Journal of the Science of Food and Agriculture J Sci Food Agric 82:781±789 (online: 2002)

DOI: 10.1002/jsfa.1095

In vitro fermentation of cereal dietary fibre

carbohydrates by probiotic and intestinal
bacteria
Ross Crittenden,* Sirpa Karppinen, Suvi Ojanen, Maija Tenkanen,
Richard Fagerström, Jaana Mättö, Maria Saarela, Tiina Mattila-Sandholm and
Kaisa Poutanen
VTT Biotechnology, PO Box 1500, FIN-02044 VTT, Finland

Abstract: A range of probiotic and other intestinal bacteria were examined for their ability to ferment
the dietary ®bre carbohydrates b-glucan, xylan, xylo-oligosaccharides (XOS) and arabinoxylan. b-
Glucan was fermented by Bacteroides spp and Clostridium beijerinckii but was not fermented by
lactobacilli, bi®dobacteria, enterococci or Escherichia coli. Unsubstituted xylan was not fermented by
any of the probiotic bacteria examined. However, many Bi®dobacterium species and Lactobacillus
brevis were able to grow to high yields using XOS. XOS were also ef®ciently fermented by some
Bacteroides isolates but not by E coli, enterococci, Clostridium dif®cile, Clostridium perfringens or by
the majority of intestinal Lactobacillus species examined. Bi®dobacterium longum strains were able to
grow well using arabinoxylan as the sole carbon source. These organisms hydrolysed and fermented
the arabinosyl residues from arabinoxylan but did not substantially utilise the xylan backbone of the
polysaccharide. Arabinoxylan was not fermented by lactobacilli, enterococci, E coli, C perfringens or
C dif®cile and has potential to be an applicable carbohydrate to complement probiotic Bif longum
strains in synbiotic combinations.
# 2002 Society of Chemical Industry

Keywords: cereal; dietary ®bre; b-glucan; xylan; arabinoxylan; xylo-oligosaccharides; fermentation; probiotic;
prebiotic; synbiotic; intestine

INTRODUCTION oligosaccharides has been demonstrated to promote

The term `dietary ®bre' encompasses a heterogeneous
range of complex plant polysaccharides that are not
substantially digested in the small intestine and pass
through to the colon. The impact of dietary ®bre on
the maintenance of human health has attracted
considerable scienti®c interest in the past three
decades. One of the many health-providing roles
attributed to cereal dietary ®bre through epidemiolo-
gical studies is the prevention of colorectal cancer.1,2
Although the mechanisms of protection afforded by
dietary ®bre are still speculative, many hypotheses
involve bene®ts stemming from fermentation of the
®bre carbohydrates by intestinal bacteria. Proposed
mechanisms include the supply of the colonic epithe-
lium with short-chain fatty acids and suppression of
microbial protein metabolism, bile acid conversion
and other toxigenic bacterial reactions.1,4±6
Some fermentable carbohydrates reaching the colon
can also provide a selective advantage for the
proliferation of particular bacterial groups within the
intestinal microbiota that are bene®cial to the host.
For example, consumption of inulin and fructo-
increases in the population of bi®dobacteria in the
colon.7 Bi®dobacteria and lactobacilli are considered
bene®cial genera within the human intestinal micro-
biota and are hence the predominant bacteria used as
probiotics. Probiotics are live microbial food supple-
ments that are consumed in order to provide a health
bene®t to the host by contributing to the maintenance
of a favourable intestinal microbial balance.8 Non-
digestible fermentable substrates that can speci®cally
promote the growth and/or activity of endogenous
probiotic bacteria within the intestinal tract are termed
prebiotics.9 There is an obvious potential for synergy
between probiotics and prebiotics, and therefore foods
containing both types of ingredients are often called
synbiotics.
Included in the major groups of bacteria involved in
carbohydrate fermentation in the intestinal tract are
species from the genera Bacteroides, Bi®dobacterium,
Clostridium and Lactobacillus.10 The types of bacteria
that can ef®ciently ferment particular dietary carbohy-
drates will in¯uence the type of primary fermentation
end-products produced as well as potentially affect the

* Correspondence to: Ross Crittenden, DSTO, PO Box 4331, Melbourne, Victoria 3001, Australia
E-mail: ross.crittenden@dsto.defence.gov.au
Contract/grant sponsor: TEKES, the National Technology Agency of Finland
(Received 6 April 2001; revised version received 22 October 2001; accepted 14 January 2002)

