International Journal of Food Microbiology 144 (2010) 104–110

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Exopolysaccharide activities from probiotic bifidobacterium: Immunomodulatory

effects (on J774A.1 macrophages) and antimicrobial properties
Ming-Hsiu Wu a,f, Tzu-Ming Pan b,1, Yu-Jen Wu c,1, Sue-Joan Chang d, Ming-Song Chang e, Chun-Yi Hu a,f,⁎
a
Department of Nutrition and Health Science, Fooyin University, Kaohsiung County, Taiwan
b
Institute of Microbiology and Biochemistry, National Taiwan University, Taipei, Taiwan
c
Department of Beauty Science, Meiho Institute of Technology, Pingtung County, Taiwan
d
Department of Biology and Institute of Biodiversity, National Cheng Kung University, Tainan, Taiwan
e
Department of Surgery, Fooyin Hospital, Pingtung County, Taiwan
f
Research and Certificate Center of Health Food, Fooyin University, Kaohsiung County, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: Exopolysaccharides (EPSs) are a metabolite of probiotics which have gained wide interest recently, but little is
Received 7 March 2010 known about their function. EPS was isolated from Bifidobacterium longum BCRC 14634 and sterilized by
Received in revised form 5 August 2010 0.22 μm filter. The proliferation of J77A.1 macrophages and their secretion of the anti-inflammatory cytokine
Accepted 6 September 2010
interleukin-10 (IL-10) was elevated after treatment with heat-killed B. longum or 5 μg/mL EPS. The endotoxin,
lipopolysaccharides (LPS), a potent inducer of pro-inflammatory cytokine tumor necrosis factor-α (TNF-α),
Keywords:
Probiotics
significantly suppressed the growth of J77A.1 cells, and induced the secretion of TNF-α from J774A.1 cells.
Exopolysaccharide (EPS) Furthermore, 24 h pretreatment with 5 μg/ml EPS suppressed 100 ng/ml LPS-induced cell growth inhibition
Cytokine and release of TNF-α from J774A.1 cells. Additional experiments showed that 80 μg/mL EPS had antimicrobial
Macrophage activity against 7 species of food-spoilage and infection bacteria. Our results suggest that EPS from B. longum
Antimicrobial test might be useful as a mild immune modulator for macrophages, contributing to the capacity of B. longum to
fight against gastrointestinal infections, and even some food-spoilage microbe.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction a role in protection against desiccation, toxic compounds, bacter-

The probiotic effects ascribed to fermented dairy products arise
not only from whole microorganisms and wall components, but also
from metabolites such as peptides and extracellular polysaccharides
(EPS) produced during fermentation. EPSs produced by lactic acid
bacteria (LAB) have gained increasing attention due to their potential
health benefits. LAB are food-grade organisms which are generally-
recognized-as-safe (GRAS), and can produce EPSs that are potentially
useful as additives to improve texture and viscosity of natural
fermented milk products and to prevent syneresis. It has also been
suggested that some EPSs produced by LAB may confer health benefits
to the consumer.
The formation of EPSs occurs in two forms depending on location:
as a capsule (capsular polysaccharides) where the polymer is closely
associated with the bacterial surface; and as slime polysaccharide
loosely associated with the bacterial surface. Such a distinction may be
difficult since some strains release capsular polysaccharide material at
the periphery (Whitfield, 1998). For bacteria, EPSs are thought to play
⁎ Corresponding author. Department of Nutrition and Health Science, Fooyin
University, Kaohsiung County, Taiwan. Tel.: +886 7 7862240; fax: +886 7 7826740.
E-mail address: mt121@mail.fy.edu.tw (C.-Y. Hu).
1
Authors worked equal contributions.
iophages, osmotic stress, and to permit adhesion to solid surfaces and
biofilm formation (De Vuyst and Degeest, 1999).
Antimicrobial components produced by lactic acid bacteria and
bifidobacteria include organic acids, hydrogen peroxide, carbon
dioxide, diacetyl, bacteriocins, and low molecular weight antimicro-
bial substances (Ouwehand and Vesterlund, 2004), but few studies
have investigated their polysaccharide metabolites, especially
exopolysaccharide.
Interleukin-10 (IL-10), a 17- to 20-kDa glycoprotein (Moore et al.,
2001), is a potent inhibitor of the production of the pro-inflammatory
cytokines such as interferon-γ (INF-γ), tumor necrosis factor-α
(TNF-α), IL-1, IL-2, IL-6, IL-8, IL-12 and IL-18, all of which are
associated with cell-mediated immunity and inflammatory responses
(Moore et al., 2001; Rousset et al., 1992). On the other hand, IL-10 is
involved in the promotion of humoral immunity (Ghildyal et al.,
1992). IL-10 stimulates secretion of Ig G, Ig M, and Ig A by B cells
(Rousset et al., 1992), but suppresses antigen-specific IgE secretion
involving allergy (Marcelletti and Katz, 1996). IL-10 enhances B cell
survival in culture and mast cell development (Go et al., 1990;
Ghildyal et al., 1992). Mice carrying a null mutation of the IL-10 gene
(IL-10−/−) show increased spontaneous development of inflamma-
tory bowel disease and extreme susceptibility to infection-induced
immunopathology (Steidler et al., 2000; Wynn et al., 1998). In

