Diets that differ in their FODMAP content alter the
colonic luminal microenvironment
Emma P Halmos,
Claus T Christophersen,
Anthony R Bird,
Susan J Shepherd,
Peter R Gibson,
Jane G Muir
▸ Additional material is
published online only. To view
please visit the journal online
Department of Gastr oenterology ,
Eastern Health Clinical School,
Monash University , Box Hill,
Victoria, Austr alia
Clinical School, Monash
University, Melbourne, Victoria,
Food Futures National
Commonwealth Scientiﬁc and
Organisation, Food, Animal
and Health Sciences, Adelaide,
South Australia, Australia
Dr Emma P Halmos,
Clinical School, Monash
University, Level 6 The Alfred
Centre, 99 Commercial Road,
Melbourne, VIC 3004,
Received 18 March 2014
Revised 9 June 2014
Accepted 24 June 2014
Published Online First
12 July 2014
To cite: Halmos EP,
Christophersen CT, Bird AR,
et al. Gut 2015;64:93–100.
Objective A low FODMAP (Fermentable
Oligosaccharides, Disaccharides, Monosaccharides And
Polyols) diet reduces symptoms of IBS, but reduction of
potential prebiotic and fermentative effects might adversely
affect the colonic microenvironment. The effects of a low
FODMAP diet with a typical Australian diet on biomarkers
of colonic health were compared in a single-blinded,
randomised, cross-over trial.
Design Twenty-seven IBS and six healthy subjects were
randomly allocated one of two 21-day provided diets,
differing only in FODMAP content (mean (95% CI) low
3.05 (1.86 to 4.25) g/day vs Australian 23.7 (16.9 to 30.6)
g/day), and then crossed over to the other diet with ≥21-
day washout period. Faeces passed over a 5-day run-in on
their habitual diet and from day 17 to day 21 of the
interventional diets were pooled, and pH, short-chain fatty
acid concentrations and bacterial abundance and diversity
Results Faecal indices were similar in IBS and healthy
subjects during habitual diets. The low FODMAP diet was
associated with higher faecal pH (7.37 (7.23 to 7.51) vs
7.16 (7.02 to 7.30); p=0.001), similar short-chain fatty
acid concentrations, greater microbial diversity and reduced
total bacterial abundance (9.63 (9.53 to 9.73) vs 9.83
(9.72 to 9.93) log
copies/g; p<0.001) compared with
the Australian diet. To indicate direction of change, in
comparison with the habitual diet the low FODMAP diet
reduced total bacterial abundance and the typical
Australian diet increased relative abundance for butyrate-
producing Clostridium cluster XIVa (median ratio 6.62;
p<0.001) and mucus-associated Akkermansia muciniphila
(19.3; p<0.001), and reduced Ruminococcus torques.
Conclusions Diets differing in FODMAP content have
marked effects on gut microbiota composition.
The implications of long-term reduction of intake of
FODMAPs require elucidation.
Trial registration numb er ACTRN12612001185853.
IBS often requires multimodal management
approaches that include psychological, dietary and
pharmacological domains. Dietary therapies are
gaining popularity as evidence of efﬁcacy for
speciﬁc diets have emerged. One strategy is to restrict
intake of poorly absorbed short-chain carbohydrates,
termed FODMAPs (F ermentable Oligosaccharides,
Disaccharides, Monosaccharides And Polyols). The
evidence-base for efﬁcacy of the low FODMAP diet
with a recent blinded placebo-
controlled cross-over study conﬁrming pr evious
studies that about 75% of patients gain clinically sig-
The low FODMAP diet is increas-
ingly being applied by health professionals in patients
with IBS as ﬁrst-line therapy.
Given the high preva-
lence and chronic nature of IBS, it is possible that
many people will restrict intake of FODMAPs over
months to years.
Signiﬁcance of this study
What is already known on this subject?
▸ A diet low in Fermentable Oligosaccharides,
Disaccharides, Monosaccharides And Polyols
(FODMAPs) reduces GI symptoms in
approximately 75% of patients with IBS.
▸ FODMAPs are fermentable substrates and some
have prebiotic effects with putative beneﬁts on
▸ A randomised parallel group study showed a
reduction of the proportion and concentration of
faecal Biﬁdobacteria spp on a dietitian-taught
low FODMAP diet compared with a habitual diet.
What are the new ﬁndings?
▸ Diets that differ in their FODMAP content are
associated with considerable changes to the
structure of the faecal microbiota.
▸ A provided low FODMAP diet reduces total
bacterial abundance but has no effect on
relative abundance of bacteria putatively
associated with colonic health.
▸ The higher FODMAP intake of the provided
typical Australian diet compared with that of the
low FODMAP or habitual diets was associated
with speciﬁc stimulation of the growth of
bacterial groups with putative health beneﬁts.
▸ No alterations in faecal short-chain fatty acid
concentrations were associated with differences
in FODMAP ingestion.
How might it impact on clinical practice in
the foreseeable future?
▸ A low FODMAP diet should not be
recommended for asymptomatic populations.
▸ Caution should be taken when recommending
the low FODMAP diet long-term.
▸ Liberalising FODMAP restriction to the level of
adequate symptom control should be exercised
to use the potential health beneﬁts of higher
FODMAP intake on the gut microbiota.
Halmos EP, et al. Gut 2015;64:93–100. doi:10.1136/gutjnl-2014-307264 93
FODMAPs are being increasingly used in food industry as
prebiotics, either formulated into various types of products or
manufactured as supplements, to promote colonic health.