# 2002 Society of Chemical Industry. J Sci Food Agric 0022±5142/2002/$30.00 781

R Crittenden et al
size and activity of speci®c bacterial populations within
the intestinal microbiota. Therefore different dietary
®bre carbohydrates fermented by different bacterial
populations may produce different functional effects
in the host. In cereals, arabinoxylans and b-glucans are
the major dietary ®bre constituents that are fermen-
table by bacteria in the human gastrointestinal
tract.11,12 The proportion, structure and molecular
weight of these polysaccharides vary between different
cereal crops.13 In general, cereal b-glucans are
unsubstituted glucose polymers with a mixture of b-
(1±3) and b-(1±4) glycosidic linkages,14 while cereal
arabinoxylans consist of a backbone of (1±4)-linked b-
D-xylose residues that are either non-, singularly or
doubly substituted with predominantly terminal a-L-
arabinose moieties.13
Although there has been considerable research into
the fermentation of non-digestible oligosaccharides by
probiotic bacteria,15 and dietary ®bre fermentation by
the consortia of intestinal bacteria as a whole,16±19
relatively few reports have focused on the fermentation
of speci®c cereal dietary ®bre polysaccharides by
individual probiotic and intestinal bacteria. Funda-
mental knowledge of the fermentative activity of
speci®c intestinal bacterial species will enable a better
understanding of the fermentation of complex carbo-
hydrates by the intestinal microbiota and the function-
ality of dietary ®bres.
In the current investigation the ability of a range of
probiotic and intestinal bacteria to ferment cereal ®bre
carbohydrates was studied. This included preliminary
examination of the mechanisms used by the bacteria
for substrate hydrolysis and fermentation. The aim
was to provide a better understanding of colonic
fermentation of speci®c ®bre polysaccharides and to
determine if particular cereal carbohydrates have the
potential to act as prebiotic substrates.
EXPERIMENTAL
Micro-organisms and growth media
The probiotic and intestinal bacteria used in this
investigation are listed in Tables 1 and 2 (see Results
section). All were revived from frozen stock cultures
stored at 80 °C by culturing on agar plates containing
10 g l 1 glucose as the carbon source. The bacteria
were incubated on the plates at 37 °C for 3 days under
anaerobic conditions, with the exception of Escherichia
coli which was incubated aerobically. The nutrient
media bases used to grow the bacteria were essentially
free of extraneous fermentable carbohydrates. For
bi®dobacteria the nutrient base was Reinforced
Clostridial Medium (RCM) which was modi®ed by
the omission of starch, glucose and agar. The base
medium for lactobacilli and enterococci was MRS (de
Man, Rogosa, Sharpe) modi®ed by the removal of
glucose. Bacteroides and clostridia were grown using
Peptone Yeast Extract Medium (PYE)20 modi®ed by
the omission of glucose and the substitution of yeast
nitrogen base (Difco, USA) in place of yeast extract. E
782
coli was grown using a minimal medium containing
20 g l 1 Trypticase casein (BBL, USA), 5 g l 1 NaCl,
4 g l 1 K2HPO4 and 1.5 g l 1 KH2PO4. All media were
sterilised by autoclaving at 121 °C for 15 min. All
cultures were tested for contamination by growing
them on agar media with the appropriate growth
nutrients (as described above) and examining colony
morphologies.
Carbon substrates
The ability of the bacteria to ferment indigestible
cereal ®bre polysaccharides as well as their component
monosaccharides was investigated. The monosacchar-
ides used were D-glucose, D-xylose and L-arabinose.
The polysaccharides examined were arabinoxylan (rye
arabinoxylan, Megazyme, Ireland), relatively unsub-
stituted xylan (from oat spelts, Sigma, USA) and b-
glucan (medium-viscosity barley b-glucan, Mega-
zyme, Ireland). Additionally, fructo-oligosaccharides
(FOS) and xylo-oligosaccharides (XOS) were investi-
gated as growth substrates. The FOS sample used was
Raftilose P95 (ORAFTI, Belgium), a mixture of b-(2±
1)-linked fructans ranging in size from degree of
polymerisation (dp) 2 to dp 6 produced from the
controlled enzymatic hydrolysis of inulin extracted
from chicory. Raftilose P95 contains only a small
amount of glucose, fructose and sucrose (<5%) as
contaminating sugars. The XOS substrate was Xylo-
oligo 70 (Suntory, Japan). This product is speci®ed to
contain approximately 70% dry weight b-xyl-(1±4)-
oligosaccharides ranging in size from dp 2 to dp 5. The
remaining carbohydrate content is predominantly
xylose (approximately 30% w/w).
Fermentations
Prior to fermentation experiments the bacteria were
pre-cultured twice in 5 ml of the appropriate nutrient
medium containing 10 g l 1 glucose as the carbon
source. Pre-cultures were incubated at 37 °C for 24 h.
Both pre-cultures and fermentations were conducted
in an anaerobic atmosphere of 85% N2, 10% H2 and
5% CO2 at 37 °C with no pH control or agitation.
Fermentations were inoculated with a 5% (v/v)
inoculum and were conducted in glass test tubes with
a working volume of 10 ml. All the bacteria listed in
Tables 1 and 2 were grown in duplicate fermentations
in the appropriate base medium containing separately
5 g l 1 glucose, xylose, arabinose, b-glucan, arabin-
oxylan and XOS. Additionally, to con®rm that
negligible growth occurred from use of extraneous
carbon sources present in the base medium, each
bacterium was also grown in a control fermentation
containing the base medium with no added carbon
source.
Bi®dobacterium longum VTT E-96664 and Bi®do-
bacterium longum VTT E-96702 were additionally
grown in 50 ml fermentations in Schott bottles with
5 g l 1 arabinose, xylose, XOS, FOS, arabinoxylan and
xylan as carbon substrates. The fermentation condi-
tions were the same as for the 10 ml fermentations.
J Sci Food Agric 82:781±789 (online: 2002)
 Fermentation of cereal ®bre carbohydrates by intestinal bacteria

