Effects of multispecies probiotic supplementation on intestinalmicrobiota in irritable bowel syndromeK. KAJANDER* ,� , L . KROGIUS-KURIKKA� , T . RINTTIL A� ,1 , H. KARJALAINEN� , A . PALVA� &
R. KORPELA* ,� ,§
*Institute of Biomedicine, Pharmacol-
ogy, Faculty of Medicine, University
of Helsinki, Helsinki; �Valio Ltd,
Research Centre, Helsinki; �Depart-
ment of Basic Veterinary Sciences,
Faculty of Veterinary Medicine, Uni-
versity of Helsinki, Helsinki; §Founda-
tion for Nutrition Research, Helsinki,
Finland
Correspondence to:
Dr R. Korpela, Institute of
Biomedicine, PO Box 63, University of
Helsinki, Helsinki FIN-00014, Finland.
E-mail: [email protected]
1Alimetrics Ltd, Helsinki, Finland.
Publication data
Submitted 25 April 2007
First decision 16 May 2007
Resubmitted 24 May 2007
Accepted 24 May 2007
SUMMARY
BackgroundA multispecies probiotic has shown beneficial effects in irritable bowelsyndrome. In addition, certain other probiotics have demonstratedadvantageous effects, but the mechanisms behind this are poorly under-stood.
AimTo investigate the mode of action of a multispecies probiotic consistingof Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Prop-ionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breveBb99 by monitoring its effects on intestinal microbiota and markers ofmicrobial activity.
MethodsA total of 55 irritable bowel syndrome patients participated in this pla-cebo-controlled double-blind trial. Subjects received either multispeciesprobiotic or placebo supplementation daily during a 6-month period.The composition of intestinal microbiota was analysed with real-timepolymerase chain reaction, short-chain fatty acids with gas chromatog-raphy and enzymes with spectrophotometer.
ResultsEach supplemented probiotic strain was detected in faecal samples.Intestinal microbiota remained stable during the trial, except for Bifido-bacterium spp., which increased in the placebo group and decreased inthe probiotic group (P = 0.028). No changes in short-chain fatty acidsoccurred. A decrease in ß-glucuronidase activity was detected in 67% of
the subjects in the probiotic group vs. 38% in the placebo group(P = 0.06).
ConclusionsFactors other than the microbial groups and metabolites studied hereinseem responsible for the alleviation of irritable bowel syndrome symp-toms by the multispecies probiotic.
Aliment Pharmacol Ther 26, 463–473
Alimentary Pharmacology & Therapeutics
ª 2007 The Authors 463
Journal compilation ª 2007 Blackwell Publishing Ltd
doi:10.1111/j.1365-2036.2007.03391.x
INTRODUCTION
The therapeutic potential and mechanisms of action of
probiotic bacteria are at the centre of substantial
research interest and activity. According to the World
Health Organization1 probiotics are defined as ‘live
micro-organisms which when administered in ade-
quate amounts, confer a health benefit on the host’.
Currently, most of the clinical data on probiotics focus
on diarrhoea as well as other gastrointestinal (GI) ill-
nesses and disturbances, but other areas outside
gastroenterology, such as allergies and infections are
also increasingly studied.2
The intestinal microbiota in healthy adults is gener-
ally considered stable over time3 with the genera Bac-
teroides, Bifidobacterium, Eubacterium, Clostridium,
Peptococcus, Peptostreptococcus and Ruminococcus
predominating.4 The microbiota in subjects with irrit-
able bowel syndrome (IBS) has been shown to be less
stable compared to healthy adults.5 No single deviance
has been identified in IBS microbiota, but various and
different alterations in the bacterial composition have,
nonetheless, been characterized by a range of tech-
niques.5–9 These findings, combined with the diversity
and complexity of IBS, may indicate that a probiotic
combination could be more efficient than a single
strain in this particular syndrome. In a recent review,
Timmerman and co-workers10 defined a multispecies
probiotic as ‘containing strains of different probiotic
species that belong to one or preferentially more gen-
era’. They suggested that multispecies probiotics may
in some conditions be more efficient than monostrain
probiotics due to, for example, enhanced intestinal
adhesion, and the production of a greater variety of
antimicrobial compounds compared with single strain
probiotics. We have previously shown that a multispe-
cies probiotic combination containing Lactobacillus
rhamnosus GG combined with Lactobacillus rhamno-
sus Lc705, Propionibacterium freudenreichii ssp. sher-
manii JS and Bifidobacterium breve Bb99 significantly
alleviated the symptoms of IBS in a 6-month placebo-
controlled intervention.11 Besides this probiotic combi-
nation, certain other probiotics have also shown posit-
ive effects in IBS.12–15 As promising clinical data
exists, understanding the functional mechanisms of
probiotic supplementation in alleviating IBS is of
paramount importance.
