FEMS Microbiology Ecology 39 (2002) 67^75
www.fems-microbiology.org
In vitro investigations of the e¡ect of probiotics and prebiotics on
selected human intestinal pathogens
Laura J. Fooks *, Glenn R. Gibson
Food Microbial Sciences Unit, School of Food Biosciences, The University of Reading, Whiteknights, Reading RG6 6BZ UK
Received 27 July 2001; received in revised form 19 October 2001; accepted 19 October 2001
First published online 10 December 2001
Abstract
This study investigated the effects of selected probiotic microorganisms, in combination with prebiotics, on certain human intestinal foodborne pathogens. Probiotics grown with different carbohydrate sources were observed to inhibit growth of Escherichia coli, Campylobacter
jejuni and Salmonella enteritidis, with the extent of inhibition varying according to the carbohydrate source provided. Prebiotics identified as
being preferentially utilised by the probiotics tested were oligofructose (FOS), inulin, xylo-oligosaccharide (XOS), and mixtures of
inulin:FOS (80:20 w/w) and FOS:XOS (50:50 w/w). Two of the probiotics, Lactobacillus plantarum and Bifidobacterium bifidum were
selected for further co-culture experiments. Each was combined with the selected prebiotics, and was observed to inhibit pathogen growth
strongly. Results suggested that acetate and lactate were directly conferring antagonistic action, which was not necessarily related to a
lowering of culture pH. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Oligosaccharides ; Lactobacillus plantarum; Bi¢dobacterium bi¢dum; Escherichia coli; Campylobacter jejuni ; Salmonella enteritidis
1. Introduction
The large bowel harbours a nutritionally and physiologically diverse range of bacteria, promoting normal intestinal function, and o¡ering the host protection against infections [1]. Disruption of the colonic £ora, due to
pathogens [2,3], dietary antigens [1] or other harmful substances [4^6] can, however, lead to intestinal dysfunction.
Lactobacillus and Bi¢dobacterium, which have a long and
safe history in the manufacture of dairy products, are
traditionally included in probiotic products to help protect
against such e¡ects [7]. Both are thought to prevent the
adherence, establishment, replication and/or virulence of
speci¢c enteropathogens [8]. A number of mechanisms
have been proposed: decreasing the luminal pH via the
production of volatile short chain fatty acids (SCFA) ;
rendering speci¢c nutrients unavailable to pathogens;
and/or producing speci¢c inhibitory compounds such as
bacteriocins [9]. In the normal intestinal £ora these mech-
* Corresponding author. Tel. : +44-1189-357220;
Fax: +44-1189-357222.
E-mail address : [email protected] (L.J. Fooks).
anisms are essential to the component bacterial populations, as a means of gaining advantage over competing
bacteria.
An ability to compete for limiting nutrients is perhaps
the most important factor that determines the composition
of the gut £ora, with species that are unable to compete
being e¡ectively eliminated from the system. One of the
primary objectives of this research was to identify a synbiotic, that is, a combination of a probiotic and prebiotic
[10], which could ultimately be used for fermentation experiments and be an e¡ective antimicrobial agent against
common enteropathogens. The range of carbohydrates
tested included non-prebiotic controls such as lactitol,
starch and dextran, and prebiotics, short and long chain
fructo-oligosaccharides, xylo-oligosaccharide (XOS) and
lactulose.
In this study, a range of probiotics, were tested using
disc/spot assays for their ability to inhibit the growth of
some common enteropathogens: Campylobacter jejuni,
Escherichia coli and Salmonella enteritidis. Each probiotic
strain tested was combined with a range of prebiotic sources in an attempt to move towards identifying synbiotic
combinations. Subsequently, two of the probiotic strains,
Lactobacillus plantarum and Bi¢dobacterium bi¢dum, combined with preferred candidate prebiotics, were examined
0168-6496 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 1 9 7 - 0
FEMSEC 1310 26-2-02
68
L.J. Fooks, G.R. Gibson / FEMS Microbiology Ecology 39 (2002) 67^75
in co-culture for their ability to inhibit growth of the enteropathogens. The mechanism underlying any antimicrobial activity was addressed by monitoring changes of pH
in the culture medium and levels of SCFA at speci¢c time
intervals, when samples were removed for enumeration of
probiotic and pathogen strains.
2. Materials and methods
2.1. Growth substrates
Oligofructose (FOS) and inulin extracted from chicory
root were supplied by Orafti Ra¤nerie (Tienen, Belgium).
Inulin was 94% pure with fructose (V1%), glucose (V1%)
and sucrose (V5%) as impurities. FOS was 98% pure with
fructose (0.9%), glucose ( 6 0.1%) and sucrose (0.9%) as
impurities. XOS 35P was supplied by Suntory Ltd. (Tokyo, Japan). The XOS was obtained by enzymatically
treating xylan derived from corncobs. The ¢nal product
contained 35% w/w oligosaccharides [11]. Growth media
and supplements were obtained from Oxoid Ltd., Basingstoke, Hampshire, UK, unless otherwise stated. All other
carbohydrates and chemicals were obtained from Sigma
Chemicals Ltd., Poole, Dorset, UK. Carbohydrates used
were FOS, inulin, inulin:FOS mixture (80:20, w/w), XOS,
FOS:XOS mixture (50:50, w/w), lactulose, lactitol, starch
and dextran.