Table 1. Growth of lactobacilli and bifidobacteria on cereal fibre carbohydrates and a commercial xylo-oligosaccharde mixture (Xylo-oligo 70)

Growth on Growth on Growth on Growth on

Organism Strain b-glucan xylan Xylo-oligo 70 arabinoxylan
Lactobacillus acidophilus VTT E-94507a ‡ ( )*
Lactobacillus amylovorus VTT E-981145a ‡ ( )*
Lactobacillus brevis PEL1b ‡‡‡ (‡)*
Lactobacillus crispatus VTT E-96729a n
Lactobacillus fermentum VTT E-78077a ‡‡ ( )*
Lactobacillus fermentum VTT E-71033a ‡
Lactobacillus johnsonii VTT E-97978a ‡ ( )*
Lactobacillus johnsonii VTT E-97797a ‡ ( )*
Lactobacillus paracasei VTT E-94510a ‡
Lactobacillus plantarum VTT E-71034a ‡
Lactobacillus plantarum VTT E-79098a ‡
Lactobacillus plantarum VTT E-78076a ‡ ( )*
Lactobacillus reuteri Ing 1c ‡ ( )*
Lactobacillus rhamnosus Lc 705d ‡
Lactobacillus rhamnosus VTT E-97800a ‡ ( )*
Lactobacillus rhamnosus VTT E-96666a ‡ ( )*
Lactobacillus salivarius VTT E-97798a ‡ ( )*
Bi®dobacterium infantis Bb-02g ‡
Bi®dobacterium adolescentis VTT E-981074a ‡‡
Bi®dobacterium adolescentis VTT E-991436a n ‡‡‡ (‡)* ‡
Bi®dobacterium angulatum ATCC 27535e n ‡‡‡ (‡)*
Bi®dobacterium bi®dum VTT E-97795a ‡
Bi®dobacterium bi®dum Bb-11g ‡
Bi®dobacterium breve VTT E-981075a ‡
Bi®dobacterium breve CIP 64.68f ‡ ( )*
Bi®dobacterium catenulatum ATCC 27539e n ‡‡ (‡)*
Bi®dobacterium gallicum ATCC 49850e n ‡‡
Bi®dobacterium infantis VTT E-97796a ‡
Bi®dobacterium lactis Bb-12g ‡‡‡ (‡)*
Bi®dobacterium lactis VTT E-97847a ‡‡‡ (‡)*
Bi®dobacterium longum VTT E-96664a ‡‡ (‡)* ‡‡‡
Bi®dobacterium longum VTT E-96702a ‡‡ (‡)* ‡‡‡
Bi®dobacterium longum CSCC 5532h ‡ ( )* ‡‡‡
Bi®dobacterium pseudocatenulatum ATCC 27919e n ‡‡ (‡)*
Bi®dobacterium pseudolongum ATCC 25526e ‡‡‡ (‡)*
Relative growth yield compared to growth on glucose: ` ' = no growth; `‡' = 0±40% of the OD on glucose; `‡‡' = 40±80% of the OD on glucose; `‡‡‡' = 80±120%
of the growth on glucose; `n' = not performed.
* (‡) and ( ) represent utilisation and non-utilisation respectively of xylo-oligosaccharides as con®rmed by HPLC analysis.
a
VTT Biotechnology, Finland.
b
University of Helsinki, Finland.
c
Ingman Foods, Finland.
d
Valio, Finland.
e
American Type Culture Collection, USA.
f
Collection of Bacterial Strains of Institut Pasteur, France.
g
Chr Hansen, Denmark.
h
CSIRO Starter Culture Collection, Australia.