Short-chain fatty acids (SCFAs), the main end-
products of colonic bacterial fermentation, are known
to play a role in gut motility and in sensitivity to
distension.16 Evidence implicates the aberrant produc-
tion of SCFAs17 as well as the excess formation of
intestinal hydrogen18 and the abnormal colonization
of the small bowel in some IBS patients.19 Moreover,
studies have demonstrated that interventions aiming at
the reduction of colonic fermentation with antibiotic
therapy or diet are helpful in some IBS patients.20, 21
All these findings suggest that one mechanism behind
probiotic effects in IBS could be the modulation of
microbiota and the colonic fermentation profile.
Consequently, the purpose of the present study was
to evaluate the effects of a multispecies probiotic com-
bination (L. rhamnosus GG, L. rhamnosus Lc705,
P. freudenreichii ssp. shermanii JS and B. breve Bb99)
on the microbiota in adult IBS patients in a double-
blind, placebo-controlled setting. Additionally, we
examined the effect of the supplementation on selec-
ted indicators of microbial activity (SCFA, bacterial
enzymes).
MATERIALS AND METHODS
Subjects and study design
The study subjects comprised a subset of a larger clin-
ical trial investigating the effects of a probiotic combi-
nation on IBS symptoms.11 Eligible subjects were
between 20 and 65 years old, fulfilled the Rome I or II
criteria for IBS,22, 23 and had undergone a colonoscopy
or a barium enema of the GI tract during the pre-
ceding 12 months. Exclusion criteria included preg-
nancy or lactation, organic intestinal diseases or
severe systemic diseases, previous major or complica-
ted abdominal surgery, severe endometriosis, dementia
or other inability to co-operate adequately, and anti-
microbial medication during the previous 2 months.
Patients with lactose intolerance were allowed to parti-
cipate if they followed their usual low-lactose or lac-
tose-free diet. For this substudy, only subjects who
fulfilled the Rome II criteria were eligible. At inclusion,
they were asked to collect faecal samples, and the par-
ticipation rate for this substudy was 79%, as 55 of the
70 subjects fulfilling the Rome II criteria participated.
The study was conducted as a randomized, double-
blind, placebo-controlled parallel group intervention.
During the 6-month study period, each subject
received daily either a probiotic capsule (Valio Ltd,
Helsinki, Finland) containing L. rhamnosus GG (ATCC
53103, LGG), L. rhamnosus Lc705 (DSM 7061, Lc705),
P. freudenreichii ssp. shermanii JS (DSM 7067, PJS)
464 K . KAJANDER et al.
ª 2007 The Authors, Aliment Pharmacol Ther 26, 463–473
Journal compilation ª 2007 Blackwell Publishing Ltd
and B. breve Bb99 (DSM 13692, Bb99) or a placebo
capsule. The total daily amount of bacteria was 8–
9 · 109 cfu ⁄ day with an equal amount of each strain.
The characteristics of each individual strain have been
described in Table 1. Compliance with the study treat-
ment was assessed by counting the remaining capsules
in each carton returned by the participants, and by
analysing the amount of the probiotic strains in faecal
samples. Consumption of other probiotic products was
not allowed during the study.
The subjects were advised to follow their usual diet-
ary habits, and not to make any changes to possible
medication. Because of a relatively long study period,
any ongoing consumption of IBS medication (mainly
commercial fibre analogues, laxatives or antidiarrhoe-
als) was allowed during the study.
Faecal samples
During the study, three faecal samples were collected
from each subject. The samples were taken before
beginning the probiotic or placebo supplementation
(baseline), and twice during the supplementation (3
and 6 months). Samples were immediately stored in
anaerobic containers from defecation onwards, frozen
within 4 h to )70 ºC, and stored therein until ana-
lysed.
Detection of probiotic strains and analysis ofmicrobiota
The quantification of the ingested probiotic strains as
well as of selected bacterial species or groups was per-
formed by real-time quantitative polymerase chain
reaction (qPCR). For the analyses, chromosomal DNA
from the faecal samples was isolated as described by
Apajalahti et al.,33 and the concentrations of the
extracted DNA were determined with the VersaFluor
Fluorometer System (Bio-Rad, Hercules, CA, USA). The
bacterial numbers were expressed per dry weight sam-
ple. To determine dry weight, 1–2 g of sample was
dried at 105 ºC for 16–18 h, cooled to room tempera-
ture in a desiccator, and weighed.
