Journal of Functional Foods 16 (2015) 211–222

Available online at www.sciencedirect.com

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

Addition of acacia gum to a FOS/inulin blend

improves its fermentation profile in the Simulator of
the Human Intestinal Microbial Ecosystem (SHIME®)

Massimo Marzorati a,*, Bingcai Qin b,1, Falk Hildebrand c,d,1, Abby Klosterbuer e,
Zamzam Roughead e, Claudia Roessle f, Florence Rochat f, Jeroen Raes c,d,g,h,
Sam Possemiers a,i
a
Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, 9000 Ghent, Belgium
b
BGI-Shenzhen, Shenzhen, China
c
VIB Department of Structural Biology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium
d
Department of Bioscience Engineering, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium
e
Nestlé R&D Center Minneapolis, 12500 Whitewater Drive, Minnetonka, MN, USA
f
Nestlé Research Centre, 1000 Lausanne, Switzerland
g
Department of Microbiology and Immunology, REGA Institute, KU Leuven, Herestraat 49, 3000 Leuven, Belgium
h
VIB Center for the Biology of Disease, KU Leuven, Herestraat 49, 3000 Leuven, Belgium
i
ProDigest BVBA, Technologiepark 3, 9052 Ghent, Belgium

A R T I C L E I N F O A B S T R A C T

Article history: The Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) was used to assess the
Received 11 November 2014 impact of the partial substitution of fructooligosaccharides (FOS) and inulin by acacia gum in
Received in revised form 21 April a fibre blend. Both blends were well fermented and the presence of acacia gum modified the
2015 intestinal fermentation of the blend from a boosted fermentation into a gradual one in the
Accepted 22 April 2015 complete colon. Confirmation of the gradual fermentation was obtained by analysis of the pH
Available online 15 May 2015 profile, measurement of the residual acacia gum fractions in the colon and by the increased
expression of specific catabolic enzymes. Both blends increased the total amount of short chain
Keywords: fatty acids (SCFAs; +42 mmol/L), propionate (+26 mmol/L) and butyrate (+9 mmol/L) and showed
Colonic fermentation bifidogenic properties. Metagenomic Illumina sequencing confirmed that the blends exerted
Gastrointestinal tract a diverse modulating activity in the different areas of the colon. Long-term repeated admin-
SHIME® istration of the prebiotic blends is needed to reach gradual changes in the gut microbial
Acacia gum community structure and activity. The partial substitution of FOS and inulin by acacia gum
Prebiotic fibre slowed the speed of the fermentation of the blend. In humans, this may translate into ben-
efits for gut health, allowing for incorporation of higher amounts of fibre into the diet.
© 2015 Elsevier Ltd. All rights reserved.