Evidence is strong for the prebiotic actions of oligosacchar-
Reduced FODMAP delivery to colonic microbiota
might, therefore, have deleterious effects on the growth of bac-
teria with potentially favourable health effects. Indeed, a rando-
mised parallel group study where the effects on faecal
microbiota of a dietitian-taught low FODMAP diet compared
with those of a habitual diet indicated a reduction of the pro-
portion and concentration of Biﬁdobacteria spp,
ﬁrst evidence for potentially unfavourable effects of the diet.
FODMAPs are also substrates for fermentation by bacteria
not considered to be prebiotic.
Bacterial fermentation of car-
bohydrates yields short-chain fatty acids (SCFAs), including
butyrate, the major energy substrate for the colonic epithe-
Butyrate is also a key regulator of colonocyte prolifer-
ation and apoptosis, and has immunomodulatory effects.
these ways, fermentable carbohydrates delivered to the colon
have potential anticarcinogenic and anti-inﬂammatory actions.
Restriction of FODMAPs delivered to the colon to reduce gas
production, subsequent luminal distension and GI symptoms
might consequently have adverse effects on colonic health.
The present study aimed to address the hypothesis that a low
FODMAP diet recommended for reduction of IBS symptoms will
have adverse effects on colonic luminal microenvironment. This
was investigated by comparing the effects of a low FODMAP diet
with those of a FODMAP content of a typical Australian diet on
faecal microbiota and biomarkers related to colonic health.
Additional comparison with faecal indices while subjects con-
sumed their own diet was also performed. The bacteria targeted
were chosen on the basis of being avid butyrate-producers with
anti-inﬂammatory association for some, traditional ‘prebiotic’ bac-
teria and representatives of mucus-associated bacteria that have
putative health-promoting or detrimental effects.
studied were participating in a randomised controlled efﬁcacy trial
of the two diets where almost all food was provided and included
patients with IBS and healthy subjects.
The study participants have been previously described in detail.
Brieﬂy, patients with IBS as deﬁned by Rome III criteria
healthy controls were recruited via advertisements and word of
mouth. Exclusion criteria comprised coeliac disease, previous
abdominal surgery, comorbid conditions such as diabetes, and
inability to understand English. No participant had previously
visited a dietitian for management of GI symptoms, and had not
used antibiotics or probiotics for 2 weeks prior to study com-
mencement. Fibre supplements, laxatives and antidiarrhoeal
medications were not taken during the trial.
The study protocol has also been recently described in detail.
Brieﬂy, for 1 week, participants recorded their habitual dietary
intake and a 5-day faecal sample was collected (as outlined below).
Participants were then randomised according to a computer-
generated order to receive 21 days of a diet low in FODMAPs or
21 days of a diet containing FODMAP content of a typical
Australian diet. Participants were blinded to the diets and almost
all food was provided.
After this 21-day diet, each participant
entered a washout period of at least 21 days in which they
resumed their usual diet and then crossed over to the alternate
diet. From day 3 to day 7 of the habitual diet and day 17 to day 21
of interventional diets, participants collected all faeces passed.
Just prior to the faecal collection (morning of day 3 of habitual
and day 17 of interventional diets), participants swallowed a
capsule containing 24 radiopaque markers (Sitzmarks, Konsyl
Pharmaceuticals, Maryland, USA). The time and date of capsule
ingestion was noted. Participants were instructed to collect each
stool in a supplied plastic container and to avoid urine contamin-
ation. The containers were sealed and immediately stored in a
portable freezer (Waeco Paciﬁc, Queensland, Australia). Each con-
tainer was marked with the date and time of stool passage. The
freezers were delivered to the laboratory within the week follow-
ing collection. Stools were X-rayed and radiopaque markers
counted to determine whole gut transit time (WGTT) based on
time of stool passage.
All participants gave written informed consent prior to study
commencement. The study protocol was approved by the Eastern
Health and Monash University Human Research and Ethics
Committees. The protocol was registered with the Australian New
Zealand Clinical Trials Registry (ACTRN12612001185853).
Participants were supplied with three main meals and three
snacks daily as previously described.
The provided food was
free of charge and delivered to participants’ homes weekly. All
food consumed was recorded in diaries; dietary adherence was
based on these records.
The nutritional contents of the interventional diets were ana-
lysed using the Foodworks program (Xyris Software Pty Ltd;
Brisbane, Queensland, Australia) except for FODMAPs, which
were analysed using high performance liquid chromatography
and enzymatic assays.
The interventional diets differed
only in FODMAP-content6 as shown in table 1. Matching of
the diets for total ﬁbre and resistant star ch was achiev ed adding
psyllium and Hi-Maize 220 (Nat ional Starch and Chemical
Table 1 The mean daily nutrition information of provided low and
typical Australian FODMAP diets
Per day Typical Australian diet Low FODMAP diet p Value
Energy (MJ) 8.17 (7.37–8.97) 8.17 (7.09–9.24) NS
Protein (g) 96.1 (84.7–107) 98.1 (83.7–113) NS
Fat (g) 71.6 (49.4–93.8) 74.4 (51.9–97.0) NS
Total carbohydrate (g) 219 (180–259) 215 (181–249) NS
Sugars (g) 120 (103–137) 122 (106–139) NS
Starch (g) 94.0 (52.8–135) 95.4 (59.7–131) NS
Total dietary fibre* (g) 29.7 (23.9–35.7) 30.4 (24.2–36.5) NS
Fibre† (g) 25.9 (21.3–30.6) 23.4 (18.7–28.2) NS
Resistant starch‡ (g) 3.74 (1.85–5.63) 6.93 (3.56–10.3) NS
Total FODMAPs (g) 23.7 (16.9–30.6) 3.05 (1.86–4.25) <0.001
Oligosaccharides (g) 5.49 (2.34–8.65) 1.57 (0.47–2.66) 0.009
Polyols (g) 4.21 (2.57–5.85) 0.20 (−0.04–0.44) 0.002
Lactose (g) 1.35 (0.20–2.49) 0.05 (−0.01–0.10) 0.033§
Fructose in excess
of glucose (g)
–17.3) 1.24 (0.41–2.07) 0.001
Diets were matched for all nutrients except daily FODMAPs, indicated in bold (paired
*Total dietary fibre comprises fibre and resistant starch.