0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2010.09.003
 M.-H. Wu et al. / International Journal of Food Microbiology 144 (2010) 104–110
contrast to IL-10, tumor necrosis factor-α (TNF-α) is a pro-
inflammatory cytokine which induces the production of terminal
inflammatory mediators, such as eicosanoid and pro-inflammatory
interleukins from inflammatory cells (Beutler and Cerami, 1989).
The chronic intestinal inflammation in Crohn's disease is thought
to be caused by dysregulated mucosal immune responses. Tradition-
ally the treatment of Crohn's disease has been the administration of
immunosuppressive or non-specific anti-inflammatory agents (Kuh-
bacher and Fölsch, 2007). More recent strategies for treatment of
Crohn's disease include attenuation of immune activation in the
gastrointestinal tract, and the use of therapeutic agents including
probiotics, IL-10, and neutralization of pro-inflammatory cytokines
(such as TNF-α) by antibodies (Bamias and Cominelli, 2006; Steidler
et al., 2000). Clinical trials of the treatment of Crohn's disease by
genetically engineered Lactococcus lactis secreting IL-10 are currently
in progress (Bamias and Cominelli, 2006). IL-10 and TNF-α are
produced by many cell types including T cells and macrophages/
monocytes (Moore et al., 2001). Macrophages recognize bacteria and
can be activated by microbial metabolites, such as polysaccharide, and
the activated macrophages kill bacteria by phagocytosis, secrete
cytokines to modulate immunity, and present antigens to helper T
cells (Serbina et al., 2008). Intestinal macrophages do not overpro-
duce pro-inflammatory cytokines in response to several inflammatory
stimuli, including microbial components (Smythies et al., 2005).
The aims of this study were to cultivate Bifidobacterium longum
BCRC 14634, produce and detect EPS, and measure the biological
activity of EPS from B. longum BCRC14634. The effect of EPS secreted
from B. longum on the activities of the murine macrophage/monocyte-
like cell line J774A.1 were investigated, including cell growth and
induction of IL-10 and TNF-α. The results of this study may provide
insight into the antimicrobial activity of EPS against pathogen and
food-spoilage bacteria.
2. Materials and methods
2.1. Bacterial strain, media, and batch fermentations
B. longum BCRC14634 was obtained from the Bioresource
Collection Research Center (BCRC), Food Industry Research and
Development Institute (Hsinchu, Taiwan), and had been isolated
from infant feces. The strain was grown in 100 mL of sterile de Man
Rogosa Sharpe (MRS) broth (De Man et al., 1960) and anaerobically
maintained by a paraffin oil overlay, rotated at 120 rpm at 37 °C.
2.2. Extraction and detection of EPS
MRS (100 mL) was inoculated (1%) with strains pregrown in MRS
medium. Broth cultures were cultivated for different times, and the
number of cells was counted with a Petroff–Hausser counting chamber
(Fisher, USA) at about 2 days of incubation using centrifuged (20 min,
at 6000 ×g) 1 mL culture samples. Two volumes of cold (4 °C) ethanol
were added to one volume of culture supernatants; the mixtures were
stored overnight at 4 °C. Precipitates were collected by centrifugation
(5 min, at 6000 ×g) and resuspended in one volume of demineralized
water. After precipitation with two volumes of cold ethanol and
centrifugation, pellets were dried at 55 °C. EPS was determined by
measuring the dry weight or total carbohydrate content of the
precipitates. The total amount of carbohydrate in the EPS was
determined using the phenol–sulfuric acid method (Dubois et al.,
1956). Pellets dissolved by four-fold volume of d′d′ water were filtered
through a 0.22 μm filter (Millipore, USA). 25 μL 80% phenol was added