Measurement of growth on glucose equals 100%:

The level of bacterial growth was determined by
measuring the optical density of samples taken
immediately following inoculation and after 24 and
48 h of fermentation. The optical densities of 200 ml
culture samples, dispensed into the wells of a 96-well
microplate, were measured at 540 nm using a Multi-
scan EX microplate reader (Labsystems, Finland).
The growth yield for each organism on the various
substrates was calculated relative to its growth yield on
glucose. The calculation was performed using the
following formula, where biomass yield from growth
biomass yield on substrate X compared to
yield on glucose ˆ ‰…A B†=…C B†Š  100%
where A is the net change in OD540 of culture grown
on substrate X, B is the net change in OD540 of culture
grown without added carbon substrate and C is the net
change in OD540 of culture grown on glucose.
In the 50 ml fermentations with Bi®dobacterium
longum VTT E-96664 and Bi®dobacterium longum
VTT E-96702 the optical density was monitored

J Sci Food Agric 82:781±789 (online: 2002) 783

R Crittenden et al

Table 2. Growth of enterococci, bacteroides, clostridia and Escherichia coli on cereal fibre carbohydrates and a commercial xylo-oligosaccharide mixture (Xylo-
oligo 70)

Growth on Growth on Growth on Growth on

Organism Strain b-glucan xylan Xylo-oligo 70 arabinoxylan
Enterococcus faecalis VTT E-97801a ‡
Enterococcus faecalis VTT E-97802a ‡‡( )*
Enterococcus faecium VTT E-981096a ‡( )*
Enterococcus faecium VTT E-981097a ‡
Bacteroides fragilis ANH 11543b ‡ ‡( )* ‡
Bacteroides fragilis RHI 3001b ‡‡ ‡‡‡(‡)* ‡
Bacteroides fragilis AHN 2981b ‡ ‡‡( )* ‡
Bacteroides fragilis AHN 2898b ‡ ‡‡( )* ‡
Bacteroides thetaiotaomicron RHI 4171b ‡ ‡‡‡(‡)* ‡
Bacteroides thetaiotaomicron AHN 1368b ‡‡‡ ‡‡ ‡
Bacteroides vulgatus RHI 3621b ‡ ‡ ‡
Bacteroides vulgatus AHN 413b ‡ ‡‡‡( )* ‡‡
Clostridium beijerinckii VTT E-97426a ‡ ‡ ‡‡‡(‡)* ‡
Clostridium perfringens VTT E-98861a n n ‡( )*
Clostridium dif®cile AHS 7589b n n ‡‡( )*
Escherichia coli VTT E-76039a ‡‡( )*
Escherichia coli IHE 13047b ‡‡( )*
Escherichia coli IHE 50817b ‡‡( )*
Escherichia coli IHE 50763b ‡‡( )*
Relative growth yield compared to growth on glucose: ` ' = no growth; `‡' = 0±40% of the OD on glucose; `‡‡' = 40±80% of the OD on glucose; `‡‡‡' = 80±120%
of the growth on glucose; `n' = not performed.
* (‡) and ( ) represent utilisation and non-utilisation respectively of xylo-oligosaccharides as con®rmed by HPLC analysis.
a
VTT Biotechnology, Finland.
b
National Public Health Institute, Finland.

throughout the fermentations, except for growth on
arabinoxylan and xylan. In fermentations with glucose,
FOS, arabinoxylan and xylan as carbon substrates the
viable counts of the bacteria were also monitored
throughout by plating 10-fold serial dilutions on
modi®ed RCM agar containing 10 g l 1 glucose as
the carbon source. The plates were incubated anaero-
bically for 3 days at 37 °C. The culture medium pH
was measured at the end of all the fermentations.
Cooled hydrolysates were diluted to between 5 and
200 mg l 1 and ®ltered using 0.45 mL ®lters. Quanti-
tative carbohydrate analysis was then conducted by
HPLC according to the method described previously,
with the exception that elution was performed using
2.5 mM NaOH up to 32 min followed by a column
wash of 100 mM NaOH/300 mM sodium acetate
followed by 300 mM NaOH.

