ARTICLE IN PRESS

International Dairy Journal 17 (2007) 770–775

www.elsevier.com/locate/idairyj

Functional activity of commercial prebiotics

J. Huebner, R.L. Wehling, R.W. Hutkins
Department of Food Science and Technology, 338 Food Industry Complex, University of Nebraska-Lincoln, Lincoln, NE 68583-0919, USA
Received 13 June 2006; accepted 3 October 2006

Abstract

This work established a quantitative score to describe the extent to which prebiotics (fructooligosaccharides, inulin, and
galactooligosaccharides) support selective growth of lactobacilli and bifidobacteria. The prebiotic activity assay was based on the
change in cell biomass after 24 h of growth of the probiotic strain on 1% prebiotic or 1% glucose relative to the change in cell biomass of
a mixture of enteric strains grown under the same conditions. From the biomass data, a prebiotic activity score was calculated for five
lactobacilli and five bifidobacteria. In general, the scores were dependent on the probiotic bacterial strain tested and the type of prebiotic
carbohydrate utilized. The highest score was obtained for Lactobacillus paracasei 1195 grown on inulin (1.17), and the lowest score was
for Bifidobacterium bifidum NCI grown on galactooligosaccharides (1.24). Results reported here provide a basis for evaluating and
optimizing combinations of probiotics and prebiotics for applications as synbiotics.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Fructooligosaccharides; Galactooligosaccharides; Inulin; Prebiotics; Probiotics

1. Introduction the form of synbiotics, are now recognized as having the

Fructooligosaccharides (FOS), inulin, galactooligosac-
charides, and other related carbohydrates have received
considerable attention due to the health benefits they are
believed to confer on the host. These so-called prebiotic
carbohydrates are defined as ‘‘nondigestible food ingredi-
ent(s) that beneficially affects host health by selectively
stimulating the growth and/or activity of one or a limited
number of bacteria in the colon’’ (Gibson & Roberfroid,
1995). Among the resident intestinal bacteria that are
stimulated by prebiotics are various lactobacilli and
bifidobacteria. Some strains of these bacteria are also
added to yogurt and other fermented dairy foods in the
form of probiotics. Ultimately, it is their ability to
metabolize prebiotic sugars, in vivo, that provides for their
selective enrichment in the gastrointestinal tract and that
result in the formation of lactic, acetic, and other short
chain organic acids that may be antagonistic to their
intestinal competitors (Wang & Gibson, 1993). Thus,
prebiotics, alone or combined with probiotic bacteria in
Corresponding author. Tel.: +1 402 472 2820; fax: +1 402 472 1693.
E-mail address: rhutkins1@unl.edu (R.W. Hutkins).
ability to influence and improve the gastrointestinal health
of humans (Tuohy, Probert, Smejkal, & Gibson, 2003).
Several studies have shown that the ability of lactobacilli
and bifidobacteria to ferment prebiotic carbohydrates is
both strain and substrate specific (Kaplan & Hutkins, 2000;
Schrezenmeir & de Vrese, 2001). In addition, it is not clear
which prebiotic carbohydrates are the most suitable
substrates for selective growth of specific strains. Recently,
several quantitative approaches were devised to determine
the functional activity of prebiotics during in vitro
fermentation conditions (Olano-Martin, Gibson, & Ras-
tall, 2002; Palframan, Gibson, & Rastall, 2003; Vulevic,
Rastall, & Gibson, 2004; Sanz, Gibson, & Rastall, 2005;
Sanz, Polemis et al., 2005). In general, these methods
provide indices that reflect the relative ability of a given
prebiotic to produce specific effects, and are based on
measurement of microbial populations, growth rates,
substrate assimilation, and/or short-chain fatty acid
production. The indices were then used to rank various
carbohydrates for their potential to stimulate growth of
specific members of a mixed microflora. However, because
fermentation of prebiotics is dependent on the bacterial
strain, rather than based on the species or genera, it is