to the prepared 1 mL sample which was washed rapidly in 2.5 mL 98%
H2SO4 after 10 min. The samples were measured at 450 nm optical
density (OD450) and glucose solution was used as standard.
105
2.3. Cell cultures and cell growth determination
Murine macrophage cell line, J774A.1 cells, were cultured in
Gibco™ Dulbecco's modified Eagle's medium (DMEM; Invitrogen,
Carlsbad, CA) supplemented with 10% heat-inactivated serum
(Hyclone, Logan, UT), 4 mM glutamate, antibiotics (50 U/ml penicillin
and 50 μg/ml streptomycin), and 3.7 g/L sodium bicarbonate (Merck,
Darmstadt Germany) in a 5% CO2 humidified incubator at 37 °C. For
determination of cell growth, 5 × 104 cells/well were grown in 12 well
plates, and were treated with EPS, heated-killed B. longum, or LPS 24 h
after plating. At the indicated time, supernatants of medium and cells
from the wells were harvested for measurement of cytokines, and the
number of viable cells was counted with a hemocytometer by trypan
blue (0.4% in PBS) exclusion staining. For experiments involving killed
bacteria preparations, B. longum were heat-killed at 70 °C for 30 min
as previously described (Cross et al., 2004), with 2.5 × 107 heat-killed
bacteria, representing a bacteria: cell ratio of 25:1.
2.4. IL-10 and TNF-α measurement
The IL-10 and TNF-α levels in the supernatants of medium from
J774A.1 cells were determined using the Quantikine® mouse IL-10
ELISA kit and the Quantikine® mouse TNF-α ELISA kit (R&D Systems
Inc, Minneapolis, MN, USA) according to the manufacturer's guide-
lines. Both of these kits employ the quantitative sandwich enzyme
immunoassay technique. The cell culture supernatants were incubat-
ed with affinity purified polyclonal antibody specific for mouse IL-10
or TNF-α pre-coated onto a 96 well microplate, and the concentration
of IL-10 was determined at 450 nm by an ELISA reader (μQuant;
Biotek Instruments, Inc, Winooski, VT, USA).
2.5. Antimicrobial activity of exopolysaccharide
The antimicrobial tests were carried out according to the growth
inhibition method as previously described (Forestier et al., 2001). The
following seven pathogens were purchased from the Bioresource
Collection and Research Center (BCRC), Food Industry Research and
Development Institute, Hsinchu City, Taiwan: E. coli BCRC10239,
Salmonella typhimurium BCRC10747 (ATCC14028), Staphylococcus
aureus BCRC10451 (ATCC6538P), Bacillus subtilis BCRC10029
(ATCC21778), Pseudomonas aeruginosa BCRC10261 (IFO3898), Vibrio
parhaemolyticus (ATCC17802), and Bacillus cereus (ATCC10361).
Strains to be tested were cultured overnight in nutrition broth
(Difco, USA), and 3% NaCl was added for Vibrio cultivation. The
supernatant was discarded, bacteria were washed once with
phosphate-buffered saline (PBS) and resuspended in LB. The assay
was performed by incubating 2 mL of this suspension (approximately
108 cells/mL) with 10 or 40 μg (20 and 80 ppm, respectively) of EPS
from B. longum BCRC14634 at 37 °C. Cells were observed under a
phase-contrast microscope (Nikon Eclipse-80i, Japan) and counted
using a Petroff–Hausser counting chamber. Bacterial strains were
stored in yeast–mannitol agar at 4 °C.
2.6. Statistical analysis
Data were analyzed by one way ANOVA test with SigmaStat
software (Systat Software Inc, Point Richmond, CA, USA). A P
value b 0.05 was considered to represent a statistically significant
difference between compared data sets.
3. Results
3.1. Bacteria cultivation and EPS production
The strain B. longum BCRC 14634 was studied in more detail, in
view of its superior growth and EPS production under the tested
106 M.-H. Wu et al. / International Journal of Food Microbiology 144 (2010) 104–110