2.2. Bacterial strains
Probiotic strains tested were L. plantarum 0407 and Lactobacillus pentosus 905 (St. Ivel European Food, Swindon,
UK), Lactobacillus reuteri SD2112 (Biogaia), Lactobacillus
acidophilus La5 and B. bi¢dum Bb12 (Chr. Hansen, Denmark). Enteropathogens used were E. coli NCIMB 9517
(National Collections of Industrial and Marine Bacteria
Ltd.), C. jejuni ATCC 11351 (American Type Culture Collection) and S. enteritidis var. danysz NCTC 4444 (National Collection of Type Cultures).
2.3. Bacterial growth conditions
Probiotic strains were grown in basal medium (30 ml)
containing, in g l31 , peptone, 2.0 ; yeast extract, 2.0; NaCl,
0.1; K2 HPO4 , 0.04; KH2 PO4 , 0.04; CaCl2 W6H2 0, 0.01;
MgSO4 W7H2 0, 0.01; NaHCO3 , 2.0; Tween 80, 2 ml; hemin
(dissolved in three drops 1 M NaOH), 0.05; vitamin K1 ,
10 Wl; cysteine^HCl, 0.5; bile salts, 0.5 with various carbohydrate sources (1% w/v) anaerobically in an anaerobic
cabinet (Don Whitley, Yorkshire, UK; 10:10:80,
H2 :CO2 :N2 ). E. coli and S. enteritidis were grown aerobically in Mueller^Hinton broth. C. jejuni was grown in
Brucella broth (Difco Laboratories, Detroit, MI, USA)
supplemented with Campylobacter growth supplement
(Oxoid) under microaerobic conditions, achieved using a
variable atmosphere incubator (VAIN; Don Whitley,
Yorkshire, UK; 4:4:10:82, H2 :O2 :CO2 :N2 ).
2.4. Disc assay technique
Tryptone soya agar (TSA; 2% w/v; Oxoid Ltd., Basingstoke, UK) (15 ml), prepared and autoclaved according to
manufacturer's instructions, was poured into sterile Petri
dishes and allowed to set. Overnight cultures (109 cfu
ml31 ) of each probiotic were centrifuged at 14 000Ug for
10 min at 4³C. Sterile ¢lter paper discs (Whatman No. 2,
5 mm) were soaked (10 Wl) in one of the culture fractions
and laid upon the agar surface. Three fractions were
tested. Probiotic cells were prepared by removing supernatant and washing the cells twice with phosphate bu¡er
(pH 7.4; 1 M) before re-suspension in 200 Wl phosphate
bu¡er. Supernatant consisted of cell-free extract following
centrifugation at 14 000Ug for 10 min. The pH of the
supernatant from each probiotic culture was recorded. Absence of cells was con¢rmed by serially diluting and plating the extract ; no growth was observed. Neutralised
supernatant was prepared by neutralising to pH 7 using
1 M NaOH.
Controls used were basal medium, and acetate and lactate solutions (10 mmol l31 , pH 4.5). Four discs were
placed on each plate, one test and three controls. Tryptic
soy soft agar (10 ml ; 0.75%) inoculated with V108 cells
(0.5 ml) of the indicator organism (E. coli, S. enteritidis) in
the late exponential phase was then poured over the surface. The plates were incubated anaerobically at 37³C for
up to 48 h. The extent of inhibition was assessed after
incubation by measuring the diameter of the clear zone
surrounding each disc. Plates were prepared in triplicate,
i.e. three per fraction, for each carbohydrate. The experimental set-up was repeated in triplicate.
2.5. Overlay assay technique
The disc assay technique was modi¢ed for testing with
C. jejuni since reliable results were not obtained with the
above technique, as its growth was restricted to the base of
the inoculated agar layer. Consequently, a disc laying on
the top agar surface was ine¡ective and 10 Wl of culture
fraction was therefore spotted onto 2% (w/v) TSA, allowed to evaporate and overlain with 0.75% (w/v) agar
inoculated with 108 cells of C. jejuni.
2.6. Growth curves ^ probiotic strains
MRS broth (10 ml) was inoculated with one cryogenic
bead of each probiotic strain and incubated overnight
under anaerobic conditions (oxygen-free nitrogen) at
37³C. Basal medium (50 ml) contained in a 50-ml batch
culture fermenter was pre-reduced overnight and 0.5 g (1%
w/v) of the selected carbohydrate added. 1 ml of each
overnight culture was then inoculated, in duplicate, into
FEMSEC 1310 26-2-02
FEMSEC 1310 26-2-02
(0.0)
(0.0)
(0.7)
(2.8)
(0.7)
(1.4)
(0.5)
(0.0)
(0.7)
(1.4)
(2.1)
(0.7)
(1.4)
(0.7)
(1.4)
(0.7)
(0.0)
(0.7)
(0.7)
(0.7)
(0.0)
(1.4)
(2.1)
(0.7)
(0.7)
(0.7)
(1.4)
(0.3)
(0.7)
(0.7)
(1.4)
(0.0)
(0.7)
(0.7)
(1.4)
(0.7)
(0.7)
(0.7)
(1.4)
(0.0)
(2.8)
(0.7)
(0.5)
(0.0)
Cultures were fractioned into cells, supernatant (S/N) and neutralised (pH 7.0) supernatant (N. S/N). Results are diameter of the inhibition zone (mm) surrounding the disc. Results are corrected for the
disc size (5 mm) for E. coli and S. enteritidis. Values in parentheses are S.E.M. of nine determinations. Asterisks denote signi¢cantly greater inhibitory e¡ect of supernatant compared to cells or neutralised supernatant.
