly Lactobacillus
and Enterococcus (Lan et al. 2003).
Resistance to simulated small intestinal juice after
sequential incubation in simulated gastric juice
of isolated LAB
High acidity in the stomach and the high concentration of
bile components in the proximal intestine of the host
influence probiotic strain selection (Hyronimus et al.
2000). In this study, the 20 isolates of LAB were selected
after the passage through the simulated gastric juice (pH
3.0) containing pepsin (3 mg ml
-1
) for 120 min at 41° C.
Then there was sequential incubation with simulated small
intestinal juice (pH 8.0) containing pancreatin (1 mg ml
-1
)
and 7% of fresh chicken bile for 6 h. Amoung these 20
isolates there was only one strain, PM1L12, isolated from
Thai indigenous chickens. However, isolates were mostly
isolated from the small intestine. There were only six
strains (KT3L20, KT2CR5, KT10L22, KT5S19, KT4S13
and PM1L12) that could survive in the sequential study by
showing the survival rate of 43.68, 37.56, 33.84, 32.89,

31.37 and 27.19%, respectively (Fig. 1). Results from this
study showed that a few strains are acid and bile tolerant.
The viable LAB cell numbers initially decreased approxi-
mately 1–2 log cfu ml
-1
for most strains. In general, the
acid tolerance of LAB depends on the pH profile of
H
?
-ATPase and on the composition of the cytoplasmic
membrane. This is largely influenced by the type of bac-
terium, the type of growth medium and the incubation
conditions (Hood and Zotolla 1988; Madureira et al. 2005).
However, all 20 isolates could survive higher than
10
6
cfu ml
-1
even after 2 h of exposure to the simulated
Table 1 Number of LAB from broilers and Thai indigenous chicken
gastrointestinal tracts
Organ Thai indigenous
chickens (isolate)
Broilers
(isolate)
Total
(isolate)
Crop 60 86 146
Small intestine 54 78 132
Large intestine 57 76 133

Cecum 55 82 137
Total 226 322 548
27
.19
7
.73
33.84
1.68
12.42
0.54
32.89
8.64
2.43
10.47
31
.
73
10.63
15
.13
43
.68
0.23
0.92
1.98
3.74
37.56
1.85
0
2

4
6
8
10
12
KT2
CR
3
KT
2
CR5
KT
2S11
KT
2S
15
KT
2
L
24
KT
3
CR
2
KT3
L
20
KT
3
CE

27
KT
3
CE
28
KT
4S13
KT
5S13
KT
5S15
KT
5
S
16
KT
5S19
KT
8
CR
4
KT
8S
16
KT
10
L19
KT
10
L

22
KT
10CE
33
PM
1
L
12
0
5
10
15
20
25
30
35
40
45
50
0h 6h %survival
Viable counts (log cfu ml
-1
)
Survival (%)
Lactic acid bacteria
Fig. 1 Survival rates of
selected LAB in the presence of
7% fresh chicken bile and
pancreatin (1 mg ml
-1

)atpH
8.0 (4 h) after sequential
incubation in simulated gastric
juice (2 h)
1340 World J Microbiol Biotechnol (2009) 25:1337–1345
123
gastric juice (pH 2.5) containing pepsin (3 mg ml
-1
) (data
not shown). In comparison to the acid tolerance of the
Lactobacillus species isolated from the gastrointestinal
tracts of swine and chicken, Lin et al. (2007) found that
L. acidophilus and L. bulgaricus from chicken were less
stable in the chicken gizzard extract (pH 2.6). However,
some L. acidophilus strains isolated from other origins such
as human digestive tract showed acid tolerance in the pH
2.5 gastric juice environment.
In order to describe selected isolates from the probiotic
point of view, resistance to pH and bile salts is of great
importance in survival and growth of bacteria in animal
gastrointestinal tracts. The second important criterion is the
resistance against bile salts that is a prerequisite for the
colonization and metabolic activity of probiotic bacteria in
the small intestine of the host (Strompfova
´
and Laukova
´
2007). The current study was based on the approach of
Madureira et al. (2005). This combined the effect of
exposure to gastric juice, followed by the effect of expo-