0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.idairyj.2006.10.006
 ARTICLE IN PRESS
J. Huebner et al. / International Dairy Journal 17 (2007) 770–775
important to understand the extent to which metabolism of
prebiotics occurs by specific strains of bacteria, especially
for those organisms whose intended use are as probiotics.
Therefore, the aims of this work were to: (1) establish a
prebiotic activity assay based on specific substrates and
strains and (2) use this assay to determine the prebiotic
activity score of various commercial prebiotics for selected
strains of lactobacilli and bifidobacteria. Ultimately, these
results may be useful in identifying combinations of
probiotics and prebiotics that could be added into dairy
and other foods.
2. Materials and methods
2.1. Bacterial strains
Bifidobacterium adolescentis 15706, B. breve 15698,
B. infantis 17930, B. longum 15708, Lactobacillus acidophi-
lus 33200, L. plantarum 4008 (American Type Culture
Collection, Rockville, MD, USA); B. bifidum NCI
(Nebraska Cultures, Inc., Walnut Creek, CA, USA);
L. acidophilus NCFM (North Carolina State University,
Raleigh, NC, USA); L. paracasei 1195 (University of
Nebraska, Lincoln, NE, USA); L. plantarum 12006
(Institute for Fermentation, Osaka, Japan); Escherichia
coli ECOR 1, E. coli ECOR 2, and E. coli ECOR 22 (E. coli
Reference Collection, University of Rochester, Rochester,
NY, USA) were used for this study. The specific test strains
of lactobacilli and bifidobacteria were selected because they
are either already established as probiotics and are used in
dairy products or they have potential probiotic properties.
The Lactobacillus and Bifidobacterium cultures were main-
tained at 80 1C in MRS Broth (Difco Laboratories,
Sparks, MD, USA) containing 15% (wt/vol) glycerol, and
E. coli cultures were maintained at 80 1C in Tryptic Soy
Broth (TSB; Difco Laboratories) containing 15% (wt/vol)
glycerol. B. bifidum NCI required 0.05% L-cysteine HCl for
growth in MRS medium. For the prebiotic activity assays,
frozen cultures were streaked onto MRS agar, for the
Lactobacillus and Bifidobacterium cultures or Tryptic Soy
Agar (TSA), for E. coli, followed by incubation at 37 1C for
24–48 h. Then, one colony from each plate was transferred
into 10 mL of MRS broth or TSB and incubated over-
night. For the E. coli strains, an additional transfer of 1%
Table 1
Commercial prebiotic carbohydrates
Commercial prebiotics Chemical structurea
NutraFlora P-95 Glua1-2-[bFru-1-2]n
Raftilose P95 Glua1-2-[bFru-1-2]n & [bFru1-2]n
Inulin-S Glua1-2-[bFru-1-2]n
Raftiline HP Glua1-2-[bFru-1-2]n
Purified GOS Glua1-4-[bGal-1-4]n
a
Glu, Glucose; Fru, Fructose; Gal, Galactose; FOS, fructooligosaccharide; GOS, galactooligosaccharide.
b
Based on manufacturer’s analysis.
c
Approximate composition after purification (see text).
771
(vol/vol) was made from a TSB overnight culture into
10 mL of M9 Minimal Medium broth (Atlas, 1993) and
incubated overnight.
2.2. Commercial prebiotics
The commercial prebiotics used in this study are
described in Table 1. The two fructooligosaccharide
(FOS) products, NutraFlora P-95 and Raftilose P95, were
obtained from GTC Nutrition (Golden, CO, USA) and
Orafti Group (Tienen, Belgium), respectively. The two
inulin products, inulin from chicory (referred to as
Inulin-S) and Raftiline HP, were obtained from Sigma-
Aldrich (St. Louis, MO, USA) and Orafti Group (Tienen,
Belgium), respectively. Oligomate 55, a galactooligosac-
charide (GOS)—containing product, was obtained from
Yakult Pharmaceutical Ind. Co., Ltd. (Minatoku, Japan).
Because the latter material contained appreciable levels of
mono- and disaccharides (about 45%), compared with the
other commercial products, the Oligomate 55 was purified
by size exclusion chromatography. Briefly, a 30% solution
was applied to a 98.5  3 cm column containing Sephadex
G-10 (Sigma-Aldrich). Fractions were eluted with water
and analyzed by refractive index measurement (Bausch &
Lomb, Rochester, NY, USA). Portions (5 mL) of the
carbohydrate-containing fractions were spotted onto silica
gel thin layer chromatography plates (Fisher Scientific,
Pittsburgh, PA, USA), along with standard solutions of
glucose, galactose, lactose, and the unpurified GOS. The
plates were developed in 1-butanol:2-propanol:ddH2O
(3:12:4), dried, sprayed with a 50% ethanolic sulfuric acid
solution, and then heated at 135 1C to visualize the dark
grey, carbohydrate-containing spots. The fractions con-
taining only GOS (corresponding to Rf values of
0.30–0.67) were pooled and lyophilized.
2.3. Prebiotic activity assay
Prebiotic activity, as it relates to this study, reflects the
ability of a given substrate to support the growth of an
organism relative to other organisms and relative to growth
on a non-prebiotic substrate, such as glucose. Therefore,
carbohydrates have a positive prebiotic activity score if
they are (1) metabolized as well as glucose by probiotic
Degree polymerization (DP) Purity (%)
2–4 97% FOSb
2–7 95% FOSb
2–60 499% Inulinb
423 (average) 499% Inulinb
2–4 499% GOSc
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772 J. Huebner et al. / International Dairy Journal 17 (2007) 770–775
strains and (2) are selectively metabolized by probiotics but