Probiotic strains
Strain-specific real-time PCR assays were developed for
the quantification of LGG, Lc705, PJS and Bb99 by
using the LightCycler (Roche, Penzberg, Germany) FRET
technique.34, 35 The standard curves used for quantifica-
tion of the strains consisted of a series of 10-fold
dilutions of target species genomic DNA between 0.1 pg
and 100 ng, equivalent to approximately 38–40 and
3.8 · 107 to 4.0 · 107 target genomes.
Microbiota
Analyses were performed with an iCycler iQ apparatus
(Bio-Rad) associated with an ICYCLER optical system
interface software (version 2.3). In the reactions we
used, an aliquot of 50 ng, 250 ng or a dilution of
1:100 of DNA from each faecal preparation. The 16S
rRNA gene targeted real-time PCR assays were per-
formed in triplicate using SYBR Green I chemistry.
The growth conditions for bacterial strains used as
positive and negative controls as well as for the pre-
paration of standards and applied oligonucleotides,
reaction mixtures and thermal cycling conditions in
real-time PCR assays are described for the Atopobium
group, Bacteroides-Prevotella-Porphyromonas, Bifido-
bacterium spp., Campylobacter spp., the Clostridium
coccoides-Eubacterium rectale group, Clostridium
difficile, the Clostridium perfringens group, the Desulf-
ovibrio desulfuricans group, Enterococcus spp.,
Table 1. Characteristics of each individual strain in the multispecies probiotic
Probiotic strain Specific characteristics
Lactobacillus rhamnosus GG (ATCC 53103) Prevention and treatment of various gastrointestinal disorders,especially diarrhoea,24, 25 immunomodulation in vitro26 and inclinical studies 27, 28
Lactobacillus rhamnosus Lc705 (DSM 7061) Inhibition of pathogen adhesion,29 production of an antimicrobialsubstance,30 binding of heavy metals and aflatoxins31, 32
Propionibacterium freudenreichii ssp. shermaniiJS (DSM 7067)
Inhibition of pathogen adhesion,29 binding of heavy metals31
Bifidobacterium breve Bb99 (DSM 13692) Inhibition of pathogen adhesion29
MICROBIOLOGICAL EFFECTS OF A PROBIOT IC 465
ª 2007 The Authors, Aliment Pharmacol Ther 26, 463–473
Journal compilation ª 2007 Blackwell Publishing Ltd
Faecalibacterium prausnitzii (formerly Fusobacterium),
Fusobacterium spp., Helicobacter-Flexispira-Wollinella,
the Lactobacillus group (forward primer: Walter
et al.,36 reverse primer: Heilig et al.37) and Veillonella
spp. by Rinttila et al.38 and for Bacteroides fragilis,
the Escherichia coli subgroup and Ruminococcus
productus-Clostridium coccoides by Malinen et al.39
The average results of triplicate samples obtained
with real-time PCR were converted to the average
estimate of target bacterial genomes per gram dry
weight faeces. The assay-specific genome sizes and
ribosomal DNA rRNA copy number for F. prausnitzii
used in the calculations are described in detail by
Malinen et al.39
Analysis of SCFA and bacterial enzymeactivities
The faecal SCFA content (acetate, butyrate, propionate,
valerate, caproate, isobutyrate, isovalerate, isocaproate)
was identified with chromatography (HP 6890, Hewlett
Packard, Palo Atto, CA USA) according to the method
described by Høverstad and co-workers.40 Concentra-
tions of SCFAs were expressed per gram dry weight of
faeces.
Bacterial enzymes (ß-glucosidase and ß-glucuroni-
dase) were analysed with a method described by
Goldin et al.41 Enzyme activities were expressed per
gram of protein, and the protein content was deter-
mined with a protein test kit (Bio-Rad Protein Assay,
Bio-Rad Laboratories).
Statistical analyses
The primary end points were the numbers of ingested
probiotic strains and selected microbial groups, and
the levels of SCFAs and bacterial enzymes were con-
sidered secondary end points. The microbial data,
SCFA levels and enzyme activities were skewed to the
right, and were logarithmically (log10 or loge) trans-
formed before the analysis. The repeated-measures
ANCOVA with baseline as covariate was used to compare
the groups at 3 and 6 months. The results appear as
baseline-adjusted mean values for both groups, and
due to logarithmic transformations, the group compar-
isons appear as probiotic ⁄ placebo ratios with 95%
confidence intervals. For the probiotic quantification
data, values below the detection limit (DL) were
replaced by DL ⁄ 2 if no more than 25% of values were
below the DL. If more than 25% of values were below
the DL, only the prevalence of the strain is shown. The
chi-squared test was used for categorical data. The
missing values in microbial (n = 3) and SCFA (n = 6)
data were imputed using the linear interpolation
method or the Last Observation Carried Forward
(LOCF)-method, for missing values at 3 and 6 months,
respectively. Possible missing baseline values or miss-
ing values in probiotic quantification data were not
estimated. SPSS (version 14.0; SPSS Inc., Chicago, IL,
USA) was used for the statistical analyses.