Chemical compounds: (+)-Arabinogalactan (PubChem CID: 24847856); Inulin (PubChem CID: 16219508).
* Corresponding author. Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, 9000 Ghent, Belgium.
Tel.: +32 9 2645976; fax: +32 9 2646248.
E-mail address: massimo.marzorati@ugent.be (M. Marzorati).
1
Authors equally contributed.
Abbreviations: SHIME , Simulator of the Human Intestinal Microbial Ecosystem; FOS , fructooligosaccharides; SCFA, short chain fatty
acid; BCFA, branched chain fatty acid; AC, TC, DC, ascending, transverse and descending colon; GIT, gastrointestinal tract; MW, molecu-
lar weight; SEC, size exclusion chromatography; AG, acacia gum; qPCR, quantitative polymerase chain reaction; KO, KEGG orthologue
group; OG, orthologue group
http://dx.doi.org/10.1016/j.jff.2015.04.039
1756-4646/© 2015 Elsevier Ltd. All rights reserved.
212 Journal of Functional Foods 16 (2015) 211–222
1. Introduction
The gastrointestinal tract (GIT) hosts the most complex mi-
crobial community in the human body (Maccaferri, Biagi, &
Brigidi, 2011). Biomedical research has shown that the balance
of this additional organ within our body is of key importance
to maintain a healthy status and that several diseases are fre-
quently correlated with a dysbiosis (Blottiere, de Vos, Ehrlich,
& Dore, 2013). In this respect, there is a great interest in de-
veloping new products (e.g. prebiotic fibres) with the aim of
modulating the gut microbiota in order to induce a positive
health effect (e.g. increased or decreased production of health
related bacterial metabolites, growth of health promoting bac-
teria, decrease in intestinal pathogens, or immune modulation)
(Al-Sheraji et al., 2013) [www.efsa.europa.eu/en/supporting/
pub/136e.htm]. This is especially important in the clinical
setting, where patients may have altered gut microbiota due
to stress, dietary changes, or medication. Prebiotics are com-
monly added to products for supplemental or complete enteral
nutrition, and identifying fibres or fibre blends that exert health
benefits without compromising gastrointestinal tolerance is of
great interest for this population. In order to bring a new product
with a specific health claim on the market, evidence of its effect
must be shown in vivo and, when possible, the mechanism of
action should be explained [http://www.efsa.europa.eu/en/nda/
ndaguidelines.htm].
Most of the in vivo studies on the gut microbiota are based
on the analysis of faecal samples due to the fact that they can
be easily collected with a non-invasive approach. However, while
faecal communities may be representative for processes oc-
curring in the distal part of the colon, they represent, at the
very best, an average of the microbiota composition and
functionalities (both luminal and mucosal) of the whole GIT
(Durban et al., 2011; Lee et al., 2013). In this respect, elucida-
tion of the mechanism of action of a fibre or a mix of fibres
in the different areas of the GIT may become a very difficult
task, making use of clinical trials with ileostomy volunteers
or animal models with humanized microbiota.
Well-designed in vitro technologies – aimed at simulating
the main physiological and microbiological processes occur-
ring in the GIT – coupled with metabolic and molecular analyses
can be an interesting toolbox to complement in vivo trials and
produce data supporting the mechanism of action of a spe-
cific product.
The Simulator of the Human Intestinal Microbial Ecosys-
tem (SHIME®) is a continuous model of the GIT that allows in-
depth longitudinal studies of the biological activity of selected
molecules in the gut under representative environmental con-
ditions and under long-term repeated administration conditions
(Marzorati et al., 2013; Possemiers, Marzorati, Verstraete, & Van
de Wiele, 2010; Sanchez et al., 2009; Van de Wiele, Boon,
Possemiers, Jacobs, & Verstraete, 2004). This model was used
to describe the behavior in the GIT of two mixtures of prebi-
otic fibres: a blend containing fructo-oligosaccharides (FOS) and
inulin (short chain, linear molecules) and a blend in which part
of the fructans present in the first blend were replaced by acacia
gum (AG), a complex fibre shown in previous studies to be slowly
fermented (Cherbut, Michel, & Lecannu, 2003). The hypothesis
was that partial substitution of FOS and inulin with AG would
alter the fermentation profile of the blend. In particular, ad-
dition of slowly fermented AG was expected to extend the
duration of fermentation relative to the other blend, and this
would be reflected by a different modulation of the microbiota
in the different areas of the colon (i.e. ascending, transverse
and descending colon) without impacting the potential pre-
biotic activity of the blend. In order to prove this, we analysed
the changes in microbiota activity and composition in each simu-
lated colon compartment during a three-week treatment period
with the 2 blends by means of metabolic analyses (i.e. short
chain fatty acids – SCFA, ammonium, pH profiling, enzymatic
activities, size exclusion chromatography) and molecular
techniques (i.e. quantitative PCR, denaturing gradient gel elec-
trophoresis, in depth metagenomic illumina sequencing).
2. Materials and methods
2.1. Products
The nutritional products used in this study were composed of
30% fat, 20% proteins and 50% carbohydrates, including a blend
of fermentable fibres. These nutritional products only dif-
fered in the composition of the fibre blend. Blend 1 (Nestlé
Health Science, Vevey, Switzerland) contained FOS and inulin
(blended in proportion of 70:30%) while in Blend 2 (Nestlé Health
Science) part of the fructans present in Blend 1 were re-
placed by AG (41% FOS, 41% acacia gum, 18% inulin). The
nutritional products were available in servings containing 3.3 g
of the fibre blends.
2.2. Simulator of the human intestinal microbial
ecosystem (SHIME®)
The reactor setup was adapted from the SHIME® (ProDigest and
Ghent University, Ghent, Belgium), representing the GIT of the
adult human, as described by Molly, Woestyne, and Verstraete
(1993). The SHIME consists of a succession of five reactors simu-
lating stomach, small intestine, ascending, transverse and
descending colon. The system has been inoculated with the
faecal microbiota from a healthy 75-year-old volunteer who had
no history of antibiotic treatment in the 6 months prior to the
faecal sample collection. Specific conditions (e.g. retention time,
pH…) and feed composition have been previously described
in Possemiers, Verthe, Uyttendaele, and Verstraete (2004). For
these experiments, a TWINSHIME® setup was used by oper-
ating two systems in parallel at the same time in order to obtain
identical environmental conditions for both systems (SHIME
1 = Blend 1; SHIME 2 = Blend 2). The experimental setup of the
SHIME run included a two-week start-up period, a two-week
control period and a three-week treatment period, as previ-
ously described (Van de Wiele et al., 2004). During the treatment
period, 3.0 g/L of the starch in the feed was removed from the
diet – to partially compensate for the additional amount of
carbon sources – and replaced with 2 servings of the fibre blends
per day.
2.3. Analytical techniques
Samples for analytical techniques (a total of 16 mL) were col-
lected three times per experimental week (i.e. Monday,
 Journal of Functional Foods 16 (2015) 211–222
Wednesday and Friday at 1 pm), divided in aliquots of 2 mL
and stored at −20 °C. By collecting 3 samples every experimen-
tal week we basically have 3 observations in what we consider
as an experimental unit (i.e. 1 week). Analyses for SCFA, am-
monium, D-lactic acid and L-lactic acid quantification, effect
of the treatment on pH were conducted as described by Terpend
and colleagues (Terpend, Possemiers, Daguet, & Marzorati, 2013).
The molecular weight (MW) profiles of polysaccharides in the
residual content were analysed by size exclusion chromatog-
raphy (SEC), as previously described (Terpend et al., 2013).
Activity for β-glucosidase, α-galactosidase, β-galactosidase, β-D-
xylosidase and α-L-arabinofuranosidase was measured by
means of colorimetric assays as described by Berg, Nord, and
Wadstrom (1978). Gas production has been measured in peni-
cillin flasks (in triplicate) during 48 h of simulated colonic
incubation, by means of a WIKA digital pressure meter (CPH6200
– S1) (LHM instrumentation, Geel, Belgium). Briefly: carbon de-
pleted medium (composition: Pepton 2 g/L; yeast extract 2 g/L;
L-cystein 0.5 g/L; NaCl 0.1 g/L; K2HPO4 40 mg/L; KH2PO4 40 mg/L;
MgSO4 7H2O 10 mg/L; CaCl2 2H2O 6.7 mg/L) has been inocu-
lated 10% with SHIME fluid collected from the ascending colon
during the second week of control. Either Blend 1 or Blend 2
have been added as a sole carbon source for bacteria at the
final concentration of 6 g/L. Gas production has been mea-
sured after 2, 4, 6, 24 and 48 h of incubation.
2.4. Molecular microbiological analyses
Samples for molecular techniques (2 aliquots of 1 mL each) were
collected once per experimental week at the end of the week
(i.e. Friday at 1 pm), for a total of five weeks from each simu-
lated colon compartment. The samples were immediately
centrifuged and the pellet stored at −20 °C. Metagenomic DNA
was extracted using the CTAB protocol as described by Boon,
Top, Verstraete, and Siciliano (2003).
Quantitative polymerase chain reaction (qPCR) for total bac-
teria, bifidobacteria, and lactobacilli were performed as reported
by Possemiers et al. (2006). The qPCR for the Firmicutes and
Bacteroidetes phyla was previously described by Guo et al.
(2008).
Denaturing gradient gel electrophoresis (DGGE) was used
to separate the PCR products for the 16S rRNA genes of total
bacteria, bifidobacteria and lactobacilli as described by Terpend
and colleagues (Terpend et al., 2013) Finally, samples from the
end of the control period (week 2) and the end of the treat-
ment period (week 3) were selected for metagenomic illumina
sequencing.
2.5. Metagenomic illumina sequencing
DNA library construction was performed following the manu-
facturer’s instruction (illumina). We constructed one paired-
end (PE) library with insert size of 350 bp for each sample. Then
high-throughput sequencing was performed using illumina
Hiseq 2000 to obtain around 240 million PE reads for all 12
samples. The reads length for each end was 90 bp. The raw reads
which contain three or more “N” or adapter contamination were
discarded.
For each sample, high quality reads were assembled to obtain
long contigs by using SOAP denovo assembler (Li et al., 2010);
213
the contigs longer than 500 bp were used to predict ORFs by
MetaGene program (Noguchi, Park, & Takagi, 2006). Mean-
while, high quality reads from each sample were aligned against
the Human Gut Gene Catalogue (Li et al., 2009; Qin et al., 2010)
by SOAP2 (Li et al., 2009) using the criterion of identity ≥90%.
We translated the aligned results to gene relative abundance
by counting the number of reads that mapped to the gene.
Based upon Human Gut Gene Catalogue’s annotation infor-
mation, genes were assigned to different taxonomy levels, KEGG
orthologue group (KO), KEGG pathway map (map) and eggNOG
orthologue group (OG). For the species profile, we utilized
Human Gut Gene Catalogue’s annotation information and
summed the relative abundance of genes from the same
species. This gross relative abundance was taken as the content
of this species in a sample to generate the species profile of
the samples. The genus profile, phylum profile, KO profile, map
profile and OG profile were constructed using the same
methods. Besides, all the redundant genes were removed from
all the 12 samples, leading to a gene-set of 201,356 genes.
Taxonomic assignment of predicted genes was carried out
using BLASTN to assign reads to a reference genome data-
base at a cut-off of 95% sequence identity and >100 bp overlap.
This assignment was used as high confidence assignment on
species level. As reference database we used all available com-
plete bacterial genomes from IMG version 3.50. The genes
annotated by KEGG were assigned into KEGG modules and Seed
pathways (Overbeek et al., 2005). The relative pathway abun-
dance of higher order functional categories was calculated from
normalized KO abundances.
2.6. Statistics
Before investigating the probability of intergroup differences,
the normality data were studied with Kolmogorov–Smirnov.
Comparison of normally distributed data was performed with
Student’s t-test for pairwise comparisons or with Wilcoxon
signed-rank test for non-normally distributed data. Statisti-
cal significance was set at P < 0.05. Calculations were performed
using the SPSS software (version 21.0, SPSS Inc., Chicago, IL,
USA). Comparison of the effect of the Blends on the meta-
bolic parameters and on qPCR data was conducted by means
of a longitudinal statistical analysis with a linear spline model,
positioning the knot on the first week of treatment (SAS, Cary,
NC, USA). The approach is based on the creation of a complex
model and the subsequent removal, step by step, of different
predictors. The difference of the maximum likelihood values
of two equations compared with the respective chi-square
allows determination whether the removed predictor had a sta-
tistical significance or not.
The obtained DGGE patterns were analysed using the
BioNumerics software v.2.0 (Applied Maths, Sint-Martens-
Latem, Belgium). A matrix of similarities for the densiometric
curves of the band patterns was calculated based on the Pearson
product–moment correlation coefficient. A composite dataset
was created by merging the information from the all the band
patterns in order to obtain a combined dendrogram – using
UPGMA linkage – containing the information from the gels on
total bacteria, bifidobacteria and lactobacilli.
For illumina data, each sample was normalized by divid-
ing each feature (species, KO abundance, etc.) within a sample
214 Journal of Functional Foods 16 (2015) 211–222