sure to bile salts on the viability of probiotic strains. After
120 min of exposure to artificial gastric juice, 0.3% (w/v)
bile salts was added to the homogenates and the incubation
was extended for a further 2 h. This approach simulated
two situations that prevailed during transit through the
gastrointestinal tract: passage through the stomach, fol-
lowed by release of bile salts in the small intestine. The pH
levels of gastric juice may vary from 2.0 to 3.5 depending
on the feeding time, the growing stage or the kind of ani-
mal (Yu and Tsen 1993). The pH in chicken proventriculus
and gizzard ranges from 2.5 to 4.74 (Malaipuang 2001) and
food ingestion can take up to 1–3 h depending on feed size.
The combined effect of a pepsin-pH solution aims at
simulating the gastric juice. However, it is not clear whe-
ther the decrease of viability conferred by the pepsin
solution at pH 2 was due to the enzyme alone, or in synergy
with low acidity (Maragkoudakis et al. 2006). In contrast to
pepsin, most strains examined in this study could survive
well in a pancreatin solution at pH 8.0 or in the presence of
fresh chicken bile (7%, v/v), simulating the chicken small
intestine environment.
Bile salts at high concentrations can rapidly dissolve
membrane lipids and cause dissociation of integral mem-
brane proteins resulting in the leakage of cell contents and
cell death (Begley et al. 2005). It has been suggested that
the major effect of bile acids would be the disaggregation
of the lipid bilayer structure of the cell membrane. Con-
jugated bile acids are less inhibitory than free bile acids
(cholic and deoxycholic acid, DCA) toward intestinal
aerobic and anaerobic bacteria. Taurine-conjugated

deoxycholic acid (TDCA) was less toxic than DCA. The
tolerance to bile salts was initially associated with the
presence of bile salt hydrolase activity (Moser and Savage
2001; Taranto et al. 2006). In the small intestine of
chicken, the total concentration of bile salts is about
10–11 mmol kg
-1
digesta and the proportion of conjugated
to unconjugated bile varies according diet. However, the
conjugated forms of chenodeoxycholic and cholic acid
dominate most frequently (Knarreborg et al. 2003; Strom-
pfova
´
and Laukova
´
2007). Lactobacillus casei NCDC 63,
L. casei VT and L. casei C1 could survive after being treated
with 2% (ox bile) for 2 h of incubation (Mishra and Prasad
2005). Lactobacillus sp. exhibited survival to bile salt and
the presence of 0.3 mg l
-1
pancreatin (Maragkoudakis et al.
2006).
Starch, protein and lipid digesting capabilities
The agar plate assays were used to study digesting capability
of the 20 isolates of LAB. In this study, sterilized skimmed
milk, tributyrin and soluble starch were used for detecting
protein, lipid and starch digestion capabilities, respectively.
There were 12 isolates (KT2CR3, KT2CR5, KT2S11,
KT2L15, KT2L24, KT4S13, KT5S13, KT5S15, KT5S16,

KT5S19, KT8CR4 and KT10CE33) which exhibited protein
digestion. However, neither starch nor lipid digestions was
detected.
Some strains of LAB are able to utilize protein, starch
and lipid (Duangchitchareon 2006; Kawai et al. 1999;
Thongsom 2004). LAB, which are able to digest starch,
protein and lipid, could enhance the health of aquatic
animals (Austin et al. 1995).
Antibacterial activity
The agar diffusion method was used to study antimicrobial
activity of the 20 isolates of LAB. All 20 isolates showed
the antimicrobial activity against Escherichia coli (with an
inhibition zone 8–25 mm in diameter), Salmonella sp. (13–
40 mm) and Staphylococcus aureus (6–24 mm). However,
one strain; PM1L12 did not show the inhibitory activity
towards E. coli (Table 2). Lan et al. (2003) reported that
the two selected probiotic strains (L. agillis JCM 1048 and
L. salivarius subsp. salicinius JCM 1230) which were
isolated from chicken, were able to inhibit growth of Sal-
monella spp. (with an inhibition zone 16–18 mm in
diameter). They were less effective for Escherichia coli (7–
8 mm) in the agar spot test.
It was shown in this study that most of the selected LAB
showed high antimicrobial activity against Salmonella sp.
The antibacterial activity of LAB may often be due to the
production of organic acids, with a consequent reduction in
pH, or to the production of hydrogen peroxide (Gonza
´
lez
et al. 2007). LAB could produce various compounds such