Ethics
The study protocol was approved by The Human Ethics
Committee of the Joint Authority for the Hospital Dis-
trict of Helsinki and Uusimaa (HUS). All the partici-
pants provided their written informed consent and
were informed that they could withdraw from the
study at any time.
RESULTS
Characteristics of participants
A total of 55 adult IBS patients who were randomized
to the main study (16 males, 39 females; mean age
46 years) participated in this substudy (Table 2). All
fulfilled the Rome II criteria for IBS, except for four
subjects, who reported slightly <12 weeks of abdom-
inal pain during the preceding year. Of the 55 IBS
patients, 28 were assigned to the probiotic group, and
27 to the placebo group. Altogether, 43 subjects (22
probiotic, 21 placebo) delivered all three samples and
completed the trial according to the protocol. Due to
insufficient amounts of sample material, all laboratory
analyses could be carried out on 40 subjects for
microbiological variables, 34 subjects for SCFAs and
42 subjects for bacterial enzymes.
Twelve participants (six probiotic, six placebo)
withdrew or were excluded from the study because
of following reasons: illness or hospitalization for
causes other than IBS (one probiotic, three placebo),
a sensation of increased GI symptoms (one probiot-
ic), a desire to use other probiotic products (one
probiotic), pregnancy (two placebo), non-compliance
(one placebo) and other reasons (three probiotic).
The mean compliance for ingesting the study capsule
was 96% (min.: 79%) as assessed by counting the
remaining capsules in each carton returned by the
participants.
466 K . KAJANDER et al.
ª 2007 The Authors, Aliment Pharmacol Ther 26, 463–473
Journal compilation ª 2007 Blackwell Publishing Ltd
Detection of probiotic strains
At baseline, 15 of 40 samples were L. rhamnosus GG-
positive (10 probiotic, five placebo). Lactobacillus
rhamnosus Lc705 was detected in three of 40 samples
(three probiotic, zero placebo), P. freudenreichii ssp.
shermanii JS in seven of 40 samples (seven probiotic,
zero placebo) and B. breve Bb99 in three of 40 samples
(three probiotic, zero placebo).
The numbers of probiotic strains at baseline and at
6 months in both studied groups appear in Figure 1.
LGG and Bb99 could be detected in the great majority
Table 2. Sociodemograph-ic and clinical characteristicsof randomized IBS patients(n = 55)
Multispecies probiotic(n = 28) Placebo (n = 27)
Age [years; mean (range)] 44 (23–65) 47 (21–65)Gender: F ⁄ M 19 (68%) ⁄ 9 (32%) 20 (74%) ⁄ 7 (26%)BMI [kg ⁄ m2; mean (range)] 25.8 (18.7–36.6) 23.9 (16.3–34.1)Predominant bowel habit, n (%)*
Diarrhoea 14 (50) 12 (44)Constipation 5 (18) 9 (33)Alternating 9 (32) 6 (22)
Regular IBS medication, n (%) 9 (32) 10 (37)Completed trial, n (%) 22 (79) 21 (78)
* According to the Rome II criteria.
citoiborPobecalP
LGG
®
LD<
3
4
5
6
7
8
9
01 enilesaB
citoiborPobecalP
LGG
®
LD<
3
4
5
6
7
8
9
01 shtnom 6 At
citoiborPobecalP
Bb9
9
LD<
3
4
5
6
7
8
9
01 enilesaB
citoiborPobecalP
Bb9
9
LD<
3
4
5
6
7
8
9
01 shtnom 6 tA
citoiborPobecalP
Lc70
5
LD<
3
4
5
6
7
8
9
01 enilesaB
citoiborPobecalP
Lc70
5
LD<
3
4
5
6
7
8
9
01 shtnom 6 tA
citoiborPobecalP
PJS
LD<
3
4
5
6
7
8
9
01 enilesaB
citoiborPobecalP
PJS
LD<
3
4
5
6
7
8
9
01 shtnom 6 tA
Figure 1. The levels (log10 of bacteria counts) of the ingested probiotic strains (LGG�, Lc705, PJS and Bb99) in the placebogroup (n = 21) and in the probiotic group (n = 19) at baseline and at the end of the study at 6 months.