by the samples’ respective total sum of reads. Higher func-
tional categories were calculated from a normalized KO
abundance matrix, but not further normalized, as described
in Hildebrand et al. (2012). Feature abundance matrices were
transformed by adding 1 to each feature and calculating log10
subsequently, avoiding negative infinite values for absent fea-
tures. All statistics were calculated using R 2.14. Functional
abundance categories were tested for significant differences
using the non-parametric Kruskal–Wallis test (p-value) that was
subsequently corrected for Multiple Testing using the
Benjamini–Hochberg (Benjamini & Hochberg, 1995) false dis-
covery rate (q-value). Functional categories with less than 10
reads over all samples were excluded from this analysis to avoid
artifacts. Post-hoc statistical testing for significant differ-
ences between all combinations of tested groupings was
conducted only if the Kruskal–Wallis test p-value was <0.2.
Wilcoxon rank-sum tests were calculated for all possible com-
binations and corrected for multiple testing using Benjamini–
Hochberg false discovery rate (q-value). If not mentioned
otherwise, tests were considered significant if they had a
p-value ≤0.05 and a q-value ≤0.1. The significance of blocked
test designs was calculated using the “independence_test” of
the “coin” R-package (Hothorn, Hornik, Van de Wiel, & Zeileis,
2006). The respective test was then used to calculate blocked
post-hoc significances, similar to the above procedure. This pro-
cedure is described in more detail in Hildebrand et al. (2013).
Non-metrical dimensional scaling (NMDS) was used to vi-
sualize inter-sample hellinger distances, as implemented in the
“vegan” 2.0.7 package (http://cran.r-project.org/web/packages/
vegan/index.html). The phylogenetic clustering was calcu-
lated from inter-sample Bray–Curtis distances from the
transformed data matrix. These distances were clustered using
centroid hierarchical clustering as implemented in the
R-package “cluster” (http://cran.r-project.org/web/packages/
cluster/index.html).
3. Results
3.1. Metabolic parameters
SCFA profiles consisted mainly of acetate, propionate and bu-
tyrate with small amounts of other acids such as isobutyric,
valeric, isovaleric and caproic acid. As shown in Table 1, both