(2.1)
(0.7)
(0.7)
(0.0)
(0.7)
16.0* (1.4)
5.5 (0.7)
11.5* (0.7)
12.5* (3.5)
13.0* (1.4)
15.5* (0.7)
6.0 (1.4)
5.5 (0.7)
4.5 (0.7)
^
10.0* (4.2)
(0.0)
(0.0)
0.5
4.0
^
2.5
8.5
5.5
6.0
1.5
^
^
2.0
(0.0)
^
4.0
^
0.5
5.0
^
0.5
1.5
4.0
4.5
4.0
(0.5)
(1.4)
(0.0)
(0.7)
^
0.5
6.0
2.0
5.5
^
2.5
6.0
6.5
7.5
6.0
^
^
2.5
1.5
^
^
1.0
0.5
^
2.5
2.0
(0.5)
(0.7)
(0.5)
0.5
2.5
0.5
^
1.5
^
3.0
1.5
^
1.5
^
4.5 (0.7)
4.0 (0.0)
5.5(0.7)
1.0 (0.0)
4.5 (2.1)
5.5 (0.7)
6.0 (1.4)
4.0 (1.4)
5.5 (0.7)
4.0 (0.0)
3.5 (0.7)
(0.7)
(1.4)
^
4.5
2.0
^
3.5
^
3.5
4.5
2.5
2.0
0.5
(1.4)
(0.7)
4.0
2.5
^
2.0
2.5
4.5
3.0
1.5
^
2.5
3.0
5.0 (0.0)
12.0* (0.0)
9.5* (0.7)
5.5* (2.1)
8.0* (0.0)
7.0 (0.0)
3.5 (0.7)
^
^
3.0 (0.0)
4.0 (1.4)
(2.1)
3.5
^
^
0.5
^
3.5
2.5
3.0
3.0
7.0
4.5
(0.7)
(1.4)
(0.7)
(3.5)
(0.7)
(0.0)
(0.7)
(0.0)
(0.7)
(1.4)
(0.0)
2.5
5.0
0.5
3.5
8.5
7.0
2.5
1.0
2.5
3.0
1.0
8.0* (1.4)
7.5* (3.5)
9.5 (0.7)
5.5 (0.7)
10.5 (0.7)
9.0 (0.0)
6.5 (0.7)
^
6.0 (1.4)
6.5* (3.5)
9.0* (2.8)
(1.4)
(2.1)
(0.7)
(0.7)
(0.0)
(0.7)
(0.0)
(0.0)
(0.0)
2.0
4.5
5.5
2.5
5.0
4.5
4.0
6.0
3.0
^
4.0
FOS
Inulin
XOS
FOS:XOS
C. jejuni
FOS
FOS:XOS
S. enteritidis FOS
Inulin
Inulin :FOS
XOS
FOS:XOS
E. coli
S/N
Cells
S/N
L. reuteri SD2112
Cells
N. S/N
S/N
L. acidophilus La5
Cells
N. S/N
S/N
L. pentosus 905
Cells
N. S/N
S/N
To assess survival of each pathogenic strain at di¡erent
initial culture pH values, overnight cultures were prepared
as previously described. Mueller^Hinton broth (50 ml) and
Brucella supplemented broth (50 ml) were prepared, and
the pH adjusted using 4 M HCl to give a range of initial
pH values from 1 to 7. These were incubated overnight
under aerobic (E. coli and S. enteritidis) or microaerobic
(C. jejuni) atmospheric conditions. 1 ml of each overnight
culture was then inoculated, in triplicate, into 50 ml of
pH-adjusted medium (Mueller^Hinton for E. coli and
S. enteritidis and Brucella for C. jejuni) and these were
incubated under the same conditions for each organism.
1 ml aliquots were removed at hourly intervals and used to
prepare a dilution series, which was plated onto a suitable
agar medium ; MacConkey No. 3 (E. coli), Brilliant green
(S. enteritidis) or CBFS+CCDA supplement (C. jejuni).
Plates were incubated for up to 48 h, when colonies
were enumerated.
L. plantarum 0407
2.8. Growth curves ^ pathogenic strains: pH e¡ect
Cells
Mueller^Hinton broth (10 ml) was inoculated with one
cryogenic bead of either E. coli or S. enteritidis and incubated overnight under aerobic conditions at 37³C. Brucella broth (50 ml) supplemented with Campylobacter growth
supplement was inoculated with one cryogenic bead of
C. jejuni and incubated overnight under microaerobic conditions at 37³C. Basal medium (50 ml), contained in a
50-ml batch culture fermenter, was pre-reduced overnight
and 0.5 g (1% w/v) of the selected carbohydrate added.
1 ml of each overnight culture (109 ^1010 cells ml31 ) was
then inoculated, in triplicate, into 50 ml carbohydrate-containing basal medium and cultures were incubated under
the same conditions for each organism. 1 ml aliquots were
removed at hourly intervals and used to prepare a dilution
series, which was plated onto a suitable agar medium
(Oxoid Manual); MacConkey No. 3 (E. coli), Brilliant
green (S. enteritidis) or Campylobacter blood-free speci¢c
(CBFS)+CCDA supplement (C. jejuni). Plates were incubated for up to 48 h, when colonies were enumerated for
each time point.