conditions. To determine growth in MRS medium, the cell number

and EPS production were determined in six-hour periods during
2 days. The greatest EPS production was 4100 ± 67.2 mg/L occurred
after 24 h, and the number of B. longum cells at that time was
1.16 × 109. The greatest cell number was 1.83 × 109 after 42 h, but EPS
at this time was 3988.4 ± 51.2 mg/L, as shown in Table 1. Based on
these results, we cultivated bacteria for 24 h to produce EPS.

3.2. EPS promoted growth and LPS suppressed proliferation of J774A.1 cells

Fig. 1A shows the growth curve of J774A.1 cells. Significant elevation

was found in the number of EPS-treated cells or heat-killed B.
longum-treated cells compared to control cells at 24–48 h. The
exponential phase of J774A.1 cell proliferation occurred between 24 h
and 72 h (Fig. 1), and cells entered stationary phase after 72 h (data not
shown). During the exponential growth phase there were, respectively,
2.1 and 2.8 fold more EPS-treated cells and heat-killed B. longum-treated
cells compared to control cells at 48 h after treatment (Fig. 1B).
Lipopolysaccharide (LPS), a component of the outer wall of Gram-
negative bacteria, is an endotoxin attributed to many of the immune
responses caused by Gram-negative pathogenic bacteria (Raetz, 1990).
Like EPS, LPS is also a polysaccharide, and LPS was subsequently
employed as a study tool to compare with the effect of EPS on J774A.1
cells. LPS suppressed proliferation of J774A.1 cells, especially during the
first 48 h, and there were ~50% fewer LPS-treated cells compared to
control cells at 48 h after exposure to LPS (Fig. 1).

3.3. Induction of IL-10 and TNF-α by EPS and heat-killed B. longum in

J774A.1 cells

Induction of TNF-α by LPS was blocked by EPS pretreatment in

J774A.1 cells IL-10 has been reported to inhibit the production of TNF-α
induced by LPS in vitro and in vivo (De Waal Malefyt et al., 1991), and to
protect against LPS toxicity (De Waal Malefyt et al., 1991; Gerard et al.,
1993). Because IL-10 secretion was slowly induced in J774A.1 cells
between 24–48 h after EPS treatment (Fig. 1B), J774A.1 cells were
treated with EPS for 24 h before LPS treatment. Pretreatment with
5 μg/ml EPS suppressed 100 ng/ml of LPS-induced cell growth inhibition
and release of TNF-α from J774A.1 cells (Fig. 4). The growth of J774A.1
cells was suppressed after LPS treatment, but EPS pretreatment for 24 h
attenuated the growth-inhibitory effect of LPS (Fig. 4A). The number of
EPS-pretreated cells was 2-fold greater than the number of LPS-treated
cells at 72 h after LPS treatment (Fig. 4A). EPS pretreatment also reduced
LPS-induced secretion of TNF-α by 4–5 fold compared to LPS treatment
alone (Fig. 4B). Interestingly, simultaneous co-treatment with EPS and
LPS did not inhibit secretion of TNF-α from J774A.1 cells (data not
shown), but EPS pretreatment for 24 h before the addition of LPS
blocked the effects of LPS on J774A.1 cells, including inhibition of cell
proliferation and secretion of TNF-α.
3.4. Morphological effects of EPS, heat-killed B. longum or LPS on J774A.1
cells
The morphology of immune cells could be changed in response to
an immune regulator, such as LPS (Raetz, 1990; Beutler and Cerami,
Fig. 1. The effect of EPS, heat-killed B. longum, and LPS on growth of J774A.1 cells. (A) J774A.1
cells were grown in 12 well plates, and were cultured in the absence (control) or presence of
EPS (5 μg/ml), heat-killed B. longum (the ratio of bacteria: cell =25:1), or LPS (100 ng/ml).
(B) The relative growth of J774A.1 cells at 48 h after exposure to EPS, heat-killed B. longum or
LPS. These results were obtained as described above, and the relative growth was the
proportion % of untreated (control) cells at 48 h. Bars, means of triplicates±S.D. *P b 0.05, as
compared with the relative growth of control cells.
1989). In this study, most J774A.1 cells were round or elliptic shape,
but the number of elongated and spindle-like cells increased slightly
increased after EPS or heat-killed B. longum treatment (Fig. 5B and E).
There were observable increases in the number of elongated J774A.1
cells with dendritic processes after LPS exposure (Fig. 5C). EPS
pretreatment also decreased the LPS-induced morphological changes
(Fig. 5F). These results indicate that EPS and heat-killed B. longum
could stimulate morphological changes in J774A.1 cells, and these
effects were distinct from those induced by LPS. EPS treatment not
only prevented LPS-induced secretion of TNF-α, but also decreased
LPS-induced morphological changes in J774A.1 cells.

Table 1
Bacteria cultivation and EPS production: Strain Bifidobacterium longum BCRC 14634 was grown in MRS medium, and the cell number and EPS production were tested in six-hour
intervals for a period of 2 days.