Table 1 – Comparison of the effect of Blend 1 and Blend 2 on the SCFA (mmol/L), lactate (mmol/L), BCFA (mmol/L) and
ammonium (mg/L) production in the different colon compartments of the TWINSHIME (ascending – AC, transverse – TC
and descending – DC colon). The data are presented per the full control period (observations per 2 weeks = 6) and per
experiment week of treatment (observations per week = 3). Data are shown as average of the observations ± standard
deviation. Comparison of the effect of the Blends was conducted by means of a longitudinal statistical analysis with a
linear spline model, positioning the knot on the first week of treatment. Significant differences are indicated with
* (P < 0.05) close to the specific parameter.
Control Treatment – week 1 Treatment – week 2 Treatment – week 3
Blend 1 Blend 2 Blend 1 Blend 2 Blend 1 Blend 2 Blend 1 Blend 2
AC
Acetate 27.4 ± 2.0 30.3 ± 0.9 37.4 ± 2.7 34.2 ± 2.4 33.3 ± 4.5 34.8 ± 2.2 33.8 ± 2.5 35.2 ± 4.3
Propionate 18.9 ± 1.6 19.2 ± 2.1 39.1 ± 4.1 43.5 ± 10.3 45.3 ± 2.3 49.3 ± 2.4 48.1 ± 7.7 48.3 ± 6.4
Butyrate* 9.9 ± 0.1 9.8 ± 0.1 15.6 ± 3.4 12.4 ± 1.2 19.0 ± 7.2 13.7 ± 1.5 23.7 ± 5.2 13.7 ± 3.7
Valerate 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
Caproate 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
BCFA 2.9 ± 0.1 2.9 ± 0.0 1.1 ± 0.5 1.5 ± 0.6 1.4 ± 0.1 1.2 ± 0.2 1.4 ± 0.2 1.3 ± 0.3
Total 59.0 ± 3.6 62.2 ± 3.1 93.3 ± 5.5 91.7 ± 11.8 99.2 ± 5.5 98.9 ± 5.6 107.3 ± 15.7 98.6 ± 14.6
Lactate* 2.4 ± 0.5 2.5 ± 0.2 9.5 ± 4.8 3.5 ± 2.1 8.0 ± 6.5 3.5 ± 1.3 7.5 ± 6.1 7.2 ± 6.5
Ammonium 352.3 ± 19.5 358.6 ± 39.7 173.1 ± 42.0 217.8 ± 51.7 168.9 ± 18.2 183.2 ± 21.5 194.1 ± 28.1 153.4 ± 34.2
TC
Acetate 31.9 ± 1.2 32.1 ± 0 35.0 ± 1.2 36.0 ± 4.5 38.1 ± 1.5 42.1 ± 2.8 37.7 ± 0.9 41.3 ± 3.0
Propionate 20.9 ± 0.6 19.9 ± 0.9 34.8 ± 11.6 40.1 ± 17.2 50.7 ± 0.8 56.9 ± 1.4 48.3 ± 2.2 51.0 ± 2.5
Butyrate* 9.7 ± 0.4 9.9 ± 0.3 19.0 ± 10.2 14.4 ± 4.4 29.2 ± 3.3 18.3 ± 2.0 25.0 ± 1.7 15.3 ± 2.3
Valerate 0.0 ± 0.0 0.0 ± 0.0 0.3 ± 0.3 0.1 ± 0.1 0.8 ± 0.3 0.2 ± 0.0 0.6 ± 0.1 0.2 ± 0.1
Caproate 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
BCFA 3.1 ± 0.1 3.1 ± 0.1 2.2 ± 0.5 2.4 ± 0.3 2.2 ± 0.2 2.1 ± 0.2 1.8 ± 0.1 1.7 ± 0.1
Total 65.5 ± 1.4 65.1 ± 0.5 91.4 ± 22.4 92.9 ± 26.0 121.0 ± 4.9 119.6 ± 1.4 113.5 ± 4.1 109.6 ± 7.6
Lactate 3.5 ± 0.3 2.8 ± 0.2 3.9 ± 0.7 2.6 ± 0.1 3.3 ± 0.6 3.1 ± 0.4 4.4 ± 2.6 3.7 ± 2.4
Ammonium 436.4 ± 26.3 410.1 ± 28.8 302.1 ± 58.9 320.2 ± 47.1 280.6 ± 23.4 266.0 ± 17.4 257.2 ± 18.5 234.6 ± 14.5
DC
Acetate 35.7 ± 0.7 33.7 ± 0.0 39.5 ± 5.7 38.7 ± 12.3 45.3 ± 2.1 50.4 ± 4.8 44.1 ± 2.8 54.4 ± 4.9
Propionate 20.9 ± 0.8 18.0 ± 1.0 32.4 ± 12.8 33.1 ± 18.9 47.3 ± 1.2 52.4 ± 2.8 47.3 ± 1.1 49.6 ± 1.6
Butyrate* 8.7 ± 0.1 8.1 ± 0.4 15.3 ± 9.0 11.0 ± 4.2 24.8 ± 2.9 16.7 ± 1.5 23.1 ± 1 .8 15.5 ± 2.2
Valerate 1.6 ± 0.2 1.5 ± 0.5 2.9 ± 1.1 2.1 ± 1.2 4.2 ± 0.2 4.0 ± 0.2 3.1 ± 1.7 2.5 ± 1.7
Caproate 1.3 ± 0.3 1.4 ± 0.7 2.3 ± 1.3 1.1 ± 0.7 4.4 ± 0.4 2.3 ± 0.1 2.3 ± 2.2 1.1 ± 1.2
BCFA 3.3 ± 0.0 3.0 ± 0.0 2.7 ± 0.3 2.5 ± 0.1 2.6 ± 0.2 2.3 ± 0.3 2.2 ± 0.1 1.9 ± 0.1
Total 71.6 ± 0.3 65.8 ± 0.1 95.1 ± 29.5 88.5 ± 37.3 128.7 ± 0.8 128.1 ± 6.9 122.1 ± 1.6 125.0 ± 8.2
Lactate 3.7 ± 0.8 2.8 ± 0.5 3.9 ± 0.3 3.0 ± 0.5 3.2 ± 0.1 3.6 ± 0.0 3.2 ± 0.5 2.6 ± 0.5
Ammonium 456.0 ± 24.5 408.0 ± 58.5 380.7 ± 50.6 346.9 ± 3.9 348.8 ± 21.9 290.5 ± 53.1 301.7 ± 18.5 230.7 ± 17.6
 Journal of Functional Foods 16 (2015) 211–222 215