Carbohydrate analysis RESULTS

Free monosaccharides, disaccharides and oligosac-
charides (up to dp 6) in the fermentation samples were
quanti®ed by HPLC with pulsed amperometric
detection using a DX 500 system (Dionex, USA)
equipped with a Dionex ED 40 electrochemical
detector. The column used was a Dionex CarboPac
PA1. Isocratic elution was conducted for the ®rst
23 min using 2.5 mM NaOH, after which a linear
gradient up to 100 mM NaOH was applied for 17 min.
The ¯ow rate throughout was 1 ml min 1 and the oven
temperature was 30 °C. External standards were used
to quantify glucose, fructose, sucrose, arabinose,
xylose and XOS (xylobiose, xylotriose, xylotetraose,
xylopentaose (Megazyme, County Wicklow, Ireland)).
The internal standard was fucose.
The concentration and monosaccharide composi-
tion of the xylan and arabinoxylan in fermentation
samples were determined by analysis of neutral sugars
following acid hydrolysis.21 The carbohydrates were
hydrolysed by boiling the samples for 2 h in 1 M H2SO4.
The ability of a range of potentially probiotic and other
intestinal bacteria to grow on indigestible carbohy-
drates from cereals is displayed in Tables 1 and 2. b-
Glucan from barley was fermented by all the Bacter-
oides isolates examined in this investigation and by
Clostridium beijerinckii. However, it did not support the
growth of any of the lactobacilli, bi®dobacteria,
enterococci or E coli strains studied when present as
the sole carbon source.
C beijerinckii was the only organism able to grow on
xylan from oat spelts, but the growth yield was
relatively low compared to growth on glucose. In
contrast, a number of bacteria were able to grow using
Xylo-oligo 70. Analysis of the sugar content of Xylo-
oligo 70 revealed that the level of free monosacchar-
ides was greater than 40% of total sugars present,
rather than the smaller proportion speci®ed by the
product supplier (Table 3). The free monosaccharides
were predominantly xylose, with a small amount of
glucose also present. HPLC analysis of the growth

784 J Sci Food Agric 82:781±789 (online: 2002)

 Fermentation of cereal ®bre carbohydrates by intestinal bacteria

Table 3. Utilisation of the carbohydrates in a commercial xylo-oligosaccharide product (Xylo-oligo 70) during fermentation by selected probiotic and intestinal
bacteria. The data represent the results of a single experiment

Xylo-oligosaccharides a
Organism Strain Ara Xyl Xyl2 Xyl3 Xyl4 Xyl5
1
Initial carbohydrate concentrations (g l )
0.04 1.19 1.71 0.29 0.09 0.01
Carbohydrate concentrations following fermentation (g l 1)
Lactobacillus brevis PEL1 0 0 0 0 0.04 0
Bi®dobacterium adolescentis VTT E-991436 0 0.74 0.04 0.01 0 0.01
Bi®dobacterium lactis VTT E-97847 0.05 1.35 0.07 0.01 0.02 0.02
Bi®dobacterium longum VTT E-96702 0.01 1.44 0.04 0.02 0.03 0.02
Bi®dobacterium longum CSCC 5532 0.01 0 1.61 0.43 0.37 0.08
Bi®dobacterium pseudolongum ATCC 25526 0.01 1.50 0.04 0.01 0.03 0.01
Bacteroides fragilis RHI 3001 0 2.74 0 0 0.10 0
Bacteroides vulgatus AHN 413 0.03 0.38 1.53 0.30 0.12 0.02
Clostridium beijerinckii VTT E-97426 0.03 0.44 0.01 0.02 0.02 0.04
Escherichia coli AHN 13047 0 0 1.56 0.27 0.08 0.01
a
Xyl2 = xylobiose; Xyl3 = xylotriose; Xyl4 = xylotetraose; Xyl5 = xylopentaose.