Items Time

6h 12 h 18 h 24 h 30 h 36 h 42 h 48 h

cells/mLa 2.50 ± × 108 5.71 × 108 1.09 × 109 1.16 × 109 1.25 × 109 1.69 × 109 1.83 × 109 1.66 × 109
EPSa (mg/mL) 2053.3 ± 24.1 3483.6 ± 58.5 3806.2 ± 74.8 4100.0 ± 67.2 4033 ± 71.1 4021.5 ± 77.4 3988.4 ± 51.2 3890.7 ± 64.5
a
For the whole-amount samples tested, each counting was a triplicate evaluation of three experiments.
 M.-H. Wu et al. / International Journal of Food Microbiology 144 (2010) 104–110
3.5. Antimicrobial activity of EPS
The antibacterial activity of EPS was examined using seven different
bacterial pathogens and food-spoilage bacteria, including the following
4 Gram-negative and 3 Gram-positive species: E. coli BCRC10239, S.
typhimurium BCRC10747 (ATCC14028), P. aeruginosa BCRC10261
(IFO3898), V. parahaemolyticus ATCC17802, S. aureus BCRC10451
(ATCC6538P), B. subtilis BCRC10029, and B. cereus ATCC10361. The
viability of these microorganisms was verified during a 7 h time course
of incubation with B. longum EPS. P. aeruginosa and V. parahaemolyticus
were not apparently inhibited by 20 ppm (μg/ml) EPS treatment
(Fig. 6A). However, all of the 7 pathogens and food-spoilage bacteria
tested were inhibited by 80 ppm EPS (Fig. 6B), suggesting this could be
an appropriate dose for their inhibition compared with 20 ppm,
although EPS could not inhibit P. aeruginosa effectively. The viability of
all these microorganisms was verified during a 7-h time course of
incubation with 80 ppm EPS from B. longum BCRC 14634. No or only a
slightly inhibitory effect was observed after 3 h of incubation with
20 ppm EPS and 80 ppm EPS, but after 5 h, the growth of all the bacterial
species was observed even in the presence of 20 ppm EPS. The level of
Fig. 2. IL-10 induction by EPS or heat-killed B. longum in J774A.1 cells. (A) The kinetics
of IL-10 secretion from J774A.1 cells following exposure to EPS or heat-killed B. longum.
J774A.1 cells were grown in 12 well plates, and the supernatants were harvested from
the cell culture after treatment with EPS (5 μg/ml) or heat-killed B. longum (the ratio of
bacteria: cell = 25:1) at the indicated time. The supernatant from J774A.1 cells not
treated with any reagent served as control. (B) Comparison of IL-10 induction by EPS or
heat-killed B. longum in J774A.1 cells at 48 h. These results were obtained as described
above at 48 h, and the concentrations of IL-10 were subsequently normalized by the
number of cells in the same sample at the same time. Bars, means of triplicates ± S.D.
*P b 0.05, as compared with the control.
107
viable bacteria remained indicated that EPS treatment did not lyse the
bacteria, but rather impaired their division.
4. Discussion
Previous studies showed that EPSs from Bifidobacterium adoles-
centis M101-4 exhibited mitogenic activity in murine splenocytes and
Peyer's patch cells (Hosono et al., 1997) and antitumoral activity in
vitro, and that phosphate groups in EPSs played an important role in
the activation of macrophages and lymphocytes (Kitazawa et al.,
1998; Nishimura-Uemura et al., 2003). In this study, we found that
EPS exposure stimulated growth and induced IL-10 secretion in
J774A.1 cells (Figs. 1, 2). EPS treatment also induced lower levels of
TNF-α secretion and slight changes in the morphology of J774A.1 cells
(Figs. 3 and 5). These results indicate that EPS from B. longum might
regulate the activities of macrophages. In contrast to EPS, LPS-induced
high levels of TNF-α secretion and suppressed the proliferation of
J774A.1 cells (Figs. 1 and 3). Notably, EPS pretreatment prevented
LPS-induced release of TNF-α from J774A.1 cells, and LPS-induced
growth inhibition (Fig. 4). Because EPS and LPS are both polysacchar-
ides, they have similar structure and interact with the same receptors.
EPS may act as an LPS blocker, but does not cause severe
immunological responses. LPS is thought to be recognized by toll-
like receptors 4 (TLR4) and CD-14 for defense against bacterial
Fig. 3. Induction of TNF-α by EPS, heat-killed B. longum, or LPS in J774A.1 cells at
different times. (A) TNF-α induction by EPS, heat-killed B. longum or LPS in J774A.1 cells
at 24 h. (B) TNF-α induction by EPS, heat-killed B. longum or LPS in J774A.1 cells at 48 h.