not by other intestinal bacteria. The assay was performed
by adding 1% (vol/vol) of an overnight culture of each
probiotic strain to separate tubes containing MRS Broth
with 1% (wt/vol) glucose or 1% (wt/vol) prebiotic. The
cultures were incubated at 37 1C under anaerobic condi-
tions (85% N2, 10% CO2, and 5% H2) in an anaerobic
chamber (Thermo Forma, Marietta, OH, USA) for
Bifidobacterium and L. acidophilus strains and at ambient
atmosphere for all other strains. After 0, 24, and 48 h of
incubation, samples were enumerated on MRS agar. In
addition, overnight cultures of E. coli strains, ECOR 1,
ECOR 2, and ECOR 22 were mixed in a 1:1:1 ratio (enteric
mixture), then added at 1% (vol/vol) to separate tubes
containing M9 broth with 1% (wt/vol) glucose or 1%
(wt/vol) prebiotic. The cultures were incubated at 37 1C at
ambient atmosphere, and enumerated on TSA after 0, 24,
and 48 h of incubation. Each assay was replicated three
times.
2.4. Prebiotic activity score
The prebiotic activity score was determined using the
following equation:
Prebiotic activity score
 cell density of L. paracasei 1195 grown on Raftilose P95,
¼ ðprobiotic log cfu mL1 on the prebiotic at 24 h
 probiotic log cfu mL1 on the prebiotic at 0 hÞ=
ðprobiotic log cfu mL1 on glucose at 24 h
probiotic log cfu mL1 on the glucose at 0 hÞ
 4008 and L. acidophilus 33200 were significantly larger
 ðenteric log cfu mL1 on the prebiotic at 24 h
 enteric log cfu mL1 on the prebiotic at 0 hÞ=
ðenteric log cfu mL1 on glucose 24 h
enteric log cfu mL1 on the glucose at 0 hÞ .
By definition, substrates with a high prebiotic activity
score support good growth of the probiotic bacteria, with
cell densities (cfu mL1) comparable with that when grown
on glucose. However, the cell densities of the enteric strains
grown on the prebiotics should, in theory, be very low
relative to growth on glucose. Using this equation, the
prebiotic activity score of a particular oligosaccharide can
be determined relative to any given strain.
2.5. Statistical analysis
Prebiotic activity assays were repeated three times and
the data were analyzed using the Statistical Analysis
System (Version 9.1, copyright 2002–2003 by SAS Institute
Inc., Cary, NC, USA). Statistical differences among the
increase in cell density values for a given strain for each
carbohydrate treatment were determined by the General
Linear Model followed by the Least Significant Difference
comparison of the means (po0.05). Statistical differences
among prebiotic activity scores were determined by the
General Linear Model with a two-way factorial experi-
mental design (probiotic and prebiotic treatments are fixed)
followed by the Least Squares Means comparison proce-
dure (po0.05).
3. Results
3.1. Growth of lactobacilli, bifidobacteria, and enteric
mixture on prebiotic carbohydrates
The increases in cell densities for five strains of
Lactobacillus and five strains of Bifidobacterium following
growth (24 h) on 1% (wt/vol) glucose or 1% (wt/vol)
prebiotic sugars are shown in Table 2. Cell densities were
also measured after 48 h, but no further increases were
observed, except for L. plantarum 12006 grown with
purified GOS and B. bifidum NCI grown with Raftilose
P95.
For a given sugar to have prebiotic activity, that sugar
should be metabolized by a test strain as well, or nearly as
well, as glucose is metabolized. For most of the test strains,
growth (as cfu mL1) on the prebiotics was less than on
glucose, with 37 of the 50 possible combinations having a
significantly lower (po0.05) increase in cell density on the
prebiotic compared to glucose. In contrast, the increase in
Inulin-S, and Raftiline HP were significantly higher
(po0.05) than for glucose. B. bifidum NCI had a
significantly higher (po0.05) increase in cell density when
grown on NutraFlora P-95 and Raftilose P95 than on
glucose. Also, the increase in cell densities of L. plantarum
(po0.05) for purified GOS than for glucose. For the
remaining strain-prebiotic combinations, there were no
significant differences compared with growth on glucose.
The other characteristic property of a prebiotic substrate
is that it should be selective and not fermented by
commensal organisms. Therefore, growth on each prebiotic
was also determined for a mixture of three enteric bacteria,
E. coli ECOR 1, ECOR 2, and ECOR 22, chosen to
represent the enteric portion of the commensal flora.
Growth of this enteric mixture on all of the prebiotics was
significantly less (po0.05) compared with growth on
glucose (Table 2).
3.2. Prebiotic activity score
Prebiotic activity scores, shown in Fig. 1, were derived
from the cell density values from Table 2. The highest
prebiotic activity scores (po0.05) were for L. paracasei
1195 paired with Inulin-S, Raftiline HP, and Raftilose P95
(1.17, 1.10, and 0.99, respectively), followed by L.
plantarum 4008, L. acidophilus 33200 and L. acidophilus
NCFM grown on purified GOS, and L. acidophilus NCFM
grown on Raftilose P95 (0.82, 0.70, 0.66, and 0.58,
respectively). In contrast, the lowest scores (po0.05) were
for B. bifidum NCI grown with purified GOS and Inulin-S
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J. Huebner et al. / International Dairy Journal 17 (2007) 770–775 773