Table 1
Plate assay inhibition of E. coli, C. jejuni and S. enteritidis by selected probiotic microorganisms grown in broth culture with a range of carbohydrate sources
2.7. Growth curves ^ pathogenic strains: carbohydrate
e¡ect
N. S/N
B. bi¢dum Bb12
N. S/N
50 ml carbohydrate-containing basal medium and cultures
were incubated under the same conditions. The optical
density at 650 nm of each culture was determined at
hourly intervals for up to 24 h. This was repeated four
times and mean values plotted. Subsequently, speci¢c
growth rates of the probiotics in each carbohydrate medium were calculated. Exponential phase growth was derived from growth plots of the probiotics (not shown).
69
7.5 (0.7)
1.5 (0.7)
^
5.5 (0.7)
9.5 (0.7)
12.5 (0.7)
2.5 (1.4)
2.5 (0.7)
2.0 (0.0)
^
1.0 (0.0)
L.J. Fooks, G.R. Gibson / FEMS Microbiology Ecology 39 (2002) 67^75
70
L.J. Fooks, G.R. Gibson / FEMS Microbiology Ecology 39 (2002) 67^75
2.9. Co-culture experiments
On the basis of disc/spot assay results, two of the probiotic strains, L. plantarum 0407 and B. bi¢dum Bb12,
were selected for further study because of their greater
ability to inhibit pathogenic organisms. Overnight cultures
(109 cells ml31 ) of each probiotic strain and each enteropathogenic strain were prepared. MRS broth (10 ml) was
inoculated with 0.01 g of lyophilised powder of each probiotic strain. The cultures were incubated overnight under
anaerobic conditions (10:10:80, H2 :CO2 :N2 ) at 37³C.
Mueller^Hinton broth (10 ml) was inoculated with one
cryogenic bead of either E. coli or S. enteritidis, and incubated overnight under aerobic conditions at 37³C. Brucella broth (50 ml) supplemented with Campylobacter
growth supplement (Oxoid) was inoculated with one cryogenic bead of C. jejuni, and incubated overnight under
microaerobic conditions at 37³C. Basal medium (50 ml)
was pre-reduced overnight and 0.5 g (1% w/v) of the selected carbohydrate added. Carbohydrates used were:
starch, FOS P95, XOS 35P, mixtures of inulin:FOS
(80:20 w/w) and FOS :XOS (50:50 w/w). 1 ml of each
overnight culture (V108 ^109 cells) was then inoculated,
in triplicate, into 50 ml carbohydrate-containing basal medium and cultures were incubated under anaerobic conditions. Agar plates (Oxoid; Beerens, 1990) for the enumeration of each organism were prepared as appropriate.
Growth media used were: Rogosa (L. plantarum), Beerens
(B. bi¢dum), MacConkey No. 3 (E. coli), CBFS+CCDA
supplement (C. jejuni) and Brilliant green (S. enteritidis).
Samples were removed at 0, 3, 6, 9 and 24 h. 1 ml was
used to prepare a dilution series, which was then plated, in
triplicate, onto the appropriate agar. For example, if
L. plantarum 0407 and E. coli 9517 were co-cultured, the
sample was plated onto Rogosa and MacConkey agar.
Plates were incubated for up to 48 h, under appropriate
atmospheric conditions (L. plantarum and B. bi¢dum : anaerobically, E. coli and S. enteritidis: aerobically, C. jejuni:
microaerobically) and colonies enumerated. The pH of the
culture medium was monitored throughout the fermentation period. Each experiment was repeated in triplicate. A
further 1 ml sample was also removed for the analysis of
SCFAs (lactate and acetate), according to the method described by Parham and Gibson [12].
Fig. 1. Growth rates (h31 ) of probiotic and enteropathogenic bacteria
utilising various carbohydrate sources. In, Inulin; In:FOS, mixture
80:20 w/w; FOS:XOS, mixture 50:50 w/w
2.10. Statistical analyses
Data were statistically analysed using pairwise Student's
t-test to evaluate signi¢cance of inhibition observed (disc/
spot assays) or changes in bacterial numbers (co-culture
experiments).
3. Results
3.1. Plate assays
Basal medium gave no inhibition of the enteropathogens tested. Acetate and lactate solutions gave inhibition
zones of 10 mm ( þ 1.8 mm) and 7 mm ( þ 0.6 mm) respectively. Use of lactulose, lactitol, starch and dextran as
carbohydrate sources was generally ine¡ective in inducing
any inhibitory e¡ects from the probiotic strains.
L. plantarum 0407 and L. pentosus 905, combined with
FOS, inulin, XOS, and mixtures of inulin:FOS and FOS:
XOS, were e¡ective in inhibiting growth of E. coli and
S. enteritidis (Table 1). The cell supernatant (cell-free extract) consistently conferred a signi¢cantly greater inhibitory e¡ect than either the cells (P 6 0.01) or neutralised
supernatant (P 6 0.05) fractions. FOS and FOS:XOS
were the only carbohydrate sources where inhibition of
C. jejuni was observed with L. plantarum, whilst with
Table 2
pH values of supernatant fraction of the overnight cultures of probiotics grown with various carbohydrate sources
Probiotic strain
FOS
L.