as organic acids, diacetyl, hydrogen peroxide, and bacte-
riocin or bactericidal proteins during lactic fermentations.
World J Microbiol Biotechnol (2009) 25:1337–1345 1341
123
Levels and types of organic acids produced during the
fermentation process depended on LAB species or strains,
culture compositions and growth conditions (Lindgren and
Dobrogosz 1990). Lactic acid is the major organic acid in
LAB fermentation where it is in equilibrium with its
undissociated and dissociated forms, and the extent of the
dissociation depends on pH. The antimicrobial effect of
organic acids lies in the reduction of pH, as well as the
undissociated form of the molecules. It has been proposed
that the low external pH causes acidification of the cell
cytoplasm, while the undissociated acid, being lipophilic,
can diffuse passively across the membrane. The undisso-
ciated acid acts by collapsing the electrochemical proton
gradient, or by altering the cell membrane permeability,
which results in disruption of substrate transport systems
(Ammor et al. 2006). In general, organic acids have a
strong inhibitory activity against gram-negative bacteria
(Makras and Vuyst 2006). The bacteriocins, generally
recognized as safe LAB by the GRAS, have generated a
great deal of attention as a novel approach to control
pathogens in food-stuffs (Savadogo et al. 2004). Hydrogen
peroxide is produced by LAB in the presence of oxygen as
a result of the action of flavoprotein oxidases or nicotin-
amide adenine dinucleotide (NADH) peroxidase. The
antimicrobial effect of hydrogen peroxide may result from
the oxidation of sulfhydryl groups causing denaturing of a

number of enzymes, and from the peroxidation of mem-
brane lipids, thus increasing membrane permeability
(Condon 1987; Kong and Davison 1980). LAB strains were
reported to inhibit the growth of pathogenic bacteria in
many studies (Ammor et al. 2006; Bernbom et al. 2006;
Collado et al. 2005; Olkowski et al. 2008; Santos et al.
2003). It may also be due to the production of bacteriocins
or bacteriocin-like compounds (Gonza
´
lez et al. 2007).
In this research, it has been demonstrated that isolated
LAB had probiotic properties against both gram-negative
and gram-positive pathogenic bacteria but the mode of
inhibition is not exactly known. Additional investigations
have to be performed to examine for the mode of action of
these LAB toward pathogens.
Strains identification
From the over all testing, there were only five isolations of
LAB that showed the best preliminary probiotic properties,
including resistance to simulated gastric and intestinal
fluids, digesting capability and antibacterial activity. There
were KT2L24, KT3L20, KT4S13, KT3CE27 and KT8S16.
These five LAB strains were isolated from the lower
intestinal tracts of broilers. These LAB were then identified
by comparing the full length of 16s rRNA sequences. The
results indicated that they were identified as Enterococcus
faecalis (100%), Enterococcus durans (99.73%), Entero-
coccus faecium (99.93%), Pediococcus pentosaceus
(99.93%) and Enterococcus faecium (99.67%), respec-
tively. The rRNA sequences were deposited in DDBJ/

EMBL/GenBank as accession numbers AB481105,
AB481101, AB481104, AB481102 and AB481103,
respectively.
Enterococci constitute part of the natural gut microflora
in mammals (De Fa’tima Silva Lopes et al. 2005; Devriese
et al. 1992). They also have good microbiologic features
such as short generation time and bacteriocin production,
but there is concern about the transmission of antimicrobial
resistance gene (Mombelli and Gismondo 2000). However,
Enterococcus such as E. faecium and Pediococcus such as
P. acidilactici are mainly bacterial strains of gram-positive
bacteria which were used in animal feed in the European
Union (EU) (Anado
´
n et al. 2006).
Pediococci are gram-positive LAB that is being used as
starters in the industrial fermentation of meat and vegeta-
bles. Some strains of the Pediococcus species produce
antimicrobial peptides that inhibit closely related LAB and
gram-positive spoilage and pathogenic bacteria (Gurira and
Buys 2005).
Table 2 Antibacterial activity of selected LAB against pathogenic
bacteria
Strains Antibacterial activity (mm)
Escherichia
coli
Salmonella
sp.
Staphylococcus
aureus