media before and following fermentation was therefore Arabinoxylan was utilised as a growth substrate
used to con®rm utilisation of the XOS for a number of most ef®ciently by strains of Bif longum (Table 1).
the organisms examined. Although the majority of the Except for Bif adolescentis E-991436 which displayed
bacteria tested were able to display some growth on slight growth on arabinoxylan, the other species of
Xylo-oligo 70, HPLC analysis showed that growth in bi®dobacteria examined could not utilise this carbo-
many cases was due to utilisation of the contaminating hydrate as a carbon substrate. Lactobacilli, entero-
monosaccharides and not consumption of XOS cocci, E coli, C perfringens and C dif®cile also failed to
(Tables 1 and 2). XOS were used by Lactobacillus grow when arabinoxylan was the sole carbon source
brevis, a number of bi®dobacteria, bacteroides and C (Tables 1 and 2). Bacteroides strains generally grew
beijerinckii. However, the oligosaccharides were not only slightly on the arabinoxylan, although one strain,
utilised by other Lactobacillus species or by the B vulgatus AHN 413, grew moderately well. B vulgatus
Enterococcus, E coli, C dif®cile and C perfringens isolates AHN 413 and C beijerinckii utilised both the arabinose
examined. and xylose components of arabinoxylan, although
A few selected examples of the quantitative analysis incompletely fermented the polysaccharide (data not
of XOS in fermentations prior to and after growth shown). Both organisms accumulated free arabinose
(Table 3) show that in addition to interspecies and xylose during fermentation of arabinoxylan,
differences in the pattern of carbohydrate utilisation, indicating that polysaccharide hydrolysis was not
differences were also observed at the strain level. For
example, Bif longum CSCC 5532 was able to utilise
xylose well but did not ferment XOS. In contrast, Bif
longum VTT E-96702 fermented XOS but did not
ferment xylose well. Indeed, free xylose accumulated
during fermentation of XOS by this strain and in
fermentations of XOS by Bif lactis VTT E-97847 and
Bif pseudolongum ATCC 25526 (Table 3). Bacteroides
fragilis RHI 3001 also hydrolysed the XOS but
accumulated free xylose in the spent medium, showing
that the substrate was not completely fermented. In
contrast, the other three strains of B fragilis examined
were unable to hydrolyse and ferment XOS (Table 2).
For some Bi®dobacterium isolates, such as Bif
angulatum ATCC 27535 and Bif longum VTT E-
96664 (Fig 1), the growth yield was high on both
xylose and Xylo-oligo 70. However, many bi®dobac-
teria, including Bif adolescentis VTT E-981074, Bif
catenulatum ATCC 27539, Bif lactis VTT E-97847
and Bif pseudolongum ATCC 25526, failed to grow to Figure 1. Comparative growth of different Bifidobacterium species on
any signi®cant degree on xylose yet displayed relatively (from left to right) xylose, arabinose, xylo-oligosaccharides (Xylo-oligo 70)
high growth yields on Xylo-oligo 70. and arabinoxylan. The data represent the results of a single experiment.

J Sci Food Agric 82:781±789 (online: 2002) 785

R Crittenden et al

rate-limiting in the fermentation. B vulgatus AHN 413

additionally accumulated xylobiose and xylotriose in
the spent medium (data not shown).
Analysis of the monosaccharide composition of
arabinoxylan fermented by two strains of Bif longum
revealed that these organisms used the arabinose side-
chain component of the polysaccharide but did not
ef®ciently utilise the xylose making up the polysac-
charide backbone (Table 4). Additionally, neither
bacterium could grow using relatively unsubstituted
xylan from oat spelts. Both strains were able to rapidly
and completely ferment free arabinose when this was
the sole carbon source at 5 g l 1 (Fig 2 and Table 4). In
contrast, free xylose was fermented slowly by Bif
longum VTT E-96664 and not fermented to any
signi®cant degree by Bif longum VTT E-96702 (Fig 2).
The speci®c growth rate of the Bif longum strains
was comparatively slow on arabinoxylan compared to
growth on arabinose, XOS and FOS (Table 5). Both
strains had higher speci®c growth rates (Table 5) and
produced slightly higher growth yields (Fig 2) on FOS
than XOS. Although Bif longum VTT E-96664 was
able to grow on xylose to yields equivalent to that seen
for growth on Xylo-oligo 70, the speci®c growth rate
on xylose was low and there was a comparatively long
lag phase for growth on this sugar. Like a number of
the other Bi®dobacterium isolates examined, Bif longum
VTT E-96702 did not grow to any signi®cant degree
using xylose but was able to grow well using XOS.