Fig. 1 – pH profiles and gas production: pH profiles expressed as the difference of the consumption NaOH and HCl on a
weekly base, in the ascending (AC), transverse (TC) and descending (DC) colon compartments of the SHIME systems treated
with Blend 1 (A) and Blend 2 (B) (observation = 1). Samples were marked as: CTRL = control, TREAT = treatment; 1, 2 or 3
indicates the specific week during either the control or treatment period in which the samples were collected, (C) Δ total gas
production related to the treatment with Blend 1 and Blend 2 ± standard deviation (n = 3). Significant differences between
the two blends have been assessed by means of a Student’s two-tailed t-test and are indicated with * for P = 0.06.

blends induced an increase in the total SCFA concentration in
all the simulated colon compartments (p < 0.01), indicating that
both products were well fermented in the GIT. Moreover, both
products induced a higher concentration of propionate and
butyrate (Table 1) and were able to move the ratio acetate–
propionate–butyrate (A/P/B) towards a higher proportion of P
and B (data not shown). When evaluating statistical differ-
ences in SCFA production between the two blends, the
butyrogenic effect of Blend 1 was higher than that of Blend 2
(approx. +10 mmol/L), which in turn led to a higher propio-
nate concentration (even if not statistically significant). Both
blends induced an increase in lactate production in the simu-
lated ascending colon compartment, with the production of
Blend 1 being 3.6 g/L higher than Blend 2 (p < 0.05) (Table 1).
Finally, both products induced a decrease in ammonium pro-
duction during the treatment period in all the simulated colon
compartments (Table 1).
The analysis of the NaOH and HCl consumption (acid and
base are used to maintain the colon compartments in the
correct pH range) showed that administration of both blends
induced acidification of the simulated colon reactors. However,
whereas this effect was limited to the simulated ascending
colon for Blend 1 (with the exception of a peak in the DC during
the first week of treatment), acidification occurred through-
out the entire simulated colon upon administration of Blend
2 (Fig. 1a and 1b). These data were confirmed by the results
of the SEC analysis. Blend 1 was quickly fermented in the simu-
lated ascending colon compartment (Appendix: Supplementary
Fig. S1) while, when dosing Blend 2, a residual part of the
product (a peak with a MW above 200,000 g/mol correspond-
ing to acacia gum) was still available for fermentation in the
simulated distal colon (Appendix: Supplementary Fig. S2).
Moreover, comparison of the samples from the different ex-
perimental weeks showed that the remaining fractions of acacia
gum decreased towards the end of the treatment period, in-
dicating a further adaptation of the microbial community
metabolism for AG.
The data related to the delta in increase/decrease (values
of the treatment period minus values of the control period) of
the activity of the enzymes potentially involved in the break-
down of the tested blends are shown in Table 2. When dosing
Blend 1, the alpha-galactosidase, alpha-L-arabinofuranosidase
and beta-galactosidase activities were proportionally mainly
enhanced in the simulated AC. In contrast, Blend 2 led to a more
216 Journal of Functional Foods 16 (2015) 211–222

Table 2 – Increase or decrease of the average enzymatic activity ± standard deviation (U/mL) calculated as the delta
between the average of the treatment period (observations = 3) and the average of the control period (observations = 2) in
the ascending (AC), transverse (TC) and descending colon (DC) of the SHIME systems treated with Blends 1 and 2. No
significant differences between the two blends were observed by means of a Wilcoxon signed-rank.
Blend 1 Blend 2
AC TC DC AC TC DC
Alpha-L-arabinofuranosidase 1.6 ± 1.1 3.4 ± 1.2 2.4 ± 1.7 0.7 ± 0.5 3.9 ± 1.7 8.2 ± 4.4
Alpha-galactosidase 7 ± 1.5 7.8 ± 3.2 5.1 ± 4 4 ± 2.4 9.5 ± 3.1 7.3 ± 3.5
Beta-galactosidase 8.4 ± 2.6 −0.7 ± 12.5 2.3 ± 6 2.4 ± 2.6 3.6 ± 9.2 4.4 ± 8.6
Beta-glucosidase 0±0 0±0 0±0 0.1 ± 0 0.1 ± 0.1 0.05 ± 0
Beta-D-xylosidase 1.2 ± 2.7 0 ± 2.0 −1 ± 2.2 1 ± 2.1 2.8 ± 2.6 2.7 ± 2.4

balanced increase of the activity of all the tested enzymes (i.e.
alpha-L-arabinofuranosidase, alpha-galactosidase, beta-
galactosidase, beta-glucosidase, beta-D-xylosidase) along the
entire simulated colonic compartment.
Finally, a short-term measurement (up to 48h) of gas pro-
duction confirmed the difference in the fermentation profile
of the two blends, with Blend 1 leading to a higher gas pro-
duction as compared to Blend 2 (p = 0.06) (Fig. 1c).
3.2. Analysis of the microbial community composition
3.2.1. Quantitative PCR and denaturing gradient gel
electrophoresis
qPCR and DGGE were used to monitor main changes in mi-
crobial groups along the simulated colon compartments.
When comparing the control period and the last two weeks
of treatment, administration of both blends resulted in a