†The low FODMAP diet was supplemented with a daily average of 3 g psyllium.
‡The low FODMAP diet was supplemented with a daily average of 5 g Hi-Maize 220
(National Starch and Chemical Company; Bridgewater, New Jersey, USA).
§While there is a significant difference in lactose, 5 g lactose per sitting is considered
well absorbed and tolerated in majority of people
FODMAP, Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols;
NS, not significant.
94 Halmos EP, et al. Gut 2015;64:93–100. doi:10.1136/gutjnl-2014-307264
Co mpany, New Jersey, USA), r espectiv ely.
For the typical Austr alian
diet, 4.4 g oligosaccharides, 2.6 g polyols and 23.7 g total
FODMAP s were consumed daily as guided by the Monash
Complete Nutritional A ssessment Q ues tionnaire.
The lo w
FODMAP diet aimed to keep oligosa ccharide, fructose in excess of
glucose and polyol content <0.5 g per sitting.
Average daily intake
was estima ted 1.6 g oligosaccharides, 0.2 g polyols and 3.1 g total
FODMAP s. Both diets wer e lo w in lactose (<5 g per sitting).
On delivery to the laboratory, the 5-day faecal samples were
defrosted, pooled and mixed, then transferred into small speci-
men containers. Samples were packed on dry ice and delivered
to the Commonwealth Scientiﬁc and Industrial Research
Organisation Animal, Food and Health Sciences (Adelaide,
South Australia), where they were thawed at 4°C, then trans-
ferred to an anaerobic chamber and aliquoted for further ana-
lysis. All samples were stored at −20°C until analysed for SCFA
and bacteria abundances. Commonwealth Scientiﬁc and
Industrial Research Organisation investigators were blinded to
the treatments, but not the patient cohorts.
Faecal contents were analysed in duplicate for SCFA by gas
chromatography and pH, as described previously.
Concentrations of the total SCFA (sum of SCFA) and individual
SCFA including branched-chain fatty acids (BCFA) were reported
as mmol/g of faecal matter.
DNA was extracted from 0.25 g faecal matter using a
repeat-bead-beating plus column method.
Total bacteria as
well as butyrate-producing bacteria Clostridium cluster IV
(Clostridium leptum group) including Faecalibacterium prausnit-
zii and Clostridium cluster XIVa (Clostridium coccoides group)
and Roseburia spp, prebiotic bacteria (Lactobacilli and
Biﬁdobacteria spp), and mucus-degrading bacteria (Akkermansia
muciniphila, Ruminococcus gnavus and Ruminococcus torques)
were analysed. These bacteria were chosen for analysis to specif-
ically examine the role of dietary FODMAPs on bacteria
thought to be markers of inﬂammation and bacteria traditionally
thought to be good markers of prebiotic effects.
Detailed methodology of the primers and optimised quantita-
tive real-time PCR conditions used are summarised in online
supplement 1 in addition to microbial diversity and lactate and
Comparison of microbiota with symptom response
In order to assess whether patterns of microbiota predicted differ-
ences in symptoms experienced during the two interventional
diets, IBS subjects were arbitrarily categorised as non-responders
or good responders as deﬁned by mean overall symptom scores in
the last 14 days of the low FODMAP diet <10 mm or >20 mm
below those in the typical Australian diet, respectively.
Whole gut transit time
WGTT was calculated as previously described.
purpose of WGTT analysis, patients with IBS were further sub-
classed as diarrhoea-predominant (IBS-D), constipation-
predominant (IBS-C), patients with IBS with existing diarrhoea
and constipation (IBS-M) and patients with IBS with neither
diarrhoea nor constipation (IBS-U).
Sample size estimations were calculated based on GI symptoms
which have been previously published.
The data from patients
with IBS and healthy subjects were examined separately and
pooled (given the similarity between the results in the two
groups). Subject age and body mass index were presented as
median (IQR). A p value ≤0.05 was considered statistically sig-
niﬁcant for data describing participant demographics, WGTT,
faecal pH and lactate and succinate. Data of faecal character-
istics were parametric, except for bacterial abundance, which
were normalised by log
conversion before further analysis. All
raw faecal characteristic data were presented as mean (95% CI).
Differences of faecal characteristics between the interventional
diets were shown as a ratio of low FODMAP compared with
typical Australian diet and did not ﬁt a normal distribution.
Hence, changes in bacterial abundance and SCFA were com-
pared between individual diets by Wilcoxon matched-pairs
signed rank test with Bonferroni corrections. Habitual diet data
comparing participant groups were analysed by unpaired t test
with Bonferroni correction; p≤0.006 was considered statistically
signiﬁcant for SCFA observations, p≤0.005 for absolute bacter-
ial abundance and p≤0.006 for relative bacterial abundance
observations. Multivariate analysis of denaturing gradient gel
electrophoresis (DGGE)-banding patterns was performed using
the Primer 6+Permanova package (PRIMER-E Limited,
Plymouth, UK), with the assumption that each DGGE band
represents one phylotype. Data were analysed using a
Bray-Curtis similarity matrix on fourth root-transformed data.
Differences between diets on the basis of DGGE-banding pat-
terns were calculated with a one-way and pairwise permanova
analysis. All statistical tests, unless speciﬁed, were analysed with
GraphPad Prism V.6 and SPSS V.20 programs.