MICROBIOLOGICAL EFFECTS OF A PROBIOT IC 467
ª 2007 The Authors, Aliment Pharmacol Ther 26, 463–473
Journal compilation ª 2007 Blackwell Publishing Ltd
of the subjects in the probiotic group at 6 months. The
prevalence rates in this group at the 3- and 6-month
sampling points were 89% (mean of log10 counts 7.0;
95% CI: 6.33–7.66) and 95% (6.7; 95% CI: 6.05–7.32)
for LGG and 89% (6.9; 95% CI: 6.38–7.42) and 79%
(6.3; 95% CI: 5.69–6.97) for Bb99. The number of sub-
jects in the probiotic group carrying Lc705 and PJS at
3 and 6 months was lower than that carrying LGG and
Bb99; Lc705 was found in 53% at both 3 and
6 months, whereas PJS was detected in 84% (7.4; 95%
CI: 6.49–8.39) at 3 months and in 63% at 6 months.
In the placebo group LGG was found in 43% at
3 months and in 24% at 6 months. The number of car-
riers of the other ingested probiotic strains was low in
the placebo group; Lc705 and Bb99 could not be
detected in any samples at 3 or 6 months, while PJS
was found in two of 21 (10%) of the samples at both 3
and 6 months.
Effects of the intervention on microbiota
To investigate the effects of the probiotic supplemen-
tation on faecal microbiota, we conducted a total of
20 real-time qPCR assays covering more than 300 dif-
ferent species. The target bacterial groups were selec-
ted due to previous reports indicating an association
between IBS and the bacteria in question, or due to
the predominant nature of the group in the gut eco-
system. The analysed bacterial groups remained highly
stable during the intervention as confirmed by the
mean numbers of bacterial groups in faecal samples at
baseline and during the supplementation. The Helicob-
acter-Flexispira-Wollinella group was not detected in
any subject, and Clostridium difficile was found in
only one subject in the probiotic group at 6 months.
Fusobacterium spp. was detected at low prevalence
(21–48%) during the study; consequently, the mean
levels for bacterial counts were not analysed.
Faecal bacterial counts at baseline and during the
intervention appear in Table 3. The numbers (as log10)
of Bifidobacterium spp. increased in the placebo group
(from 9.2 at baseline to 9.8 at 3 months and to 9.8 at
6 months), while the number decreased in the probiot-
ic group (from 9.6 to 9.4 and 9.1, respectively). The
baseline-adjusted probiotic ⁄ placebo ratio was 0.23
during the supplementation period (P = 0.028 for dif-
ference between groups). As an exploratory analysis,
we performed a subgroup analysis. Subjects were divi-
ded into two groups at baseline: those with bifidobac-
teria below the median value, and those with
bifidobacteria above the median value (data not
shown). The probiotic appeared to decrease bifidobac-
teria significantly only in those subjects who had
above median counts at baseline (baseline-adjusted
probiotic ⁄ placebo ratio 0.14, 95% CI: 0.02–0.78,
P = 0.027), whereas in subjects below the median
counts, we detected no significant difference between
groups (ratio 0.75, 0.06–9.06, P = 0.810).
The Atopobium group tended to be slightly lower
with probiotic supplementation (10.3 vs. 10.6;
P = 0.10). The number of other analysed bacterial
groups showed no changes in either group during the
intervention.
Effect of the intervention on SCFAs andbacterial enzymes
No significant differences occurred between the study
groups in the levels of acetate, butyrate, propionate,
valerate, isobutyrate or isovalerate. Isocaproate was
below the detection limit (1 mg ⁄ 100 g) in all samples,
and caproate was found in such low prevalence that
no mean values could be calculated. Concentrations of
SCFAs in the faeces at baseline and during the probi-
otic supplementation appear in Table 4.
The baseline level of ß-glucosidase (nmol ⁄ min ⁄ mg
protein) was 15.2 (range: 8.3–25.2) in the probiotic
group and 13.8 (7.5–23.3) in the placebo group. The
corresponding values for ß-glucuronidase were 7.7
(2.9–20.4) and 6.4 (2.7–11.5), respectively. During the
intervention, the baseline-adjusted geometric mean val-
ues were 15.9 and 15.0 for ß-glucosidase and 6.7 and
6.5 for ß-glucuronidase in the probiotic group and pla-
cebo group, respectively. The corresponding probiot-
ic ⁄ placebo ratios were non-significant: 1.1 (95% CI:
0.96–1.18, P = 0.219) and 1.0 (0.85–1.24, P = 0.798).