L.
L.
L.
B.
4.29
4.35
4.31
4.54
4.27
plantarum 0407
pentosus 905
acidophilus La5
reuteri SD2112
bi¢dum Bb12
In
( þ 0.05)
( þ 0.08)
( þ 0.06)
( þ 0.03)
( þ 0.02)
4.52
4.77
5.10
5.26
4.49
( þ 0.04)
( þ 0.05)
( þ 0.02)
( þ 0.04)
( þ 0.06)
In:FOS
XOS
5.00
5.02
5.38
5.24
5.72
4.92
4.83
5.15
5.85
4.47
( þ 0.03)
( þ 0.03)
( þ 0.04)
( þ 0.08)
( þ 0.06)
( þ 0.02)
( þ 0.09)
( þ 0.03)
( þ 0.06)
( þ 0.03)
FOS:XOS
Lactulose
Lactitol
Starch
Dextran
4.78
4.72
4.63
4.89
4.17
5.98
6.14
6.27
6.23
6.08
6.55
6.39
6.09
6.86
6.11
6.56
6.54
6.33
6.78
6.05
6.73
6.84
6.41
6.67
6.18
( þ 0.03)
( þ 0.05)
( þ 0.07)
( þ 0.05)
( þ 0.03)
( þ 0.04)
( þ 0.05)
( þ 0.04)
( þ 0.07)
( þ 0.03)
( þ 0.05)
( þ 0.04)
( þ 0.05)
( þ 0.02)
( þ 0.04)
( þ 0.04)
( þ 0.06)
( þ 0.08)
( þ 0.04)
( þ 0.01)
( þ 0.07)
( þ 0.03)
( þ 0.05)
( þ 0.04)
( þ 0.03)
Values in parentheses are average þ S.E.M. of triplicate determinations. In: Inulin; In:FOS, mixture 80:20 w/w; FOS:XOS, mixture 50:50 w/w.
FEMSEC 1310 26-2-02
L.J. Fooks, G.R. Gibson / FEMS Microbiology Ecology 39 (2002) 67^75
71
Fig. 2. Survival of (a) E. coli, (b) C. jejuni and (c) S. enteritidis at di¡erent initial culture pH values. Samples were removed at hourly intervals and
enumerated using on selected agars. Data are averaged ( þ S.E.M.) from triplicate determinations.
L. pentosus, the most e¡ective carbohydrate sources were
FOS, inulin, XOS and mixtures thereof. Inhibition was
signi¢cant for FOS (P 6 0.01) and FOS:XOS (P 6 0.05).
The improved antimicrobial e¤cacy of the probiotic with
these carbohydrate sources was repeated with L. acidophilus La5 and L. reuteri SD2112 although the magnitude of
inhibition was less than that of the other two lactobacilli.
In contrast, B. bi¢dum Bb12 was most e¡ective, combinations with FOS, inulin, XOS and related mixtures imparting high inhibition levels against all three enteropathogens.
Again, the cell-free fraction of the Bb12 culture was most
e¡ective at inhibiting pathogen growth, which was signi¢cant compared to the other cell fractions (P 6 0.05).
The pH of the cell-free extract was recorded for each
probiotic strain grown with the various carbohydrates
(Table 2). pH was approximately 7.0 prior to inoculation.
In general, the largest decrease in pH was observed when
FOS and the mixture of FOS:XOS (50:50) was used as the
carbohydrate source, with the variation being strain-dependent.
3.2. Probiotic growth curves
Highest growth rates of the lactobacilli were obtained in
media that contained glucose as the carbon and energy
source. For the bi¢dobacterial strain, the mixture of
FOS :XOS gave optimal growth. L. plantarum 0407 and
B. bi¢dum Bb12 generally grew faster than the other
strains, regardless of the carbohydrate source used. In
general, the oligosaccharides FOS, XOS or their mixtures
were fermented preferably to all other carbohydrate sources (Fig. 1).