Table 2
Increase in cell density between time 0 and time 24 hb, reported as log10(cfu mL1)7standard deviation, for bacterial cultures grown with various
carbohydrates

Bacterial Culture Glucose NutraFlora P-95 Raftilose P95 Inulin-S Raftiline HP Purified GOS
a a a
L. paracasei 1195 1.6670.14 1.8870.13 2.4870.26 2.6370.31 2.4870.27 0.9670.07a
L. plantarum 4008 2.2770.03 2.1170.11 1.9970.07 1.5470.07a 1.8070.39a 2.9170.22a
L. plantarum 12006 1.8870.12 1.0870.02a 0.9270.04a 0.4670.10a 0.4170.10a 1.5270.06a
L. acidophilus NCFM 2.0470.14 1.9870.03 2.2070.11 0.8970.32a 0.9270.02a 2.2970.19
L. acidophilus 33200 1.6670.07 0.6970.06a 0.3370.12a 0.4370.07a 0.3370.02a 1.9870.07a
B. breve 15698 2.1370.08 1.9170.04a 1.9270.06a 1.5970.03a 1.5970.07a 1.5570.02a
B. infantis 17930 2.2070.05 1.9370.05a 1.8470.06a 1.6670.12a 1.5670.09a 1.6770.08a
B. adolescentis 15706 2.0870.11 1.8370.03a 1.8170.10a 1.6170.14a 1.5770.05a 1.6470.02a
B. longum 15708 2.1070.02 1.9370.03a 1.8370.02a 1.6770.02a 1.6170.10a 1.5270.08a
B. bifidum NCI 1.4870.04 1.7270.04a 1.7070.07a 1.1170.08a 0.3970.12a 1.1470.21a
Enteric mixture 1.9470.11 1.3370.07a 0.9870.06a 0.8170.04a 0.7770.01a 0.9070.04a
a
Mean value (7standard deviation) for prebiotic-grown cells differ significantly (po0.05) from glucose-grown cells for the specific bacterial culture.
b
Increase in cell density between time 0 and time 48 h reported for L. plantarum 12006 grown on GOS and B. bifidum NCI grown on Raftilose P95.