DISCUSSION
Knowledge of the fermentative capacity of individual
species within the intestinal microbiota assists in the
understanding of mechanisms of polysaccharide fer-
mentation in the human colon and the effect different
non-digestible carbohydrates have on population Figure 2. Growth curves for two strains of Bifidobacterium longum (A, VTT
dynamics in the intestinal microbiota. In the current E-96702; B, VTT E-96664) during the fermentation of carbohydrate
investigation, numerically dominant saccharolytic in- substrates present in cereals: circles, no carbon; open squares, arabinose;
testinal bacterial species were examined for their full squares, fructo-oligosaccharides; full triangles, xylose; open triangles,
ability to grow using the major fermentable polysac- xylo-oligosaccharides. The data represent the results of a single
experiment.
charide components of cereal dietary ®bres as carbon
substrates. Additionally, growth on these substrates by
the potentially harmful intestinal bacteria C dif®cile, C plant polysaccharides by Bacteroides spp,22±24 the
perfringens and E coli was investigated. Bacteroides isolates examined in the current investiga-
In agreement with other studies of utilisation of tion were able to grow using arabinoxylan as a

Following Following
fermentation fermentation
Initial by Bif longum by Bif longum
Substrate concentration VTT E-96664 VTT E-96702
Arabinose 4.55 0 0
Table 4. Utilisation of the free
monosaccharides arabinose and
Xylose 4.93 0.09 4.75
xylose and the polysaccharides Arabinoxylan (monosaccharide content)
arabinoxylan and xylan in Xylose 2.28 1.84 2.34
fermentations by two strains of Arabinose 1.42 0.21 0.23
Bifidobacterium longum. Sugar Xylan (monosaccharide content)
concentrations are in g l 1. The data Xylose 3.85 3.71 3.96
represent the results of a single Arabinose 0.42 0.34 0.34
experiment