3.3. Enteropathogen growth curves
Each of the three enteropathogens was tested for its
FEMSEC 1310 26-2-02
72
L.J. Fooks, G.R. Gibson / FEMS Microbiology Ecology 39 (2002) 67^75
Table 3
Inhibition of enteropathogens by probiotic L. plantarum 0407 in co-culture experiments
Carbohydrate
source
Change in numbers, pH or concentration (0-24 h)
Starch
E. coli
C. jejuni
S. enteritidis
Probiotic
numbers
Pathogen
numbers
Culture pH
[Acetate] mM
[Lactate] mM
Probiotic
numbers
Pathogen
numbers
Culture pH
[Acetate] mM
[Lactate] mM
Probiotic
numbers
Pathogen
numbers
Culture pH
[Acetate] mM
[Lactate] mM
FOS
9
Inulin:FOS
9
10
FOS:XOS
XOS
+1.62U10
( þ 1.33U109 )
+4.50U109
( þ 4.33U108 )
+0.03 ( þ 0.04)
+2.32 ( þ 0.33)
+16.64 ( þ 2.12)
+9.98U1012 ( þ 0.00)
+2.14U10
( þ 2.00U109 )
38.25U108 **
( þ 2.83U107 )
31.15 ( þ 0.06)
+2.24 ( þ 0.18)
+25.38 ( þ 0.55)
+1.00U1013 ( þ 0.00)
+1.35U10
( þ 2.83U109 )
+3.27U109
( þ 1.31U109 )
31.02 ( þ 0.03)
+2.35 ( þ 0.47)
+9.77 ( þ 2.02)
+1.00U1013 ( þ 0.00)
+1.40U10
( þ 2.02U107 )
32.37U107
( þ 6.66U107 )
31.00 ( þ 0.24)
+1.23 ( þ 0.04)
+23.06 ( þ 0.51)
+1.00U1013 ( þ 0.00)
+9.40U108
( þ 2.11U109 )
32.25U108
( þ 1.98U107 )
30.94 ( þ 0.22)
+1.20 ( þ 0.09)
+17.62 ( þ 0.75)
+1.00U1013 ( þ 0.00)
31.29U107
( þ 1.78U105 )
30.41 ( þ .021)
+2.73 ( þ 0.33)
+17.39 ( þ 2.86)
+1.21U1010
( þ 7.36U109 )
+7.42U1010
( þ 8.08U109 )
30.35 ( þ 0.04)
+2.58 ( þ 0.43)
+11.55 ( þ 5.36)
32.09U109 *** ( þ 0.00) 31.74U109 ( þ 0.00)
31.30U109 ( þ 0.00)
31.89U109 ( þ 0.00)
32.20 ( þ 0.06)
+2.26 ( þ 0.13)
+20.17 ( þ 0.41)
+2.15U1010
( þ 1.70U1010 )
37.25U1010 ***
( þ 0.00)
+1.79 ( þ 0.04)
+0.94 ( þ 0.10)
+20.89 ( þ 0.70)
32.33 ( þ 0.25)
+1.87 ( þ 0.30)
+21.27 ( þ 0.89)
+1.76U108
( þ 5.65U107 )
32.68U107
( þ 1.04U107 )
31.87 ( þ 0.08)
+2.04 ( þ 0.16)
+20.74 ( þ 0.38)
32.13 ( þ 0.18)
+1.92 ( þ 0.05)
+20.68 ( þ 1.29)
+6.10U109
( þ 9.19U108 )
37.15U106
( þ 2.26U106 )
31.43 ( þ 0.03)
+1.81 ( þ 0.05)
+13.46 ( þ 0.33)
31.99 ( þ 0.14)
+2.27 ( þ 0.11)
+11.37 ( þ 1.12)
+2.20U1010
( þ 1.48U1010 )
38.08U108
( þ 2.09U102 )
31.55 ( þ 0.04)
+1.52 ( þ 0.22)
+11.06 ( þ 1.62)
8
Data presented are changes in parameter between 0 and 24 h inoculation. Probiotic and enteropathogen numbers were enumerated at various time
points after inoculation. Results are cfu ml31 ( þ S.E.M.) averaged from six determinations. For culture pH and acetate and lactate concentrations
(mM), 1-ml samples were removed at speci¢ed intervals and parameters determined. Results are average ( þ S.E.M.) of triplicate determinations. + and
3 denote an increase or decrease respectively of the described parameter. A signi¢cant decrease of pathogen numbers from baseline (0 h fermentation)
is denoted; **P 6 0.01, ***P 6 0.001.
ability to grow with various carbohydrate sources (Fig. 1).
E. coli grew well on glucose. S. enteritidis also utilised
glucose e¡ectively, although growth rate was approximately half that observed for E. coli. C. jejuni failed to
grow on any of the carbohydrates. The e¡ect of pH on
growth of the pathogens was also investigated. Initial medium pH of 3 or below completely inhibited growth of
E. coli (Fig. 2a) and of C. jejuni (Fig. 2b) and S. enteritidis
(Fig. 2c) at initial pH values of 4 and below.
3.4. Co-culture investigation
Both L. plantarum 0407 (Table 3) and B. bi¢dum Bb12
(Table 4) survived in all culture conditions tested such
that, generally, their numbers after 24 h fermentation
were maintained. B. bi¢dum numbers were enhanced by
1-log value in the presence of FOS and up to 2 logs
when a FOS:XOS (50:50) mixture was used as the carbohydrate source. An increase in probiotic numbers after 24 h
fermentation was observed when both L. plantarum and
B. bi¢dum were grown with C. jejuni, again regardless of
the carbohydrate source used.
L. plantarum combined with FOS was the most e¡ective
at inhibiting pathogen growth (Table 3). A signi¢cant,
6-log (P 6 0.01) decrease in E. coli numbers was observed
when FOS was used, whilst after the same time period,
C. jejuni and S. enteritidis were undetectable (P 6 0.001).
B. bi¢dum combined with FOS:XOS proved an e¡ective
synbiotic combination (Table 4). C. jejuni and S. enteritidis
were decreased to below detectable levels (P 6 0.001),
whilst a 2-log decrease in E. coli numbers was observed.
Changes in pH during the 24-h fermentation were observed but were not signi¢cant for either L. plantarum
0407 (Table 3) or B. bi¢dum Bb12 (Table 4). No de¢nitive
correlation could be made between the observed decrease
in pH and the extent of inhibition. Culture pH decrease
was generally observed after fermentation for 3 h, yet a
lowering of pathogen numbers was not observed until fermentation for 9^24 h. Furthermore, when B. bi¢dum Bb12
was co-cultured with E. coli in the presence of XOS, a pH
decrease was observed, even though there was no evidence
of antimicrobial activity.