1.50
NutraFlora P-95
Raftilose P95
Inulin-S
1.00
Raftiline HP
Purified GOS
Prebiotic activity score

0.50

0.00
L. paracasei 1195

L. plantarum 4008

L. plantarum 12006

L. acidophilus NCFM

L. acidophilus 33200

B. breve 15698

B. infantis 17930

B. adolescentis 15706

B. longum 15708

B. bifidum NCI
-0.50

-1.00

-1.50
Bacteria

Fig. 1. Prebiotic activity scores of various bacteria grown on commercial prebiotics.

(1.24 and 1.17, respectively), followed by L. acidophilus acidophilus NCFM for all of the fructooligosaccharides
33200 grown on Raftilose P95 and B. bifidum NCI grown and inulin type prebiotics, but a similar score for
on Raftiline HP (0.70 and 0.67, respectively). Also, purified GOS.
L. plantarum 12006 and L. acidophilus 33200 had prebiotic
activity scores below zero when grown on all the prebiotics,
except purified GOS. A low or negative prebiotic activity 4. Discussion
score was obtained if the test strain grew less well (based on
cell densities) on the prebiotic compared with that on Prebiotic carbohydrates are, by definition, metabolized
glucose and/or had less growth on the prebiotic than the only by selected members of the gastrointestinal tract.
growth of the enteric mixture on the prebiotic carbohy- Accordingly, these sugars have the ability to influence the
drate. population of the gastrointestinal tract due to their
Strains within the same species had significantly different selective utilization. Organisms that rapidly ferment pre-
prebiotic activity scores for the same prebiotic. For biotic sugars are enriched, presumably at the expense of
example, L. plantarum 4008 had significantly higher scores those that do not. The effectiveness of a prebiotic depends,
(po0.05) compared with L. plantarum 12006 for all of the therefore on its ability to be selectively fermented by and to
prebiotics tested. In addition, L. acidophilus 33200 had support growth of specific targeted organisms. The goal of
significantly lower scores (po0.05) compared with L. this study, therefore, was to quantify the extent to which
 ARTICLE IN PRESS
774 J. Huebner et al. / International Dairy Journal 17 (2007) 770–775
prebiotic sugars express this activity using selected strains
of Lactobacillus and Bifidobacterium.
In vitro studies by Olano-Martin et al. (2002), Palframan
et al. (2003), Vulevic et al. (2004), Sanz, Gibson et al.
(2005), and Sanz, Polemis et al. (2005) have also developed
equations to quantitatively evaluate the ability of carbohy-
drates to have prebiotic effects. In these studies, prebiotic
indices were based on changes in bacterial populations (of
specific genera), substrate assimilation, growth rates, and/
or short chain fatty acid production, with each character-
istic assigned a positive or negative effect on the calculated
index. Unlike previous studies, our method to assess
prebiotic activity evaluates the combination of a prebiotic
with specific strains of putative probiotic bacteria. Within
the equation, the change in growth of the test strain on the
prebiotic is compared with the growth on glucose and to
the growth of a mixture of commensal bacteria on the
prebiotic and glucose. This method is comparably more
simple than previous methods because fecal samples are
not required. In addition, it is a relatively quick way to
evaluate a prebiotic’s ability to be utilized by specific
strains of bacteria.
As might be expected, given the known metabolic
diversity of the lactobacilli, there was considerable varia-
tion in prebiotic activity scores for the different pre-
biotics utilized by a single probiotic strain. For example,
L. paracasei 1195 had significantly higher scores (po0.05)
for the inulin type prebiotics and Raftilose P95 compared
with purified GOS. In contrast, L. plantarum 12006 and
L. acidophilus 33200 had significantly higher scores