786 J Sci Food Agric 82:781±789 (online: 2002)


Speci®c growth
Substrate
Glucose
Arabinose
Xylose
XOSa
Table 5. Growth of two strains of FOSa
Bifidobacterium longum on a variety of Arabinoxylan
carbohydrates present in cereals. The Xylan
data represent the results of a single
a
experiment Abbreviations FOS and XOS refer to fructo-oligosaccharides and xylo-oligosaccharides respectively.
substrate, although growth yields were considerably
lower than for growth on glucose. All the Bif longum
strains and one of the Bif adolescentis strains examined
grew using rye arabinoxylan. Examination of the
arabinoxylan following fermentation by the Bif longum
strains showed that these bacteria almost completely
utilised the arabinose component of the polysacchar-
ide but did not ef®ciently utilise the xylose backbone.
Additionally, this species did not grow when relatively
unsubstituted xylan (from oat spelts) was the sole
carbon source. This indicates that Bif longum produces
one or more a-L-arabinofuranosidases able to cleave
the arabinose side-chains from arabinoxylan but does
not produce signi®cant b-D-xylanase activity able to
hydrolyse the xylan backbone and make this portion of
the polysaccharide available for fermentation. An
arabinofuranosidase has previously been isolated from
Bif adolescentis (DSM 20083) which was able to
ferment arabinoxylan.25 In the current investigation,
utilisation of arabinoxylan by Bif adolescentis appeared
to be strain-variable. Bif longum has also been observed
to grow using wheat arabinoxylan,26 which indicates
that arabinoxylan from different cereal sources may be
fermented by this species.
It is probable that hydrolysis of the arabinosyl
groups from polymeric arabinoxylan occurs extracel-
lularly with the arabinose released from the polysac-
charide then imported into the cell. Consistent with
this hypothesis, all the Bi®dobacterium strains able to
grow using arabinoxylan were able to grow well using
free arabinose as a carbon source. In contrast,
utilisation of free xylose and XOS was variable among
these organisms.
In the current investigation, arabinoxylan was not
fermented by the other species of bi®dobacteria
examined or by any of the lactobacilli. Importantly,
it was not fermented by the potentially deleterious
intestinal bacteria E coli, C perfringens and C dif®cile.
Arabinoxylan may therefore prove to be an applicable
polysaccharide to complement Bif longum probiotics in
synbiotic combinations.
Xylan was not utilised as a carbon source by any of
the lactobacilli or bi®dobacteria examined in the
current investigation. A previous study27 also demon-
strated that growth on xylan was rare amongst
bi®dobacteria, although some strains of Bif catenula-
J Sci Food Agric 82:781±789 (online: 2002)
Fermentation of cereal ®bre carbohydrates by intestinal bacteria
Bif longum VTT E-96664 Bif longum VTT E-96702
Final Speci®c growth Final
rate (h 1) culture pH rate (h 1) culture pH
0.36 4.32 0.33 4.33
0.26 4.28 0.32 4.28
0.10 4.26 0.03 6.38
0.19 4.52 0.21 4.50
0.31 4.89 0.23 5.09
0.09 5.59 0.09 5.61
0.01 6.35 0 6.54
tum and Bif pseudocatenulatum did utilise this substrate.
In contrast to growth on xylan, many Bi®dobacterium
isolates were able to ef®ciently ferment xylo-oligosac-
charides. Indigestible XOS have previously been
observed to provide a suitable growth substrate for
bi®dobacteria26,28±30 and have been proposed as
potential prebiotics. In feeding trials in rodents29 and
in a small, linear, uncontrolled trial in humans,28
feeding of XOS appeared to relatively selectively
increase the numbers of bi®dobacteria in faeces.
However, larger and more rigorously controlled cross-
over trials are required to con®rm this effect. In the
current study and in other in vitro studies,26,28±30 XOS
were utilised well by some Bacteroides isolates in
addition to bi®dobacteria. In contrast to the observa-
tions of Jaskari et al,31 no utilisation of XOS was
observed for the C dif®cile isolate AHS 7589 in the
current investigation. This supports the observation by
Okazaki et al 32 that C dif®cile does not ferment XOS.
In the current study, C perfringens, E coli, Enterococcus
faecalis, Enterococcus faecium and the majority of
Lactobacillus species were unable to ef®ciently utilise
XOS as a carbon source. The results of this study and
other in vitro studies26,28±30 indicate that XOS are
ef®ciently fermented by only a limited number of the
dominant bacterial species in the intestinal tract. The
results of the current investigation support the notion
that further assessment of these oligosaccharides as
potential prebiotic substrates is warranted.
Interestingly, a number of the bi®dobacteria grew to
high cell yields on XOS but did not grow well on
xylose. This suggests that some bi®dobacteria import
XOS before hydrolysing them and do not possess
ef®cient membrane transport mechanisms for free
xylose. Similar phenomena have been observed in
bi®dobacteria for other non-digestible oligosacchar-
ides and their constituent monosaccharides31±33 and
may re¯ect evolutionary adaptation to the negligible
availability of free simple sugars in the colon. Import-
ing oligosaccharides before hydrolysing them may also
minimise loss of substrate to competing cross-feeding
bacteria in the intestinal ecosystem. In the intestinal
milieu it is possible that bi®dobacteria could utilise
XOS released from xylan by the action of xylanases
produced by other intestinal bacteria.
Some lactobacilli and bi®dobacteria have been
787
R Crittenden et al
shown to grow well using b-gluco-oligosaccharides
produced from partially hydrolysed cereal b-glu-
can.30,34 However, in the current investigation, none
of the lactobacilli or bi®dobacteria examined could
utilise barley b-glucan as the sole carbon source,
indicating that they do not possess glycosidases
capable of hydrolysing this polysaccharide. Therefore
mixed linkage b-glucans present in cereals are unlikely
to directly provide a carbon source to promote the
proliferation of probiotic bacteria in the intestinal
tract. However, lactobacilli and bi®dobacteria may be
able to cross-feed on oligomers resulting from the
hydrolysis of b-glucans by other intestinal bacteria
such as bacteroides, which grew using this polymer in
the current investigation and have previously been
shown to hydrolyse b-glucans.35
In terms of health functionality, larger, more slowly
fermentable polysaccharides may provide an advan-
tage over the rapidly fermented oligosaccharides
currently used as prebiotics, in that they provide a
carbohydrate source for SCFA production and sup-
pression of protein metabolism more distally in the
colon. In vitro fermentation studies using faecal
inocula have demonstrated that arabinoxylan is
fermented relatively slowly compared to inulin and
b-glucan,36 and studies in pigs37,38 indicate that
arabinoxylan is relatively slowly fermented in the
intestinal tract. In the current investigation the speci®c
growth rate of Bif longum on arabinoxylan was low
relative to growth on FOS and XOS. While the current
study suggests that arabinoxylan from rye may have
potential as a prebiotic substrate for the proliferation
of Bif longum, a numerically dominant Bi®dobacterium
species in the adult human colon,39 pure culture in
vitro studies cannot predict which bacterial species will
play the major role in fermentation of complex
carbohydrates in the intestinal tract. Polysaccharide
degradation and fermentation in the colon is almost
certainly a co-operative process involving consortia of
different bacterial species. To assess the prebiotic
potential of arabinoxylan and XOS, further studies are
required to investigate the fermentation of these
carbohydrates in the intestinal milieu where substrate
availabilities, nutrient competition and cross-feeding,
and bacterial population levels and niches will
in¯uence bacterial metabolism, population dynamics
and functional effects in the host.
ACKNOWLEDGEMENT
This work was supported by TEKES, the National
Technology Agency of Finland.
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