However, the probiotic tended to decrease ß-glucuroni-
dase activity: 67% of subjects in the probiotic group vs.
38% in the placebo group had a decrease in activity at
6 months (P = 0.06). No significant modification of ß-
glucosidase activity occurred during the intervention.
DISCUSSION
This study applied molecular and biochemical methods
to investigate the microbiological effects of a multispe-
cies probiotic (L. rhamnosus GG, L. rhamnosus Lc705,
P. freudenreichii ssp. shermanii JS and B. breve Bb99),
which has previously shown promising value in
alleviating IBS symptoms.11 All supplemented strains
468 K . KAJANDER et al.
ª 2007 The Authors, Aliment Pharmacol Ther 26, 463–473
Journal compilation ª 2007 Blackwell Publishing Ltd
Tabl
e3.
Fae
cal
bac
teri
alco
unts
(log
10
of
bac
teri
aper
gra
mdry
wei
ght
of
faec
es)
atbas
elin
ean
dduri
ng
the
pro
bio
tic
(n=
22)
or
pla
cebo
(n=
21)
supple
men
tation
(fae
cal
sam
ple
sw
ere
collec
ted
at3
and
6m
onth
sduri
ng
the
supple
men
tation)
Bas
elin
eBas
elin
e-ad
just
edm
argin
alm
eans
duri
ng
supple
men
tation
(3m
onth
s–6
month
s)Rat
ioPro
bio
tic
⁄Pla
cebo
Pro
bio
tic
mea
n(m
in–m
ax)
Pla
cebo
mea
n(m
in–m
ax)
Pro
bio
tic
adj.
mea
n(9
5%
CI)
Pla
cebo
adj.
mea
n(9
5%
CI)
Mea
n(9
5%
CI)
P-v
alue*
Ato
pob
ium
gro
up
10.5
(9.6
1–11.3
1)
10.2
(7.4
1–11.7
1)
10.3
(10.0
8–10.5
1)
10.6
(10.3
3–10.7
7)
0.5
5(0
.27–1.1
3)
0.1
03
Bac
tero
ides
frag
ilis
7.5
(4.6
9–9.9
0)
6.5
(4.6
9–10.4
9)
6.6
(6.0
8–7.2
0)
6.9
(6.3
1–7.4
6)
0.5
6(0
.09–3.6
5)
0.5
37
Bac
tero
ides
–Pre
vote
lla–
Por
phyr
omon
as10.7
(8.9
7–11.6
0)
10.6
(9.1
9–11.1
5)
10.6
(10.4
4–10.7
4)
10.5
(10.3
3–10.6
4)
1.3
(0.7
7–2.0
9)
0.3
36
Bifi
doba
cter
ium
spp.
9.6
(7.7
9–10.6
9)
9.2
(5.0
9–11.5
2)
9.2
(8.8
2–9.6
1)
9.9
(9.4
5–10.2
6)
0.2
3(0
.06–0.8
5)
0.0
28
Cam
pyl
obac
ter
spp.
5.5
(4.6
1–7.6
6)
5.4
(4.6
1–6.6
5)
5.4
(5.0
7–5.6
3)
5.2
(4.8
6–5.4
4)
1.6
(0.6
3–4.0
7)
0.3
14
Clo
stri
dium
cocc
oide
s-Euba
cter
ium
rect
ale
gro
up
11.5
(10.6
2–12.2
8)
11.4
(11.0
2–11.9
0)
11.4
(11.2
5–11.5
1)
11.4
(11.2
4–11.5
0)
1.0
(0.6
7–1.5
9)
0.8
84
Clo
stri
dium
per
frin
gens
gro
up
5.9
(4.2
2–8.6
4)
5.5
(4.2
2–7.1
7)
5.8
(5.3
9–6.2
5)
6.0
(5.5
4–6.4
2)
0.6
9(0
.17–2.8
6)
0.5
99
Des
ulfov
ibri
ode
sulfuri
cans
gro
up
6.9
(4.9
2–8.9
7)
7.0
(4.9
2–8.8
7)
7.0
(6.7
3–7.3
1)
7.1
(6.8
3–7.4
3)
0.7
8(0
.30–2.0
5)
0.6
05
Ente
roco
ccus
spp.