Thirty-eight participants (30 IBS and 8 healthy controls) com-
pleted the study. Of the six IBS subjects who ceased the typical
Table 2 Comparison of subject demographics and habitual diet
characteristics between IBS and healthy cohorts
controls (n=6) p Value
Female* 21 (78%) 5 (83%) NS
Age (years)† 43 (29–54) 31 (23–61) NS
Body mass index (kg/m
)† 24 (23–27) 24 (23–29) NS
Habitual dietary intake
Energy (MJ) 9.1 (8.3 to 10) 8.3 (7.1 to 9.5) NS
Protein (g) 94.7 (84.3 to 105) 91.4 (72.3 to 110) NS
Fat ( g) 87.6 (76.9 to 98.3) 81.7 (63.2 to 100) NS
Carbohydrate (g) 236 (207 to 266) 204 (144 to 264) NS
Sugars (g) 110 (88.8 to 130) 93.4 (58.4 to 128) NS
Starch (g) 140 (124 to 157) 118 (76.8 to 160) NS
Fibre (g) 24.1 (21.2 to 27.0) 22.5 (17.6 to 27.3) NS
Total FODMAPs (g)‡ 16.6 (14.2 to 18.9) 18.2 (11.9 to 24.9) NS
Oligosaccharides (g) 3.9 (3.3 to 4.4) 3.9 (3.2 to 4.7) NS
Polyols (g) 1.6 (1.2 to 2.1) 2.5 (1.1 to 4.0) NS
Lactose (g) 11.1 (8.7 to 13.5) 11.8 (6.1 to 17.4) NS
Fructose (g) 18.7 (14.1 to 23.3) 16.5 (7.1 to 25.9) NS
Glucose ( g) 24.0 (17.7 to 30.3) 22.6 (12.9 to 32.4) NS
Data are presented as mean (95% CI) and compared by unpaired t test except where
*n (percentage of total ); Fisher’s exact analysis used.
‡Total FODMAP content does not include fructose in excess of glucose which cannot
be estimated from Foodworks program.
FODMAP, Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols;
NS, not significant.
Halmos EP, et al. Gut 2015;64:93–100. doi:10.1136/gutjnl-2014-307264 95
Australian diet early due to unbearable symptoms, four com-
pleted faecal collection prior to exiting the study (range 7–
12 days). One IBS and two healthy subjects did not complete
faecal collection. Thus, data from 27 IBS and 6 healthy subjects
were included in analysis. As shown in table 2, the cohorts were
well matched for sex, age and body mass index. Their habitual
diets were also matched in nutrients including FODMAP
content (table 2). Dietary adherence during the interventional
diets was good with all participants adhering to the typical
Australian diet, and 81% of IBS and all healthy participants
adhering to the low FODMAP diet for at least 81% of the
Faecal analysis during habitual diet
Faecal pH, lactate, succinate, SCFA and absolute and relative
bacterial abundance in IBS and healthy subjects during their
habitual diet are shown in table 3. Besides lower isobutyrate and
isovalerate concentrations in patients with IBS when compared
with healthy subjects, there were no differences in other mea-
sured indices. On the habitual diets, WGTT was signiﬁcantly
slower in healthy (67.1 (47.0 to 87.2) h) participants when
compared with IB S-D (31.9 (22.4 to 41.4) h; p=0.001) and
IBS-M subjects (40.6 (21.5 to 59.6) h; p=0.034) (see online
Comparison of biochemical indices on habitual and
As there were few differences in faecal measures during the
habitual diet, IBS and healthy cohorts were combined. Faecal
pH was 0.2 units higher on the low FODMAP in comparison
with the habitual and typical Australian diets (p=0.008). No
differences were seen in total or speciﬁc faecal SCFA or succin-
ate between diets (table 4 and ﬁgure 1A). Only one participant
had complete data for faecal lactate, so could not be analysed.
Molar proportions of the major SCFA were also unchanged
(data not shown).
Absolute and relative abundance of bacteria are shown in
table 5. When analysed as absolute abundance, the low
FODMAP diet had a lower total bacterial load compared with
habitual and typical Australian diets (table 5 and ﬁgure 1B).
Furthermore, absolute abundance of the butyrate-producing
bacteria, the prebiotic bacteria, Biﬁdobacteria spp and the
mucus-associated bacterium, A. muciniphila, was greater on the
typical Australian diet compared with the other two diets
(table 5 and ﬁgure 1B). In terms of relative abundance, the
typical Australian diet increased Clostridium cluster XIVa and A.
muciniphila in comparison with habitual and low FODMAP
diets, but decreased R. torques in comparison with the low
FODMAP diet (table 5 and ﬁgure 1C). Microbial diversity in
the Clostridium cluster XIV was greater in subjects on the low
FODMAP compared with the typical Australian diet and the
habitual diet (table 5). Similar patterns were seen when data
were separated into IBS and healthy cohorts.
Neither dietary changes nor the habitual diet pattern in abso-
lute and relative bacterial abundances predicted symptomatic
difference between the interventional diets in non-responders
and good responders (see online supplement 3).
Altering dietary FODMAPs did not affect WGTT in any
subject cohort, including speciﬁc IBS subtypes. The healthy sub-
jects had a slower WGTT when compared with IBS-D and
IBS-M subjects on all diets as well as IBS-C subjects during the
interventional diets (see online supplement 2). No correlation
between WGTT and the composition of the gut microbiota were
observed (data not shown).
The low FODMAP diet has good evidence of efﬁcacy for
symptom management in patients with IBS.