J774A.1 cells were grown in 12 well plates, and the supernatants were harvested from
cells cultured in the absence (control) or presence of EPS (5 μg/ml), heat-killed B.
longum (the ratio of bacteria: cell = 25:1) or LPS (100 ng/ml) at the indicated time.
TNF-αconcentrations were normalized by the number of cells in the same sample at the
same time. Bars, means of triplicates ± S.D. *P b 0.05, as compared with the control.
108 M.-H. Wu et al. / International Journal of Food Microbiology 144 (2010) 104–110
Fig. 4. EPS pretreatment blocked the effect of LPS on J774A.1 cells. (A) EPS pretreatment
blocked the effect of LPS on growth of J774A.1 cells. J774A.1 cells with or without LPS
(100 ng/ml) treatment at 24 h after exposure to EPS (5 μg/ml). Points, means of triplicates
± S.D. (B) EPS pretreatment blocked LPS-induced secretion of TNF-α from J774A.1 cells.
TNF-α concentrations were normalized by the number of cells in the same sample at the
same time. Bars, means of triplicates± S.D. *P b 0.05, as compared with the control.
Fig. 5. The morphology of J774A.1 cells at 48 h after exposure to MRS, EPS, heat-killed B. longum, or LPS. (A) The control cells were not treated with any reagents. (B) The cells were
treated with EPS (5 μg/ml). (C) The cells were treated with LPS (100 ng/ml). (D) The cells were treated with MRS medium (the same volume of heat-killed B. longum). (E) The cells
were treated with heat-killed B. longum (the ratio of bacteria: cell = 25:1). (F) The cells were pretreated with EPS for 24 h and exposure to LPS (100 ng/ml) for further 24 h. The
morphology of J774A.1 cells was examined under an inverted microscope (×400).
infection, and to stimulate the activation of immune cells (Jerala,
2007).
The morphological appearances of LPS-treated J774A.1 cells were
also distinct from those of EPS-treated cells, and elongated cells with
dendritic processes were more abundant after LPS treatment
compared with untreated cells and EPS-treated cells (Fig. 5). These
findings suggest that large round macrophages possess phagocytic
activity while elongated macrophages with dendritic processes do not
(Wilkins and Jones, 1996). Because chronic bowel inflammation in
Crohn's disease is associated with impaired macrophage function
(Mpofu et al., 2007), EPS from B. longum might be applied to enhance
macrophage function in the gastrointestinal tract.
EPS may be more advantageous than treatment with whole
microorganisms in food processing because it is more resistant to high
temperature, acidic, and alkaline conditions of food processes. For
example, EPS may be used as a food additive, such as pasting agent, to
improve the mouth feel and flavor of dietary formula for the therapy
of chronic intestinal inflammation.
Forestier et al. (2001) tested the antimicrobial activity of
Lactobacillus casei subp. rhamnosus (Lc35) cell-free supernatant
against 9 human pathogenic bacteria, and the volume ratio of Lc35
supernatant to pathogenic bacteria broth was 1:1. In the present
study, we examined the antimicrobial activity of 80 ppm EPS from
Bifidobacteria longum against 4 pathogenic bacteria and 3 food-
spoilage bacteria. Our results suggest that the antimicrobial property
of Lc35 supernatant might be due to metabolites from Lc35, such as
the exopolysaccharide of Lc35.
Chiu et al. (2008) used supernatants of probiotic LAB strains with
activity against Samonella invasion in mouse intestinal epithelium
cells, and revealed the strains may be used for vegetable processing.
About the growth percentage of P. aeruginosa BCRC10261 treating
with 20 and 80 ppm EPS, the growth percentage was 661.63 ± 88.37%
and respectively for 7 h treatment of EPS, meaning it had seven fold of
bacteria compared with the control group, though the growth
percentage of 80 ppm EPS treatment 97.75 ± 6.72%. It revealed EPS
may be not a proper preservative against P. aeruginosa. As to V.
parhaemolyticus (ATCC17802), the growth percentage was still over
100% (413.95 ± 81.69%for 7 h) after treating with 20 ppm of EPS, but
the growth percentage reduced when treated with 80 ppm of EPS
(23.86 ± 3.41%for 7 h), revealed that EPS could be an natural
 M.-H. Wu et al. / International Journal of Food Microbiology 144 (2010) 104–110 109