Fig. 2 – Quantitative PCR results: qPCR data from each bacterial group presented per experimental week in each colon
compartment. (A) Total bacteria; (B) Bacteroidetes; (C) Firmicutes; (D) Bifidobacteria; (E) Lactobacilli. Samples were marked
as: CTRL = control, TREAT = treatment; 1, 2 or 3 indicates the specific week during either the control or treatment period in
which the samples were collected. For each bar is indicated the standard error of the technical replicate of the qPCR (n = 3).
* = the difference from the average of the control is statistically significant according to a t-test (P < 0.05) (n ≥ 6). To compare
the effect of the 2 Blends, a linear spline model was applied, with the knot positioned on the first week of treatment.
Samples with different letters are statistically different.
 Journal of Functional Foods 16 (2015) 211–222 217

statistically significant increase of lactobacilli (p < 0.01) and
bifidobacteria (p < 0.05) in all colon compartments (Fig. 2). Ad-
ministration of both blends increased the counts of the number
of total bacteria and bacteria belonging to the phylum of
Firmicutes. However, when considering the first experimen-
tal weeks, Blend 2 initially led to a decrease in the 16S rRNA
genes copy number for total bacteria in the simulated AC. This
decrease mainly correlated with the decrease of the domi-
nant Firmicutes and Bacteroidetes phyla in the same colon
compartment. Finally, Blend 2 induced also a statistically sig-
nificant increase in Bacteroidetes in the simulated descending
colon (Fig. 2).
To compare the effect of the 2 blends on the different bac-
terial groups, we applied a longitudinal statistical approach that
allowed comparison of the different trends induced by the treat-
ments. A linear spline model was used to fit the data positioning
the knot on the first week of treatment, in order to take into
account the extent of delay in the treatment effect.
Both blends showed a bifidogenic effect. When comparing
the effect of the blends, the increase in bifidobacteria induced
by Blend 1 was statistically higher than Blend 2. Conversely,
Blend 2 induced a higher increase in lactobacilli in the simu-
lated ascending colon as compared to Blend 1.
DGGE analyses on total bacteria, bifidobacteria and lacto-
bacilli were used to evaluate the qualitative changes that
potentially occurred within the structure of the microbial com-
munity during the treatment period. Data are presented as a
cluster analysis conducted on a composite dataset of the three
gels using the unweighted pair group with mathematical av-
erages (UPGMA) and distance matrices of each DGGE gel, based
on the Pearson correlation similarity coefficients (Fig. 3). For
both blends, samples from treatment week 1 tended to cluster

100
15

20

25

30

35

40

75

80

85

95
45

50

55

60

65

70

90
CTRL2 TC
CTRL2 DC
CTRL2 AC
CTRL1 TC
CTRL1 DC
CTRL1 AC
TREAT1 TC
TREAT1 DC
TREAT1 AC
TREAT2 TC
TREAT2 DC
TREAT2 AC
TREAT3 AC
TREAT3 TC
TREAT3 DC

b
100
70

72

74

76

78

80

82

84

86

88

90

92

94

96

98

CTRL2 DC
CTRL2 AC
CTRL2 TC
CTRL1 TC
CTRL1 DC
CTRL1 AC
TREAT1 TC
TREAT1 DC
TREAT1 AC
TREAT2 AC
TREAT2 TC
TREAT2 DC
TREAT3 DC
TREAT3 AC
TREAT3 TC