FODMAPs, especially oligosaccharides, have shown positive
effects on the colonic microenvironment and microbiota in
a low FODMAP diet might impact nega-
tively on colonic health. The present study investigated its
effects in 33 subjects compared with those of a carefully
matched diet representing the typical FODMAP intake in
Australia on markers linked to colonic health through the assess-
ment of WGTT, soluble luminal microenvironment and faecal
microbiota. Marked differences in absolute and relative bacterial
abundance and diversity, but not SCFA or transit were observed.
Table 3 Faecal pH, lactate, succinate, SCFA, absolute and relative
bacterial abundance and bacterial diversity on pooled 5-day
samples in IBS and healthy subjects during their habitual diet
(n=6) p Value
pH 7.19 (7.07–7.31) 7.17 (6.63–7.72) 0.916
Lactate (g/100 g)* 0.02 (0.02–0.03) 0.02 (0–0.04) 0.375
Succinate (g/100 g)* 0.03 (0.02–0.03) 0.04 (0.02–0.06) 0.063
SCFA concentration (mmol/g)
Total SCFA 81.9 (72.3–91.5) 93.4 (58.6–128) 0.334
Butyrate 15.6 (13.0–18.1) 18.9 (9.61–28.3) 0.283
Propionate 15.8 (13.6–18.0) 17.6 (9.53–25.6) 0.526
Acetate 42.5 (37.4–47.5) 44.7 (27.5–61.9) 0.715
Isobutyrate 1.92 (1.66–2.17) 3.10 (2.52–3.68) <0.001
Isovalerate 2.99 (2.56–3.43) 5.06 (3.92–6.19) <0.001
Valerate 2.28 (1.73–2.83) 3.70 (1.62–5.78) 0.044
Caproate† 0.95 (0.58–1.32) 0.46 (−0.12–1.04) 0.175
Absolute abundance (Log
copies of 16S rRNA gene/g)
Total bacteria 9.85 (9.72–9.98) 9.82 (9.61–10.0) 0.861
Clostridium cluster IV 8.40 (8.20–8.59) 8.37 (7.97–8.76) 0.892
7.86 (7.68–8.05) 7.76 (7.12–8.39) 0.643
Clostridium cluster XIVa 8.26 (8.11–8.41) 8.04 (7.66–8.41) 0.193
7.64 (7.46–7.83) 7.50 (6.90–8.10) 0.518
Lactobacilli 6.27 (6.03–6.51) 5.94 (5.49–6.39) 0.219
Bifidobacteria 7.61 (7.37–7.86) 8.07 (7.55–8.58) 0.104
4.07 (3.42–4.72) 4.50 (2.53–6.47) 0.571
Ruminococcus gnavus 7.20 (7.06–7.34) 6.96 (6.70–7.22) 0.129
Ruminococcus torques 6.09 (5.83–6.34) 6.74 (6.37–7.11) 0.025
Relative abundance (percentage of total bacteria)
Clostridium cluster IV 4.05 (3.34–4.77) 3.73 (2.39–5.07) 0.687
F. prausnitzii 1.31 (0.89–1.74) 1.18 (0.19–2.16) 0.778
Clostridium cluster XIVa 2.81 (2.40–3.22) 1.82 (0.97–2.67) 0.037
Roseburia 0.82 (0.57–1.06) 0.67 (0.13–1.22) 0.609
Lactobacilli 0.07 (0.01–0.13) 0.02 (0–0.03) 0.412
Bifidobacteria 1.33 (0.53–2.13) 2.18 (0.48–3.87) 0.348
A. muciniphila 0.01 (0–
0.02) 0.01 (0–0.03) 0.756
R. gnavus 0.30 (0.20–0.40) 0.16 (0.04–0.28) 0.204
R. torques 0.03 (0.01–0.06) 0.12 (0.01–0.23) 0.008
Diversity (Shannon index)
Clostridium cluster XIV 1.83 (1.67–1.98) 1.64 (1.35–1.93) 0.310
Differences in cohorts were analysed by unpaired t test. Statistically significant
differences are shown in bold based upon p≤0.05 for pH, lactate and succinate,
p≤0.006 for SCFA concentrations, p≤0.005 for absolute and p≤0.006 for relative
bacterial abundance after Bonferroni correction.
*Due to difficulties in analysis, for lactate, IBS n=8 and healthy controls n=2.
†Due to difficulties in analysis, for caproate, IBS n=18 and healthy controls n=5.
SCFA, short-chain fatty acid.
96 Halmos EP, et al. Gut 2015;64:93–100. doi:10.1136/gutjnl-2014-307264
Comparisons with data during the participants’ habitual diet
were also made.
Colonic luminal concentrations of SCFA are of major import-
ance to gut health given their role in secretion, absorption,
motility and epithelial health. Because they are products of bac-
terial fermentation, a change in the delivery of fermentable sub-
strates to the colon is anticipated to alter the concentrations and
output of faecal SCFA. The low FODMAP diet reduced total
bacterial abundance in the faeces by an average 47% in compari-
son with the typical Australian diet, which in turn could pos-
sibly lower SCFA production. However, faecal SCFA
concentration was unaffected by the FODMAP content of the
diet, although faecal pH was higher on the low FODMAP diet.
This apparently paradoxical situation requires explanation.
While SCFAs are the major anions in the large bowel, other bac-
terial metabolites not measured or alterations in protein catabol-
ism may have contributed to the lower faecal pH associated
with FODMAP intake. The higher average resistant starch
content of the low FODMAP compared with that of the typical
Australian diet (table 1) might have compensated for the lower
FODMAP content as suggested by an animal trial comparing
resistant starch with fructo-oligosaccharides (FOS).
the difference in resistant starch consumed in the present study
was small (3.2 g/d) and all previous studies have shown that at
least 16 g/d is required to alter faecal SCFA concentration, but
only when combined with wheat bran.