KT2CR3 17.0 ± 1.0 19.7 ± 0.6 11.7 ± 1.2
KT2CR5 18.3 ± 2.1 26.7 ± 1.2 15.3 ± 1.5
KT2S11 24.3 ± 0.6 15.0 ± 1.7 16.3 ± 2.1
KT2S15 17.0 ± 2.0 25.7 ± 2.1 16.7 ± 2.3
KT2L24 16.3 ± 1.2 17.0 ± 1.0 11.0 ± 1.0
KT3CR2 11.7 ± 0.6 36.3 ± 1.2 20.7 ± 1.2
KT3L20 25.7 ± 1.5 25.3 ± 1.5 16.7 ± 1.5
KT3CE27 17.0 ± 1.0 28.0 ± 0.0 21.0 ± 1.0
KT3CE28 08.3 ± 0.6 13.7 ± 0.6 06.7 ± 0.6
KT4S13 25.3 ± 1.2 26.7 ± 0.6 13.7 ± 0.6
KT5S13 14.0 ± 1.0 29.3 ± 2.3 14.7 ± 0.6
KT5S15 15.3 ± 1.5 29.7 ± 2.3 15.7 ± 0.6
KT5S16 18.0 ± 1.0 28.3 ± 2.1 14.3 ±
1.2
KT5S19 18.0 ± 1.0 24.7 ± 0.6 15.7 ± 1.2
KT8CR4 18.0 ± 2.0 40.0 ± 2.6 24.0 ± 1.0
KT8S16 21.0 ± 1.0 26.3 ± 0.6 14.0 ± 2.0
KT10L19 13.3 ± 1.5 26.0 ± 2.0 15.3 ± 0.6
KT10L22 15.3 ± 0.6 35.7 ± 1.2 17.0 ± 1.0
KT10CE33 08.7 ± 0.6 26.0 ± 1.7 18.7 ± 1.5
PM1L12 – 22.3 ± 1.5 09.3 ± 0.6
Each value in the table is the mean ± standard deviation of three trials
‘–’ Represents the absence of an inhibition efficiency
1342 World J Microbiol Biotechnol (2009) 25:1337–1345
123
Survival of free and encapsulated probiotic LAB during
sequential incubation in simulated gastric and intestinal
juices
The survival rate of free cell and microencapsulated
E. durans KT3L20 using simulated small intestine juice

after sequential use of simulated gastric juice were inves-
tigated. The extrusion technique exhibited higher survival
rates than the emulsion technique and free cell, respec-
tively (Table 3). The encapsulation techniques could
protect these E. durans KT3L20 effectively from high acid
and bile conditions. Muthukumarasamy et al. (2006) found
that microencapsulation using alginated solutions by
extrusion or emulsion techniques provided greater protec-
tion against gastric juice for L. reuteri. The extrusion
technique was better able to protect cells.
In this study there were 1.4 log decreases in viable cells
of encapsulated E. durans KT3L20 after 2 h of incubation
with simulated gastric juice (pH 2.5) compared to 3 log
decrease in the free cells. Chandramouli et al. (2004)
reported the survival of L. acidophilus CSCC 2409. There
was a two log decrease in encapsulated cells after 3 h
incubation at pH 2, compared to a four log decrease in the
free cells under similar conditions. Microencapsulation of
L. casei NCDC-298 in alginate beads resulted in better
survival than free cells after incubation in a simulated
gastric and intestinal bile salt solution (Mandal et al. 2006).
Microencapsulation is able to protect the bacterial cell
against harsh environments such as during passage through
the acidic pH of the stomach (Muthukumarasamy et al.
2006). This study indicated that the immobilization of this
culture with alginate could enhance bacterial survival
under simulated intestinal condition. Higher survival was
also reported when probiotic immobilized in alginate beads
were incubated in simulated gastric and bile salt solution
(Chandramouli et al. 2004; Krasaekoopt et al. 2004; Lee

and Heo 2000; Mandal et al. 2006).
Alginate gels are stable in low pH solutions but swell in
weakly basic solutions. While the ionotropic alginate gel
formed by Ca
2?
crosslinking of carboxylate groups is
insoluble at low pH, exposure to neutral pH or higher
solubilizes the alginate (Annan et al. 2008). Alginate cap-
sules and microspheres can be used to protect cells from
the acidity of gastric juices while allowing subsequent
release in the basic environment of intestinal fluids.
Conclusions
Enterococcus faecalis KT2L24, Enterococcus durans
KT3L20, Enterococcus faecium KT4S13, Pediococcus
pentosaceus KT3CE27 and Enterococcus faecium KT8S16
were found in vitro to possess desirable probiotic properties.
These strains are good candidates for further investigation in
vivo studies to elucidate their potential heath benefits and
their application as promising probiotic strains in the feed
industry.
Acknowledgments This work was financially supported by Prince
of Songkla University through Contract No. AGR5122020037S,
Faculty of Agro-Industry and Graduate school, Prince of Songkla
University.
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