Fig. 3 – DGGE dendrograms: Clustering analysis for a composite dataset, created by the combination of the information
contained within the DGGE gels for total bacteria, lactobacilli and bifidobacteria from Blend 1 (a) and Blend 2 (b) SHIME
experiments. The analysis on the composite dataset was performed based on the Pearson product–moment correlation
coefficient and the result is presented as dendrogram by using UPGMA linkage. Samples were marked as: CTRL = control,
TREAT = treatment; 1, 2 or 3 indicates the specific week during either the control or treatment period in which the samples
were collected for DNA extraction; AC, TC or DC indicates respectively ascending, transverse and descending colon.
218 Journal of Functional Foods 16 (2015) 211–222
separately from the other treatment samples and more closely
to the samples from the control period, confirming that long-
term administrations of the blends are needed to induce gradual
changes towards the development of a new community
structure.
3.3. Illumina deep sequencing
Analysis of the illumina data to evaluate the composition of
the microbial community confirmed a different modulating
effect of the 2 blends in the different simulated colon com-
partments. The number of phylogenetically unclassified reads
had a decreasing trend when comparing the AC with TC and
DC in the control period (Appendix: Supplementary Fig. S3).
Moreover, a decreasing trend was also observed in each simu-
lated colon compartment when comparing control vs.
treatment. Hierarchical clustering of phylogenetic families
showed a separation of treatment and control samples, as well
as a clustering of gut sectors (Fig. 4a). Following the treat-
ment, AC sectors were more separated from the other colon
compartments and showed a closer clustering, while DC
samples showed the least stable clustering (indicative of the
fact that the 2 blends had a different effect in the distal colon).
The analysis of the functional genes associated to the dif-
ferent simulated colon compartments is shown in Fig. 4b, c and
d. A NMDS ordination of the KO abundances showed a clear
separation between treated and control samples on axis 1
(Fig. 4b). On the contrary, the simulated gut sectors are mostly
close to each other under all conditions and show a similar
distribution along axis 2 independently of the treatment.
However, in a test blocked for the type of treatment and control,
it is possible to observe that there were differences along the
simulated colon (Fig. 4c). For instance, while the cell struc-
ture biosynthesis indicated a higher growth rate in the
simulated proximal colon and in the control samples, fermen-
tation and amino-acid degradation increased mainly in the
simulated distal colon and were higher for the samples from
the treatment period. Significant functional differences were
observed also when comparing control vs. treatment (analy-
sis blocked for the gut region – Fig. 4d). Following the treatment
with both blends, it was possible to observe a higher abun-
dance of genes involved in fermentation (e.g. glutamate
degradation via hydroxyglutarate; pyruvate fermentation to
ethanol; pyruvate fermentation to propionate via acrylate
pathway; and acetyl-CoA fermentation to butyrate), protein and
lipid degradation (e.g. triacylglycerol degradation; fatty acid beta-
oxidation; acetoacetate degradation to acetyl CoA) and
carboxylates degradation. Finally, during the treatment with
Blend 1 and Blend 2, glycolysis was substituted by the faster,
but less energy-yielding Entner–Doudoroff pathway (Fig. 4d).
This effect was stronger in the distal colon for Blend 2.
4. Discussion
The objective of this study was to compare the effect of long-
term repeated administration of an enteral feed containing two
different fibre blends on the composition and metabolism of
the gut microbiota.
Blend 1 is a common ingredient mixture in enteral nutri-
tion formulas, and has been previously tested in vivo. In these
studies, it was associated with significantly increased lacto-
bacilli in preadolescent cancer patients (n = 67), a bifidogenic
effect in children (n = 140) following antibiotic treatment, and
an enhanced IgG antibody response to vaccination in infants
(Klosterbuer, Roughead, & Slavin, 2011). Due to its composi-
tion of readily fermented fructans (i.e. FOS and inulin), Blend
1 is predominantly fermented in the proximal colon
(Macfarlane, Gibson, & Cummings, 1992). Moreover, the single
ingredients of the blend have also been positively associated
to biomarkers of large bowel health in rat studies (Paturi et al.,
2015). Although this blend has an established prebiotic benefit,
low amounts of fibre have to be dosed in order to minimize
potential gastrointestinal discomfort due to rapid fermenta-
tion (Barrett & Gibson, 2012). In order to extend the fermentation
along the colon it was decided to replace part of FOS and inulin
with AG. The latter, due to its more complex structure (a poly-
saccharide with high MW), has been recently shown to be
fermented more distally in the colon (Terpend et al., 2013). It
was therefore hypothesized that the addition of AG would
extend positive physiological effects to the distal colon without
significantly impacting the prebiotic activity of the original
blend. Moreover, the prolonged fermentation was also sus-
pected to beneficially impact the tolerance of the fibre blend.
By means of a dynamic, multi-compartment gastrointes-
tinal simulator representative for the conditions of the human
ascending, transverse and descending colon, we were able to
show that the two blends had a different fermentation profile
and induced different colonic region-specific effects (Fig. 5).
Indeed, the partial substitution of FOS and inulin with AG re-
sulted in a more gradual fermentation of the blend along the
entire simulated colon. This main conclusion was supported
by several analyses conducted on the samples collected from
the different areas of the simulated colon compartments. SEC
analysis and the analysis of the pH profiles showed that while
Blend 1 was mainly fermented in the proximal colon, Blend 2
was more gradually fermented, with some fractions of the
product being still available in the distal colon. SEC analysis
also showed that the efficiency of bacteria using the AG added
in Blend 2 increased during the 3 weeks of treatment. These
data and the adaptation property are in line with what was
described by Terpend et al. (2013) on the fermentation of AG.
Moreover, the lower gas production induced by Blend 2 was
further evidence of the gradual fermentation of this product
(Fig. 1c). In line with these findings, Koecher, Thomas, and Slavin
(2015) showed – in a randomized, double-blind, crossover trial
with 20 healthy subjects – that the consumption of a blend very
similar to Blend 2 used in this work (i.e. a mix of FOS, inulin,
AG and pea hull fibre) led to less negative symptoms related
to bowel urgency and no problem with gas production and
bloating symptoms.
The SCFA data gave indications of a different but benefi-
cial fermentation profile for the two blends, especially when
considering the net production in the simulated TC compart-
ment (Fig. 5). When evaluating differences in SCFA production
between control and treatment, the standard deviation of the
average of the 3 samples collected during the first week of treat-
ment resulted to be high (Table 1). This relates to the adaptation
period the bacteria needed to modulate their metabolism to
 Journal of Functional Foods 16 (2015) 211–222 219

a) b)
0.5

0.15
AC1_TREAT
0.4

0.10
AC2_CTRL
AC1_CTRL AC2_TREAT
0.3

NMDS D2 (27.79%)
0.05
0.2

TC1_TREAT

0.00
TC2_TREAT
TC1_CTRL
TC2_CTRL

−0.05
0.1

DC1_CTRL DC2_TREAT
DC1_TREAT

−0.10
0.0

AC2_TREAT
TC2_TREAT

DC2_TREAT
AC2_CTRL

AC1_CTRL

TC1_CTRL

DC1_CTRL

DC1_TREAT

TC1_TREAT

AC1_TREAT
TC2_CTRL

DC2_CTRL

−0.15
DC2_CTRL
−0.15 −0.10 −0.05 0.00 0.05 0.10 0.15
NMDS D1 (55.24%)

c) cell−structure−biosynthesis fermentation amino−acid−degradation

p= 0.02237 p= 0.01608 p= 0.0186
0.0044

AC
TC
0.0020

DC
log10 Read Count

0.0040
0.0038

0.0016

0.0030
0.0032

d1

d2
d2

d1
d1

L
d2

TR

TR
en

TR
en
en
en
en
en
Bl

Bl

Bl
Bl

Bl

C
Bl

C
d) fatty−acid−and−lipid−degradation
p= 0.0221
protein−degradation
p= 0.01413
fermentation
p= 0.01574