Changes in colonic
transit will inﬂuence faecal SCFA excretion, but WGTT was
similar in each dietary period. This ﬁnding is consistent with
previous data indicating that non-fermented or poorly fermen-
ted dietary ﬁbres are more effective faecal bulking agents and so
have a greater effect on hastening transit than readily fermented
Although the methodology used measures
whole gut transit, it is likely that this reﬂects large bowel transit
as this is the longest duration. Indeed, faecal weight was not
altered by FODMAP ingestion,
which might have been
expected by such a reduction in total bacterial abundance.
Supplementation of ﬁbre and resistant starch to the low
FODMAP diet may have concealed the expected change. The
most likely explanation for the apparent paradox is, because
FODMAPs are fermented in the proximal colon, that faecal
concentrations of SCFA are poor markers of colonic fermenta-
tion. It is also known that more than 95% of SCFAs are rapidly
absorbed and metabolised.
It is likely that changes in the structure of the microbiome
will translate into functional changes, although the nature of
such a relationship remains undeﬁned. The interventional diets
were associated with several differences in faecal microbiota.
Absolute abundances overall and of butyrate and prebiotic bac-
teria were less in association with the low FODMAP diet. This
is not surprising as these bacteria metabolise carbohydrates,
and a reduction of such substrates should lead to reduced pro-
liferation non-speciﬁcally. Of the mucus-degrading bacterial
species, R. gnavus and A. muciniphila were reduced in total
abundance. As total bacterial abundance was altered on the
interventional diets, the effects of the diets on relative abun-
dance of speciﬁc bacteria were of more importance. Three bac-
terial groups of putative functional importance were targeted.
First, Clostridium cluster XIVa showed a sevenfold difference
between the two diets and this wide difference was observed in
all participants (IBS or healthy). This observation is consistent
with those in animal and human studies showing increased
Clostridium cluster XIVa in faeces and digesta after consump-
tion of oligosaccharides, or foods containing FODMAPs
Second, traditionally ‘prebiotic’ bacteria, namely
Biﬁdobacterium spp, was similar between the two diets. The
only previous study to investigate prebiotic bacteria in associ-
ation with a low FODMAP diet identi ﬁed a difference in abso-
lute and relative abundance of faecal Biﬁdobacterium spp in
patients with IB S compared with those in a parallel UK popula-
tion consuming their habitual diet.
One possible explanation
for this discrepancy might be that the habitual diet in the UK
population contained a higher amount of galacto-oligosacchar-
ides (GOS) (mean (95% CI) 2.0 (1.4 to 2.5) g/d) and total oligo-
saccharides than provided by the typical Australian (GOS 1.01
(0.09 to 1.94) g/d) and habitual diets (GOS 0.76 (0.55 to 0.96)
g/d) of this Australian cohort. As oligosaccharides (including
GOS) are thought to inﬂuence faecal concentrations of
the greater decline from the habitual UK
to the low FODMAP diet may have been responsible for the
altered relative abundance in the UK subjects.
The third bacterial group studied was mucus-degrading bac-
teria, speciﬁcally A. muciniphila and two Ruminococcus spp.
Simplistically, such bacteria are able to adhere to mucus and
feed off glycans and mucin proteins as part of the mucus
secreted by the gut epithelium.
Extensive degradation of the
mucous layer might be detrimental by impairing gut barrier
function. On the other hand, such foraging bacteria may
provide substrates for other bacteria important for development
of a healthy mucus-associated microbiota.
The diets were asso-
ciated with marked differences in the relative abundance of
A. muciniphila, (lower in the low FODMAP arm) and R. torques
(higher in the low FODMAP arm). The pattern observed was
similar as the difference seen in patients with IBD compared
Similarly in mice
and rats inoculated with
ingestion of oligosaccharides increase the
excretion of A. muciniphila. However, the mechanism is uncer-
tain as oligosaccharides do not directly promote the growth of
A. muciniphila in vitro.
Faecal abundance appears to reﬂect
distal colonic mucosal abundance,
but whether it reﬂects
mucus-degrading microbiota in the proximal colon is uncertain
Table 4 Faecal pH, succinate, total and specific SCFA (mmol/g) on
pooled 5-day faecal samples after following a habitual diet for
5 days and low FODMAP and typical Australian diets for 17–21 days
in cross-over trial (n=33)
Measure Australian diet Low FODMAP diet p Value Habitual diet
pH 7.16 (7.02–7.30) 7.37*(7.23–7.51) 0.001 7.18 (7.07–7.31)
Succinate† 0.03 (0.02–0.04) 0.03 (0.02–0.03) 0.178 0.03 (0.03–0.04)
Total SCFA 74.7 (65.9–83.4) 77.6 (68.8–86.5) 0.208 84.0 (74.8–93.2)
Butyrate 14.0 (11.8–16.2) 13.5 (11.3–15.7) 0.672 16.2 (13.7–18.6)
Propionate 14.4 (12.4–16.4) 15.2 (13.4–16.9) 0.145 16.2 (14.0–18.2)
Acetate 38.6 (34.1–43.1) 40.9 (36.1–45.6) 0.126 42.9 (38.2–47.6)
Isobutyrate 2.01 (1.66–2.37) 2.07 (1.69–2.45) 0.836 2.13 (1.86–2.41)
Isovalerate 3.15 (2.52–3.78) 3.22 (2.54–3.91) 0.857 3.37 (2.89–3.85)
Valerate 2.25 (1.82–2.67) 2.29 (1.90–2.69) 0.974 2.54 (1.97–3.10)
Caproate‡ 1.03 (0.64–1.42) 1.13 (0.74–1.52) 0.454 1.20 (0.85–1.55)
Interventional diets were analysed by Wilcoxon matched-pairs signed rank test.