Fig. 6. Percentage of different microbes growth after treating with EPS. Different bacteria, including E. coli, Salmonella typhimurium, Staphylococcus aureus, Bacillus subtilis,
Pseudomonas aeruginosa, Vibrio parhaemolyticus, Bacillus cereus, were treated with 20 (A) or 80 ppm (μg/ml) (B) of EPS from B. longum at 37 °C. The relative bacteria growth was the
proportion (%) of untreated (control) cells at indicated time. *P b 0.05, as compared with the relative growth of control bacteria at the same time.

preservative adding in some processing seafood against V. parhaemo- Acknowledgements

lyticus (Fig. 6).
The vast majority of immunomodulatory probiotic bacteria are
gram-positive lactic acid bacteria, lactobacilli, and bifidobacteria
(Cross et al., 2004). The mode of action of bifidobacteria and lactic
acid bacteria in the gastrointestinal tract appears to be non-specific,
increasing immune responsiveness to a wide variety of antigens (De
Roos and Katan, 2000). A previous study showed (Ouwehand et al.,
1999) that probiotics exert effects through interactions between
lymphoid tissue and intact microorganisms, fragments thereof or
metabolites produced in situ. Bacterial cell wall breakdown products
may play an important role in a number of homeostatic mechanisms
as well as in non-specific immunity (Erickson and Hubbard, 2000;
Kankaanpa et al., 2003).
Our results suggest that EPS from B. longum might be a mild
immune modulator for macrophages. This study also showed that
EPS from B. longum might increase the capacity of the host to fight
against gastrointestinal infections, and has potential application as a
natural preservative against food-spoilage bacteria. Our data also
indicate that EPS from B. longum might play roles involving the
cross-talk between probiotics and macrophages in the gastrointes-
tinal tract.
This work was supported by grant NSC 97-2313-B-242-002 from
the National Science Council, Taiwan, and 97 Cooperative Projects of
Fooyin Hospital and University AH91014, Kaohsiung County, Taiwan.
References
Bamias, G., Cominelli, F., 2006. Novel strategies to attenuate immune activation in
Crohn's disease. Current Opinion in Pharmacology 6, 401–407.
Beutler, B., Cerami, A., 1989. The biology of cachectin/TNF—a primary mediator of the
host response. Annual Review of Immunology 7, 625–655.
Chiu, H.H., Tsai, C.C., Hsih, H.Y., Tsen, H.Y., 2008. Screening from pickled vegetables the
potential probiotic strains of lactic acid bacteria able to inhibit the Salmonella
invasion in mice. Journal of Applied Microbiology 104, 605–612.
Cross, M.L., Ganner, A., Teilab, D., Fray, L.M., 2004. Patterns of cytokine induction by
Gram-positive and Gram-negative probiotic bacteria. FEMS Immunology and
Medical Microbiology 42, 173–180.
De Man, J.C., Rogosa, M., Sharpe, M.E., 1960. A medium for the cultivation of lactobacilli.
The Journal of Applied Bacteriology 23, 130–135.
De Roos, N.M., Katan, M.B., 2000. Effects of probiotic bacteria on diarrhea, lipid
metabolism, and carcinogenesis: a review of papers published between 1988 and
1998. The American Journal of Clinical Nutrition 71, 405–411.
De Vuyst, L., Degeest, B., 1999. Heteropolysaccharides from lactic acid bacteria. FEMS
Microbiology Reviews 23, 153–177.
110 M.-H. Wu et al. / International Journal of Food Microbiology 144 (2010) 104–110
De Waal Malefyt, R., Abrams, J., Bennett, B., Figdor, C.G., Vries, J.E., 1991. Interleukin 10 (IL-
10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10
produced by monocytes. The Journal of Experimental Medicine 174, 1209–1220.
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for
determination of sugars and related substances. Analytical Chemistry 38, 350–356.
Erickson, K.L., Hubbard, N.E., 2000. Probiotic immunomodulation in health and disease.
The Journal of Nutrition 130, 403S–409S.
Forestier, C., De Champs, C., Vatoux, C., Joly, B., 2001. Probiotic activities of Lactobacillus
casei rhamnosus: in vitro adherence to intestinal cells and antimicrobial properties.
Research in Microbiology 152, 167–173.
Gerard, C., Bruyns, C., Marchant, A., Abramowicz, D., Vandenabeele, P., Delvaux, A., Fiers,
W., Goldman, M., Velu, T., 1993. Interleukin 10 reduces the release of tumor
necrosis factor and prevents lethality in experimental endotoxemia. The Journal of
Experimental Medicine 177, 547–550.
Ghildyal, N., McNeil, H.P., Stechschulte, S., Austen, K.F., Silberstein, D., Gurish, M.F.,