(po0.05) for GOS compared with the other commercial
prebiotics. In fact, it was observed that even strains within
a single species (e.g., L. plantarum strains 4008 and 12006)
had significantly different prebiotic activity scores, indicat-
ing that differences in the metabolic capacity of related
strains apparently exist. Utilization of prebiotics by lactic
acid and related bacteria requires the presence of specific
hydrolysis and transport systems for the particular
prebiotic (Barrangou, Altermann, Hutkins, Cano, &
Klaenhammer, 2003; Gopal, Sullivan, & Smart, 2001;
Imamura, Hisamitsu, & Kobashi, 1994; Kaplan & Hutkins,
2003; Muramatsu, Onodera, Kikuchi, & Shiomi, 1992,
1994; Perrin, Warchol, Grill, & Schneider, 2001; Rabiu,
Jay, Gibson, & Rastall, 2001). Therefore, genes coding for
these metabolic systems may be present or absent in the
different strains, resulting in varied prebiotic activity
scores. In contrast to the metabolic diversity observed for
the lactobacilli, the prebiotic activity scores for the
bifidobacteria on the different prebiotics varied relatively
little (except for B. bifidum), with most scores ranging from
0.2 to 0.4. In previous studies using mixed flora samples
(Palframan et al., 2003), the highest prebiotic index scores
were generally obtained for GOS (as well as pectic,
isomaltooligosaccharides and soybean oligosaccharides),
compared with fructooligosaccharides and inulin.
The prebiotic activity scores reported in this study reflect
the extent to which a given carbohydrate would promote
selective growth of specific organisms in the presence of
competitors unable to utilize that particular carbohydrate.
Thus, these scores provide a rational basis for identifying
synbiotics for incorporation into dairy and other foods
(Chow, 2002; Fooks & Gibson, 2002; Rastall & Maitin,
2002). However, it is important to recognize two important
factors. First, in the gastrointestinal tract, commensal
organisms likely exist that, unlike the E. coli strains used in
this study, will have some ability to utilize prebiotic
carbohydrates (Hartemink, Van Laere, & Rombouts,
1997). Indeed, despite the definition of prebiotics as being
fermented by a ‘‘limited number’’ of colonic bacteria, it
now appears that Bacteroides and other resident members
of intestinal microflora have the metabolic capacity to
metabolize these substrates (Van der Meulen, Makras,
Verbrugghe, Adriany, & De Vuyst, 2006). In addition, the
specific means by which metabolism of prebiotic carbohy-
drates occurs is also relevant with respect to their prebiotic
activity. If, for example, a particular organism initiates
metabolism of an oligosaccharide via extracellular hydro-
lysis, the products (mono- or disaccharides) that are
released may then ‘‘cross-feed’’ other organisms. In pure
culture, the sugar would appear to be prebiotic and would
have a prebiotic activity score, but in a mixed culture
environment, even ‘‘nonfermentors’’ would still have access
to fermentable hydrolysis products.
5. Conclusions
Quantitative prebiotic activity scores that describe the
extent to which five prebiotics support selective growth of
five lactobacilli and five bifidobacteria were determined.
The highest score was obtained for L. paracasei 1195
grown on inulin, while the lowest score was for B. bifidum
NCI grown on galactooligosaccharides. This study pro-
vides a basis for the evaluation of combinations of
probiotic and prebiotic ingredients for applications as
synbiotics in dairy and other foods.
Acknowledgements
This work was supported by a grant through the United
States Department of Agriculture Cooperative State
Research, Education, and Extension Service (# 2004-
35503-14118). The authors would like to thank GTC
Nutrition, Orafti Group, and Yakult for their generous
donation of commercial prebiotics.
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