Statistically significant differences are shown in bold and based upon p≤0.05 for
faecal pH and p<0.006 for SCFA concentrations after Bonferroni correction.
Differences between habitual diet and interventional diets are indicated with an
*p=0.004 compared with habitual diet; Wilcoxon matched-pairs signed rank test.
†Due to difficulties in analysis for succinate, n=18.
‡Due to difficulties in analysis for caproate, n=15.
FODMAP, Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols;
SCFA, short-chain fatty acid.
Halmos EP, et al. Gut 2015;64:93–100. doi:10.1136/gutjnl-2014-307264 97
especially when caecal abundance of A. muciniphila was
reduced in association with FOS-induced increase in faecal
excretion of A. muciniphila in rodents.
Most evidence would
suggest that mucus-associated A. muciniphila have favourable
effects, perhaps via the provision of substrates such as acetate
and propionate for the support of a healthy consortium of bac-
teria adjacent to the epithelium.
The study of the effect of the
low FODMAP diet on mucus-degrading microbiota in the unin-
ﬂamed proximal and distal colon is required to resolve this
The diets differing in FODMAP content were also associated
with differences in the diversity of a cluster of bacteria including
many butyrate-producers, with greater diversity on the low
FODMAP diet. Reduced diversity is a common ﬁnding in dis-
eased colons, particular in association with IBD, where diversity
is inversely proportional to the degree of intestinal inﬂamma-
However, the focus was speciﬁcally on the Clostridium
cluster XIVa, which includes a large number of butyrate-
producing bacteria. The abundance of this group (Clostridium
cluster XIVa) declined signiﬁcantly on the low FODMAP diet in
IBS and healthy subjects. These two ﬁndings together may rep-
resent an alteration in dynamics of this cluster of bacteria from
a cluster with fewer and more dominant species to one with
more but less dominant species on the low FODMAP diet.
However, functional and health signiﬁcances cannot be attribu-
ted to this difference in diversity at the present time.
The key question regarding the differences in microbiota
between diets that vary in their FODMAP intake is what is
increased and what is decreased. The characterisation of faecal
microbiota in the same participants while taking their habitual
diet provided that opportunity. As anticipated, bacterial abun-
dance was reduced in association with the low FODMAP diet.
However, the marked changes in relative abundance of
Clostridium cluster XIVa and A. municiphila reﬂected an increase
Figure 1 Comparison of faecal indices with the two interventional diets in subjects with IBS and healthy subjects. (A) Changes in pH, total and
major short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs); (B) total and speciﬁc absolute bacterial abundance; and (C) relative
bacterial abundance. All data are presented as a ratio of low Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols (FODMAP)
to typical Australian diet and analysed by Wilcoxon matched-pairs signed rank test. Statistically signiﬁcant differences between the diets are
indicated with an asterisk based upon p≤0.05 for faecal pH, p≤0.006 for SCFA concentrations p≤0.005 for absolute and p≤0.006 for relative
bacterial abundance after Bonferroni correction.
98 Halmos EP, et al. Gut 2015;64:93–100. doi:10.1136/gutjnl-2014-307264
in association with the Australian diet rather than a decrease in
the low FODMAP diet. This could be arguably described as a
prebiotic effect of the former diet. It is also reasonable to postu-
late that such a difference resided in the modest differences in
oligosaccharide intake in the typical Australian diet compared
with the habitual diet (approximately 1.6 g), although these two
indices were measured using different methods. In concert with
more oligosaccharides in the typical Australian than habitual diet,
there was a small increase in GI symptoms.
Hence, the low
FODMAP diet reduced absolute abundance of faecal bacteria,
but did not have an ‘antiprebiotic’ effect.
The present study was not powered to compare faecal micro-
biota and biochemical indices in healthy subjects with those
who have IBS. However, similar trends in microbiota and SCFA
concentrations were noted. The only exceptions were the higher
faecal concentrations of the BCFA. BCFAs are products of
protein degradation, fermented increasingly through progression
to the distal colon,
and associated with increased genotoxicity
and possibly less cytotoxicity.
Reasons for differences in the
faecal BCFA concentrations, such as differences in microbiota
associated with protein fermentation, were not speciﬁcally inves-
tigated. However, dietary components with potential to increase
faecal BCFAs, such as protein and calcium, which are thought
to alter genotoxicity and cytotoxicity, respectively,
similar between subject groups. Altering the dietary FODMAP
content showed a similar lack of response in SCFAs across the
Post hoc analyses of faecal microbiota were performed to gain
some insight as to whether changes in the microbiota might
predict clinical response in patients with IBS (see online supple-
ment 3). No differences were observed between non-responders
and good responders indicating that the symptoms and micro-
biota were probably not directly associated in response to diet.
Similarly, faecal content of SCFA and BCFA were not associated
with response (data not shown).
In conclusion, this study is the ﬁrst randomised controlled
trial to compare faecal soluble milieu and microbiota in sub-
jects with IBS while following two controlled diets differing in
their FODMAP content in a cross-over design. There was a
higher faecal pH, but the concentrations of faecal SCFAs were
not different. In contrast, marked changes in the microbiota
were found. The low FODMAP diet was associated with lower
absolute abundance of total bacteria, butyrate-producing bac-
teria, prebiotic bacteria and A. muciniphila and R. gnavus.
Marked lower relative abundances of Clostridium cluster XIVa
and A. muciniphila,andasigniﬁcantly higher abundance of
R. torques were also observed. Finally, bacterial taxonomic diver-
sity of a large cluster of primarily butyrate-producers was greater
on the low FODMAP diet. Comparison with faecal microbiota
on habitual diet indicated that the low FODMAP intake was
associated with reduced absolute abundance of bacteria, but the
higher FODMAP intake associated with the typical Australian
diet showed evidence of speciﬁc stimulation of the growth of
bacterial groups with putative health beneﬁts. The functional sig-
niﬁcance and health implications of such changes might lead to
caution about reducing FODMAP intake in the longer term.