Somerville, L.L., Stevens, R.L., 1992. IL-10 induces transcription of the gene for mouse
mast cell protease-1, a serine protease preferentially expressed in mucosal mast cells
of Trichinella spiralis-infected mice. Journal of Immunology 149, 2123–2129.
Go, N.F., Castle, B.E., Barrett, R., Kastelein, R., Dang, W., Mosmann, T.R., Moore, K.W.,
Howard, M., 1990. Interleukin 10, a novel B cell stimulatory factor: unresponsiveness
of X chromosome-linked immunodeficiency B cells. The Journal of Experimental
Medicine 172, 1625–1631.
Hosono, J., Lee, A., Amenati, M., Natsume, M., Hirayama, T., Adachi, S., Kaminogawa, 1997.
Characterization of a water-soluble polysaccharide fraction with immunopotentiating
activity from Bifidobacterium adolescentis M101-4. Bioscience, Biotechnology, and
Biochemistry 61, 312–316.
Jerala, R., 2007. Structural biology of the LPS recognition. International Journal of
Medical Microbiology 297, 353–363.
Kankaanpa, P., Sutasb, Y., Salminena, S., Isolaurib, E., 2003. Homogenates derived from
probiotic bacteria provide downregulatory signals for peripheral blood mononuclear
cells. Food Chemistry 83, 269–277.
Kitazawa, H., Harata, T., Uemura, J., Saito, T., Kaneko, T., Itoh, T., 1998. Phosphate group
requirement for mitogenic activation of lymphocytes by an extracellular phosphopo-
lysaccharide from Lactobacillus delbrueckii subsp. bulgaricus. International Journal of
Food Microbiology 40, 169–175.
Kuhbacher, T., Fölsch, U.R., 2007. Practical guidelines for the treatment of infl ammatory
bowel disease. World Journal of Gastroenterology 13, 1149–1155.
Marcelletti, J.F., Katz, D.H., 1996. IL-10 stimulates murine antigen-driven antibody
responses in vitro by regulating helper cell subset participation. Cell Immunology
167, 86–98.
Moore, K.W., de Waal Malefyt, R., Coffman, R.L., O'Garra, A., 2001. Interleukin-10 and
the interleukin-10 receptor. Annual Review of Immunology 19, 683–765.
Mpofu, C.M., Campbell, B.J., Subramanian, S., Marshall-Clarke, S., Hart, C.A., Cross, A.,
Roberts, C.L., McGoldrick, A., Edwards, S.W., Rhodes, J.M., 2007. Microbial mannan
inhibits bacterial killing by macrophages: a possible pathogenic mechanism for
Crohn's disease. Gastroenterology 133, 1487–1498.
Nishimura-Uemura, J., Kitazawa, H., Kawai, Y., Itoh, T., Oda, M., Saito, T., 2003.
Functional alteration of murine macrophages stimulated with extracellular
polysaccharides from Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1. Food
Microbiology 20, 267–273.
Ouwehand, A.C., Vesterlund, S., 2004. Antimicrobial components from lactic acid bacteria.
In: Salminen, S., VonWright, A., Ouwehand, A. (Eds.), Lactic Acid Bacteria —
Microbiological and Functional Aspects. Marcel Dekker, Inc., New York, pp. 375–395.
Ouwehand, A.C., Kirjavainen, P.V., Shortt, C., Salminen, S., 1999. Probiotics: mechanisms
and established effects. International Dairy Journal 9, 43–52.
Raetz, C.R., 1990. Biochemistry of endotoxins. Annual Review of Biochemistry 59,
129–170.
Rousset, F., Garcia, E., Defrance, T., Peronne, C., Vezzio, N., Hsu, D.H., Kastelein, R., Moore,
K.W., Banchereau, J., 1992. Interleukin 10 is a potent growth and differentiation
factor for activated human B lymphocytes. Proceedings of the National Academy of
Sciences of the United States of America 89, 1890–1893.
Serbina, N.V., Jia, T., Hohl, T.M., Pamer, E.G., 2008. Monocyte-mediated defense against
microbial pathogens. Annual Review of Immunology 26, 421–452.
Smythies, L.E., Sellers, M., Clements, R.H., Mosteller-Barnum, M., Meng, G., Benjamin, W.H.,
Orenstein, J.M., Smith, P.D., 2005. Human intestinal macrophages display profound
inflammatory anergy despite avid phagocytic and bacteriocidal activity. The Journal of
Clinical Investigation 115, 66–75.
Steidler, L., Hans, W., Schotte, L., Neirynck, S., Obermeier, F., Falk, W., Fiers, W., Remaut,
E., 2000. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10.
Science 289, 1352–1355.
Whitfield, C., 1998. Bacterial extracellular polysaccharides. Canadian Journal of
Microbiology 34, 415–420.
Wilkins, B.S., Jones, D.B., 1996. Contribution of monocyte/macrophage differentiation
to the stromal layer in human long-term bone marrow cultures. Biologicals 24,
313–318.
Wynn, T.A., Cheever, A.W., Williams, M.E., Hieny, S., Caspar, P., Kuhn, R., Muller, W.,
Sher, A., 1998. IL-10 regulates liver pathology in acute murine Schistosomiasis
mansoni but is not required for immune down-modulation of chronic disease.
Journal of Immunology 160, 4473–4480.