With L. plantarum, the ¢nal concentration of lactate
varied depending on the carbohydrate source provided,
ranging from 12^17 mM with starch and inulin :FOS, to
20^25 mM with FOS and FOS:XOS (Table 3). For
B. bi¢dum, acetate concentrations were consistently higher
than levels of lactate by the end of the fermentation period
(Table 4). Both FOS and FOS :XOS gave rise to higher
levels of acetate, reaching approximately 20^30 mM 24 h
after inoculation.
FEMSEC 1310 26-2-02
L.J. Fooks, G.R. Gibson / FEMS Microbiology Ecology 39 (2002) 67^75
73
Table 4
Inhibition of enteropathogens by probiotic B. bi¢dum Bb12 in co-culture experiments
Change in numbers, pH or concentration (0^24 h)
Carbohydrate source
Starch
E. coli
C. jejuni
S. enteritidis
Probiotic
numbers
Pathogen
numbers
Culture pH
[Acetate] mM
[Lactate] mM
Probiotic
numbers
Pathogen
numbers
Culture pH
[Acetate] mM
[Lactate] mM
Probiotic
numbers
Pathogen
numbers
Culture pH
[Acetate] mM
[Lactate] mM
FOS
7
+1.69U10
( þ 5.99U107 )
32.56U108
( þ 2.61U108 )
+0.16 ( þ 0.13)
+14.02 ( þ 0.57)
+9.05 ( þ 1.34)
+1.00U1013
( þ 1.11U105 )
31.26U108
( þ 4.92U107 )
30.49 ( þ 0.73)
+10.82 ( þ 1.85)
+10.93 ( þ 0.92)
+3.68U108 ( þ 0.00)
31.66U108
( þ 1.11U105 )
+0.09 ( þ 0.16)
+10.65 ( þ 1.25)
+11.40 ( þ 1.50)
Inulin :FOS
9
7
FOS:XOS
9
+2.02U108
( þ 6.78U107 )
36.68U107
( þ 4.08U107 )
30.97 ( þ 0.21)
+11.15 ( þ 1.18)
+18.29 ( þ 1.40)
+1.00U1013 ( þ 0.00)
+1.89U10
( þ 1.20U109 )
31.92U107
( þ 6.46U106 )
31.46 ( þ 0.09)
+28.39 ( þ 1.32)
+12.64 ( þ 0.72)
+1.00U1013 ( þ 0.00)
+5.27U10
( þ 3.09U107 )
34.80U107
( þ 4.89U107 )
31.02 ( þ 0.52)
+22.70 ( þ 1.07)
+14.52 ( þ 0.53)
+1.00U1013 ( þ 0.00)
32.29U108 **
( þ 1.24U109 )
32.20 ( þ 0.18)
+25.82 ( þ 0.97)
+12.80 ( þ 1.92)
+1.42U109
( þ 3.91U108 )
31.36U109 ( þ 0.00)
32.38U108 ** ( þ 0.00) 32.77U108 *** ( þ 0.00) 32.07U108
( þ 5.03U108 )
32.06 ( þ 0.19)
32.43 ( þ 0.26)
31.97 ( þ 0.51)
+21.71 ( þ 2.05)
+26.19 ( þ 1.04)
+15.19 ( þ 2.49)
+14.23 ( þ 2.00)
+16.50 ( þ 1.28)
+11.01 ( þ 1.27)
+8.84U107
+8.75U108
+1.94U107
( þ 5.95U107 )
( þ 2.07U108 )
( þ 9.00U106 )
33.19U107
31.70U109 *** ( þ 0.00) 33.05U108
( þ 1.99U107 )
( þ 2.10U108 )
31.38 ( þ 0.64)
31.37 ( þ 0.13)
30.96 ( þ 0.12)
+22.98 ( þ 1.23)
+26.26 ( þ 3.03)
+12.81 ( þ 3.98)
+17.12 ( þ 2.25)
+17.45 ( þ 3.20)
+11.49 ( þ 1.62)
31.59 ( þ 0.08)
+21.55 ( þ 1.97)
+13.08 ( þ 0.25)
+1.26U10
( þ 2.18U108 )
31.25U106
( þ 6.13U105 )
30.77 ( þ 0.12)
+14.63 ( þ 0.47)
+29.11 ( þ 2.38)
+1.00U1013 ( þ 0.00)
XOS
Data presented are changes in parameter between 0 and 24 h inoculation. Probiotic and enteropathogen numbers were enumerated at various time
points after inoculation. Results are cfu ml31 ( þ S.E.M.) averaged from six determinations. For culture pH and acetate and lactate concentrations
(mM), 1-ml samples were removed at speci¢ed intervals and parameters determined. Results are average ( þ S.E.M.) of triplicate determinations. + and
3 denote an increase or decrease respectively of the described parameter. A signi¢cant decrease of pathogen numbers from baseline (0 h fermentation)
is denoted; **P 6 0.01, ***P 6 0.001.