7.8
(6.2
1–8.8
6)
7.8
(6.6
8–9.3
9)
7.7
(7.4
7–7.9
6)
7.8
(7.5
3–8.0
2)
0.8
8(0
.40–1.9
4)
0.7
39
Esc
her
ichia
coli
subgro
up
6.9
(4.8
0–8.6
1)
7.7
(4.8
0–10.5
6)
6.7
(6.3
6–7.1
1)
7.1
(6.7
0–7.4
7)
0.4
5(0
.13–1.5
9)
0.2
07
Fae
caliba
cter
ium
pra
usn
itzi
i9.1
(7.7
4–10.0
4)
9.0
(7.6
8–9.5
2)
8.9
(8.6
9–9.0
6)
8.8
(8.6
4–9.0
3)
1.1
(0.5
9–2.0
5)
0.7
63
Lact
obac
illu
sgro
up
8.2
(6.5
5–9.3
6)
7.9
(4.9
9–9.3
7)
8.2
(7.8
9–8.4
6)
8.0
(7.7
0–8.2
7)
1.5
(0.6
1–3.9
3)
0.3
53
Rum
inoc
occu
spro
duct
us–
C.co
ccoi
des
6.8
(4.8
0–8.2
6)
6.9
(5.7
9–9.2
8)
6.9
(6.7
1–7.1
1)
6.8
(6.5
4–6.9
6)
1.5
(0.7
4–2.8
4)
0.2
67
Vei
llon
ella
spp.
8.3
(6.4
3–9.6
2)
8.2
(4.9
2–9.9
2)
8.1
(7.8
3–8.3
8)
7.9
(7.6
4–8.2
0)
1.5
(0.6
2–3.8
0)
0.3
45
*A
NO
VA
for
repea
ted
mea
sure
men
ts,usi
ng
logar
ithm
ical
ly(log
10)
tran
sform
edval
ues
and
bas
elin
eas
aco
var
iate
.Bold
indic
ates
stat
istica
lly
signifi
cant
val
ue.
MICROBIOLOGICAL EFFECTS OF A PROBIOT IC 469
ª 2007 The Authors, Aliment Pharmacol Ther 26, 463–473
Journal compilation ª 2007 Blackwell Publishing Ltd
could be detected in the faecal samples. The analysed
bacterial groups remained relatively stable during the
supplementation, with the exception of Bifidobacterium
spp., which increased in the placebo group and
decreased in the probiotic group. No changes in the lev-
els of faecal SCFAs occurred in either group during the
intervention, whereas the probiotic tended to decrease
ß-glucuronidase activity.
Part of the favourable effects of probiotics in IBS is
attributed to the modulation of microbiota and its
metabolism.42 In this study, we detected the ingested
probiotics in faecal samples, but otherwise the quanti-
fied bacterial populations and measured microbial me-
tabolites remained stable. The results of an earlier
study in infants agrees with our findings concerning
the survival of the probiotic strains in the GI tract.43
Our results suggest that the reduction in IBS symptoms
by the consumption of the probiotic combination may,
in addition to alterations in the overall microbiota,
result from direct effects of the administered strains.
One should bear in mind that according to current
estimations; however, the human intestine is colonized
by a complex microbiota comprising more than 1000
species,44 and our study included analyses on 20 bac-
terial species or groups.
Few randomized-controlled trials on IBS and probiot-
ics have incorporated microbial variables, and the pre-
sent study is, to our knowledge, the first one to measure
SCFAs and enzymes. Furthermore, the number of bac-
terial groups studied was more extensive than in any
other IBS intervention reported thus far. In a study on
L. plantarum 299v, colonization of the strain in the
majority of the subjects was found, but no other micro-
bial effects were discovered, except for an increase in
Enterococcus in the placebo group.14 A concurrent
decrease in pain and flatulence was observed, yet the
administration of L. plantarum 299v failed to relieve
IBS symptoms or alter colonic fermentation in another
setting.45 The breath hydrogen level was, however,
lower in the Lactobacillus group after lactulose inges-
tion. In another study, O’Mahony and colleagues12 con-
firmed the detection of supplemented strains of
Bifidobacterium and Lactobacillus in faecal samples, but
carried out no other bacterial analyses. In addition to
IBS, the microbial effects of a similar multispecies pro-
biotic, as in this trial, were investigated during H. pylori
eradication.46 The eradication induced long-term dis-
turbances in the microbiota, which probiotic consump-
tion appeared slightly to counteract.