Liberalising FODMAP restriction to the level of adequate
symptom control should be exercised. The low FODMAP diet
should not be recommended for asymptomatic populations.
Acknowledgements The authors thank Gina Dimitrakopoulos and Debbie King
(Monash University) for their assistance with food preparation and packaging, Kelly
Liels, Ourania Rosella and Rosemary Rose (Monash University) for analysis of
FODMAP content of meals, Simone Peters and Chu Kion Yao (Monash University) for
statistical analysis, and Jennifer Giles (CSIRO) for molecular microbiological analysis.
Contributors Study concept and design: EPH, SJS, PRG, JGM; recruitment, enrolment
and assessment of participants: EPH, JGM; acquisition of data: EPH; analysis and
interpretation of data: EPH, CTC, ARB, PRG; study supervision: SJS, PRG, JGM; drafting
of the manuscript: EPH, CTC, ARB, PRG; approval of ﬁnal draft: all authors.
Funding This study was supported by the National Health and Medical Research
Council (NHMRC) of Australia and the Les and Eva Erdi Foundation. EPH was
Table 5 Absolute and relative bacterial abundance and bacterial diversity on pooled faecal samples after following a habitual diet for 5 days
and low FODMAP and typical Australian diets for 17–21 days in cross-over trial (n=33)
Measure Bacteria Australian diet Low FODMAP diet p Value Habitual diet
Absolute abundance (Log
copies of 16S rRNA gene/g) Total bacteria 9.83 (9.72–9.93) 9.63*(9.53–9.73) <0. 001 9.85 (9.73–9.96)
Clostridium cluster IV 8.33 (8.15–8.52) 8.05*(7.88–8.23) <0. 001 8.39 (8.23–8.56)
Faecalibacterium prausnitzii 7.72 (7.49–7.95) 7.45*(7.25–7.65) <0. 001 7.84 (7.67–8.01)
Clostridium cluster XIVa 9.05*(8.93–9.16) 8.03 (7.91–8.15) <0.001 8.22 (8.09 –8.36)
Roseburia 7.72 (7.59–7.85) 7.49 (7.34–7.63) <0.001 7.62 (7.45–7.79)
Lactobacilli 6.35 (6.20–6.50) 6.08 (5.91–6.24) 0.003 6.21 (6.00–6.42)
Bifidobacteria 7.71 (7.53–
7.88) 7.30*(7.11–7.50) <0.001 7.70 (7.48–7.91)
Akkermansia muciniphila† 5.46*(4.88–6.04) 4.29 (3.58–4.99) <0.001 4.29 (3.67–4.92)
Ruminococcus gnavus 7.26 (7.14–7.37) 7.10 (6.96–7.25) 0.002 7.16 (7.04–7.28)
Ruminococcus torques 6.08 (5.85–6.31) 6.23 (6.07–6.39) 0.140 6.20 (5.97–6.44)
Relative abundance (percentage of total bacteria) Clostridium cluster IV 4.00 (3.21–4.71) 3.32 (2.70–3.94) 0.108 3.99 (3.39–4.60)
F. prausnitzii 1.11 (0.82–1.40) 0.95 (0.69–1.22) 0.108 1.29 (0.92–1.66)
Clostridium cluster XIVa 18.1*(15.4–20.8) 2.72 (2.33–3.12) <0.001 2.63 (2.26 –3.01)
Roseburia 0.85 (0.585–1.11) 0.82 (0.68–0.96) 0.153 0.79 (0.58–1.00)
Lactobacilli 0.05 (0.03–0.06) 0.04 (0.03–0.05) 0.634 0.06 (0.01–0.11)
Bifidobacteria 1.33 (0.74–1.92) 0.87 (0.47–1.27) 0.028 1.48 (0.79–2.18)
A. muciniphila† 0.10*(0.03–0.16) 0.02 (0.01–0.03) <0.001 0.01 (0–0.02)
R. gnavus 0.37 (0.23–0.50) 0.41 (0.27–0.53) 0.480 0.27 (0.19–0.36)
R. torques 0.04 (0.02–0.06) 0.06 (0.04–0.09) 0.001 0.05 (0.02–0.08)
Diversity (Shannon index) Clostridium cluster XIV 1.47 (1.39–1.55) 1.79‡ (1.70–1.89) <0.001 1.60 (1.46–1.73)
Interventional diets were analysed by Wilcoxon matched-pairs signed rank test. Statistically significant differences are shown in bold and based upon p≤0.05 for bacterial diversity,
p≤0.005 for absolute and p≤0.006 for relative bacterial abundance after Bonferroni correction. Differences between habitual diet and interventional diets are indicated with an asterisk.
*p<0.001 compared with habitual diet; Wilcoxon matched-pairs signed rank test.†Due to difficulties in microbial analysis for A. muciniphila, three IBS subjects could not be included;
‡p<0.05 compared with habitual diet; pairwise permanova test.
FODMAP, Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols.
Halmos EP, et al. Gut 2015;64:93–100. doi:10.1136/gutjnl-2014-307264 99
supported by a scholarship from the Faculty of Medicine, Nursing and Health
Sciences, Monash University.
Competing interests SJS has published a book on food intolerances and several
cookbooks related to the topic of the manuscript. PRG has published a book on food
intolerances. There were no conﬂicts of interest to declare for EPH, CTC, ARB, JGM.
Patient consent Obtained.
Ethics approval Eastern Health and Monash University Human Research and
Provenance and peer review Not commissioned; externally peer reviewed.
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