4. Discussion
All of the probiotic strains tested using plate assays
o¡ered some inhibition of each of the pathogenic strains,
E. coli, S. enteritidis and C. jejuni. The extent of inhibition
was dependent on the probiotic strain, such that L. plantarum 0407 and B. bi¢dum Bb12 tended to inhibit pathogen growth to a greater extent than that observed for the
other strains included, particularly with E. coli. These results, in this respect, compound the ¢ndings of a number
of investigations, which used similar pure culture in vitro
methodologies to establish the antimicrobial potential of
lactobacilli and bi¢dobacteria. Jacobsen et al. [13] used the
spot assay to examine the capabilities of 47 strains of
Lactobacillus spp. to inhibit a range of pathogenic organisms, including E. coli and Salmonella typhimurium.
Twenty eight of the strains tested inhibited E. coli growth,
whilst 12 of the 47 strains tested inhibited growth of
S. typhimurium. A plethora of information surrounds the
proposed probiotic activity of L. reuteri [14], primarily
ascribed to production of an antimicrobial protein called
reuterin [15,16], whose synthesis requires glycerol in the
growth medium [17]. This was omitted from media used
in the current investigation, since it is not known how
much glycerol would be present in the gut, to become
available for utilisation by L. reuteri, in the production
of reuterin [14]. Six B. bi¢dum strains, tested using well
di¡usion assays, inhibited growth of E. coli and to a lesser
extent, Salmonella typhosa [18,19]. Gibson and Wang [20]
reported the strain-dependent ability of eight di¡erent species of bi¢dobacteria, including B. bi¢dum, to inhibit the
growth of a range of pathogenic bacteria, including E. coli,
a Salmonella spp., and a Campylobacter spp.
The antimicrobial potential exhibited by each of the
probiotics used here appeared to depend on the carbohydrate source used. FOS, inulin, XOS, and mixtures of
FOS :XOS (50:50 w/w) and inulin:FOS (80:20 w/w) all
caused greater inhibition than lactulose, lactitol, starch
and dextran, perhaps suggesting a structure-to-function
relationship in terms of the prebiotic used. The type of
bond linking the component monomers, in view of speci¢c
cleavage enzymes being required for fermentation of the
carbohydrate, may e¡ect fermentation rate, and thereby
determine the speed at which potential inhibitory metabolic end products are released. Chain length of the carbohydrate is also likely to be a contributory factor, since long
chain oligosaccharides, with multiple branching, require
more enzymatic hydrolysis by the organisms before its
complete fermentation.
The major metabolic end products of lactobacilli and
bi¢dobacterial fermentations are acetate and lactate [21],
leading to a decrease in the culture pH. In both plate assay
and co-culture experiments, the pH of the cell-free extract
was lower when FOS, inulin and XOS, and mixtures there-
FEMSEC 1310 26-2-02
74
L.J. Fooks, G.R. Gibson / FEMS Microbiology Ecology 39 (2002) 67^75
of were provided as the carbohydrate sources. The extent
of inhibition with these prebiotic carbohydrates was often
correspondingly increased, suggesting that a possible
mechanism of antimicrobial action may be attributable
to the low culture pH. However, since some results contradicted this ¢nding, it cannot be de¢nitively stated that low
pH acts as the principal inhibitory parameter. The enhanced antimicrobial e¡ect of FOS and FOS :XOS may
be attributed to the action of the synbiotic since numbers
of the pathogen decreased in co-cultures, but not in monocultures of the pathogen. Con¢rmatory evidence of the
inability of E. coli, C. jejuni and S. enteritidis to withstand
an acidic environment was obtained (Fig. 2) where these
bacteria failed to proliferate at the pH environment
(pH95.0) normally induced by bi¢dobacterial and lactobacilli fermentation. Similar ¢ndings have been observed
for B. bi¢dum 1452 [19], an L. plantarum strain [22] and a
bi¢dobacterial strain B. longum [23], where a decrease in
pH during incubation time increased the antagonistic
properties against E. coli, Staphylococcus aureus, Klebsiella
pneumoniae, and a number of clostridia and bacteroides
species, including Clostridium perfringens and Bacteroides
fragilis.
The toxicity of fermentation acids at a low pH has been
traditionally explained by the transmembrane £ux of undissociated acids, dissociation of the acids in the more
alkaline cytoplasm, and metabolic uncoupling [24]. However, Russell and Diez-Gonzalez [25] suggested that the
mechanistic explanation lies with the pH gradient-mediated anion accumulation within the bacterial cytoplasm.
Fermentation acid dissociation in the more alkaline interior causes an accumulation of the anionic species, and
this accumulation is dependent on a pH gradient across
the membrane. Where culture pH of the cell-free extract
decreased to pH 4.17, when short chain carbohydrates had
been utilised, this would theoretically induce a considerable pH gradient across the pathogenic organism membrane, thus allowing accumulation of the fermentation
acids in the cytoplasm. Furthermore, since the cell-free
extract promoted the greatest bactericidal e¡ect, it can
be assumed that the fermentation acids accumulated in
this fraction.
In conclusion, this study has shown that lactobacilli and
bi¢dobacteria species can inhibit some important pathogenic species. This antagonism was in£uenced by the carbohydrate provided in vitro. The inhibitory mechanism
underlying the e¡ect has been initially addressed, and a
strong case has been presented for the production of
SCFA as the underlying mechanism of inhibition of enteropathogens.
Acknowledgements
L.J.F. was a recipient of a PhD studentship from St.
Ivel European Food, Wootton Bassett, UK.
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