Blend1
0.00055

Blend2
0.0020

CONTROL
log10 Read Count

4e−05
0.00035 0.00045

2e−05

0.0016
0e+00
AC

TC

AC

TC

AC

TC

C
D

carboxylates−deg glycolysis−variants MC0098:Entner−Doudoroff pathway I

p= 0.01246 p= 0.02788 p= 0.01246
3.0e−05
0.00075
log10 Read Count
0.0010

1.5e−05
0.00065
0.0008

0.0e+00

AC

TC

C
AC

TC

C
AC

TC

D
D
D

Fig. 4 – 16S rRNA-targeted Illumina sequencing data on samples from the end of control and treatment periods:
(a) hierarchical clustering based on phylogenetic families and (b) NMDS (Non-metrical dimensional scaling) ordination of
the KO abundances for samples belonging to ascending (AC), transverse (TC) and descending (DC) colon compartments of
the SHIME treated with Blend 1 and Blend 2 during the last week of control (CTRL) and the last week of treatment (TREAT)
period. Each colon compartment is indicated with numbers 1 or 2 depending on which Blend was tested. D1 and D2 refer to
the first 2 dimensions of the NMDS analysis. (c) Selected significant functional differences between AC, TC and DC in a test
blocked for the type of treatment.(d) Significant functional differences between Blends 1 and 2 and the control samples, in a
test blocked for the colon region.
220 Journal of Functional Foods 16 (2015) 211–222
Fig. 5 – Schematic summary of the blends effect in the
colon: Summary of the effects that Blend 1 and Blend 2
induced in the different areas of the simulated colon with
respect to: pH, ammonium (NH3), positive net production of
acetate (A), propionate (P) and butyrate (B) (the relative
letter is present in a colon section only if the treatment
induced a positive net production of the specific SCFA),
glycolysis (GL), Entner–Doudoroff pathway (ED),
bifidobacteria (Bif), lactobacilli (Lactos) and residual amount
of substrate to be fermented (*).
the new nutritional environment. In parallel with the above,
a decrease in ammonium production was observed during the
treatment with both blends, indicative of the fact that both
products were well-fermented by bacteria.
In this study, an illumina metagenomic approach has been
used for the first time in a SHIME experiment. The analysis of
the functional genes at DNA level (i.e. potentiality of the mi-
crobial metabolic activity) confirmed that differences occurred
between the two blends in the simulated colon compart-
ments. Among them, the most interesting effect is that related
to the switch between the energy pathways of glycolysis and
Entner–Doudoroff (ED). ED is commonly used by bacteria when
carbon sources are not limiting in a system for carbohydrate
metabolism: it is faster even if less energy-yielding (1 ATP/
glucose vs. 2 ATP/glucose of the glycolysis) (Conway, 1992). For
both blends, glycolysis decreased while the ED pathway in-
creased and this effect was colon-sector specific (Fig. 4d). More
specifically, with Blend 1 – which was fermented more proxi-
mally – the decrease in genes involved in glycolysis was stronger
in the simulated AC. On the contrary, with Blend 2, the de-
crease in glycolysis was more balanced and extended also to
the simulated DC. An opposite and complementary effect was
observed for the genes involved in the ED pathway (i.e. the in-
crease for Blend 2 in the distal colon was bigger than that induced
by Blend 1).
The analyses on the microbiota (qPCR, DGGE and illumina)
further confirmed a different modulating effect of the 2 blends
in the simulated colon compartments. In fact, the hierarchical
clustering of phylogenetic families showed that the TC and DC
sectors of the SHIME treated with Blend 2 tended to cluster
much closer following the treatment as compared to the re-
spective control period, indicative of a modulation of the
microbiota induced by the treatment itself. Moreover, when
comparing the 2 SHIME systems, the least stable clustering was
associated with the DC sectors. This was again indicative of
the fact that the two blends had a differential effect in the distal
colon. DGGE analyses showed that long-term repeated admin-
istrations of the blends are needed to reach gradual changes
in the gut microbial community structure. The increased con-
centration of Bacteroidetes in the simulated DC upon Blend 2
administration further confirmed the more gradual fermen-
tation of AG in the distal colon. Bacteroidetes are considered
as very important saccharolytic fermenting bacteria, as a large
part of the proteins codified by this phylum goes to breaking
down polysaccharides and metabolizing their sugars (Karlsson,
Ussery, Nielsen, & Nookaew, 2011). Also bacteria belonging to
the phylum of Firmicutes – frequent users of the metabolic in-
termediates produced by the metabolism of Bacteroidetes –
showed a similar trend. Both blends had a positive effect on
bifidobacteria and lactobacilli – two genera indicated as po-
tentially beneficial bacteria – showing that the new composition
of Blend 2 supported this potential health-promoting effect
(Ventura et al., 2007).
The aim of this work was to show that the structural char-
acteristics of AG (i.e. a polysaccharide with high MW) were
linked to a distal fermentation of the product. In this type of
studies, the relevance of the data in terms of potential
interindividual variability (i.e. different effect of the test product
due to a different composition of the gut microbiota) is always
questionable. In this respect, we can conclude that the distal
fermentation of AG appears to be independent of the tested
microbiota as we were able to show this specific fermenta-
tion pathway working with a mix of faecal samples from three
different donors (Terpend et al., 2013), with a new indepen-
dent donor in this study and also with a third donor as shown
in Appendix: Supplementary Fig. S4 (unpublished results).
Moreover, most studies on gut microbiota are based on the
analysis of faecal samples because they are easily collected in
a non-invasive manner (Durban et al., 2011). However, the rep-
resentativeness of these samples for the microbial processes
occurring along the colon may be questioned (e.g. SCFA that
are produced in the ascending colon are readily absorbed or
used in cross-feeding reactions). This work shows that the use
of in vitro models provides a tool for looking at potential changes
in other colonic areas that may not be or are poorly repre-
sented by a faecal sample (i.e. proximal colon). This approach
represents an interesting preliminary or complementary tool
to in vivo trials in order to elucidate the potential mechanism
of action of a given product.
5. Conclusions
This work suggests that inulin/FOS and acacia gum may exert
an interesting complementary effect. In fact, the partial re-
placement of FOS and inulin by acacia gum changed the
intestinal fermentation profile from boost fermentation in the
 Journal of Functional Foods 16 (2015) 211–222
proximal colon into a more gradual fermentation in the com-
plete colon. Blend 2 extended the benefits of fermentation (e.g.
decreased ammonium production, acidification, SCFA produc-
tion) to the distal colon, and at the same time attenuated gas
production. Further in vivo studies are needed to link the outcome
of this study to the possibility of a greater provision of fibre in
enteral products, with potentially reduced risk of intolerance.
Acknowledgments
The authors would like to thank Tim Lacoere for his support
on graphical design, and Eva Ackermann and Terri Born for their
work on preparing the fibre samples.
M.M., F.H. and J.R. are funded by the Research Foundation
Flanders (FWO, Belgium). F.H. and J.R. are also funded by the
Brussels Institute for Research and Innovation, the EU Frame-
work 7 programme (MetaCardis Health-F4-2012-305312), VIB,
the REGA institute and KU Leuven. Support for this study was
provided by Nestlé Health Science S.A. (Vevey, Switzerland).
Appendix: Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.jff.2015.04.039.
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