Compared to single strains, multispecies probiotics
may have more diversified effects on IBS symptoms
because of an ability to colonize several niches of the
GI tract, a wider range of functional characteristics and
synergistic effects between the strains. In this multispe-
cies probiotic L. rhamnosus GG, the most extensively
studied probiotic strain worldwide, was combined with
one Lactobacillus strain, one Propionibacterium strain
and one Bifidobacterium strain. The advantageous
effects of L. rhamnosus GG have been well documented
in various GI disturbances,24, 25 but the strain alone
has not been successful in IBS.47 Recently, certain spe-
cies of Bifidobacterium, Lactobacillus and Streptococcus
were shown to provoke different effects on gut motil-
ity,48 indicating that a combination of different species
Table 4. Faecal short chain fatty acids (lmol per gram dry weight of faeces) at baseline and during the probiotic (n = 22)or placebo (n = 18) supplementation (faecal samples were collected at 3 and 6 months during the supplementation)
Baseline
Baseline-adjusted marginalmean values duringsupplementation (3–6 months) Ratio probiotic ⁄ placebo
Probiotic geom.mean (min–max)
Placebo geom.mean (min–max)
Probiotic adj.G mean (95% CI)
Placebo adj.G mean (95% CI) Mean (95% CI) P-value*
Acetate 204 (59.1–596.9) 236 (92.5–678.1) 197 (155–250) 216 (166–281) 0.81 (0.35–1.84) 0.605Butyrate 67.8 (14.0–197.1) 90.7 (21.1–259.8) 60.6 (45.4–80.8) 61.5 (44.7–84.7) 0.96 (0.35–2.62) 0.941Propionate 59.1 (15.0–294.8) 68.7 (20.3–240.5) 63.5 (47.5–85.0) 65.0 (47.1–89.8) 0.95 (0.35–2.58) 0.913Valerate 8.3 (3.0–15.8) 9.5 (0.5–27.3) 7.9 (6.14–10.39) 10.2 (7.64–13.66) 0.57 (0.23–1.40) 0.213Isobutyrate 7.2 (4.0–15.6) 5.7 (0.5–15.9) 6.6 (5.11–8.53) 6.0 (4.54–8.01) 1.2 (0.51–2.99) 0.636Isovalerate 9.4 (4.4–16.9) 9.3 (3.1–23.5) 8.5 (7.04–10.31) 10.7 (8.63–13.15) 0.60 (0.31–1.15) 0.119
* ANOVA for repeated measurements, using logarithmically (loge) transformed values and baseline as a covariate.
470 K . KAJANDER et al.
ª 2007 The Authors, Aliment Pharmacol Ther 26, 463–473
Journal compilation ª 2007 Blackwell Publishing Ltd
may give a broader effect on motility disturbances.
Regarding synergistic effects, especially adhesion may
be remarkably increased in this multispecies probiotic,
as all strains alone are adherent to mucus,29 but the
presence of L. rhamnosus GG more than doubles the
adhesion of a Bifidobacterium and a Propionibacteri-
um.49, 50 Moreover, it appears that reduction of patho-
gen adhesion is another feature that is strengthened in
this multispecies probiotic compared to effects by the
individual strains.29
A disadvantage of our study was that the probiotic
strains and L. rhamnosus GG in particular, were also
detected within the placebo group at baseline as well
as during the trial. This finding clearly reflects the wide
commercial availability and exceptionally high con-
sumption of probiotic products in Finland. Unexpect-
edly, we observed an increase in bifidobacterial
numbers in the placebo group and a decrease in the
probiotic group. Upon closer examination, only those
with initially high levels of bifidobacteria appeared to
have suffered a decrease in the bacterial counts. Com-
petition and growth inhibition may have arisen
between the administered Bifidobacterium strain and
certain other bifidobacteria species ⁄ strains, or subjects
in the highest percentiles may be more sensitive to
temporal intraindividual variations in bacterial levels.
In a trial in which infants were supplemented with the
same multispecies probiotic combination, as in the pre-
sent study, no major changes occurred in the total bifi-
dobacterial counts.43 Bifidobacteria are among the
most commonly used probiotics, and their ingestion in
adults has usually resulted in either an increased51, 52
or unchanged53 level of Bifidobacterium spp.
Our results do not support the hypothesis that the
modulation of SCFA production is involved in IBS
symptom reduction. We observed the stable production
of the major SCFAs acetate, propionate and butyrate
that followed a previously shown normal molar ratio
of approximately 60:20:20.54 The measurement of fae-
cal SCFAs is, on the other hand complicated, as their
absorption in the colon involves a very efficient pro-
cess with only 10–20% being excreted in the faeces.55
Luminal samples would be required in order to deter-
mine the exact production rate of SCFAs in the colon.
Bacterial enzyme activities have not been associated
with IBS ethiopathology, but a slight reduction in fae-
cal ß-glucuronidase may have an overall beneficial
effect on the GI tract, as the enzyme is thought to
hydrolyse precarcinogenic compounds into carcino-
gens.56
Our results, combined with those of other groups,
suggest that more emphasis should be placed on the
study of novel mechanisms behind probiotic function-
ality in IBS. In addition, microbiota-related markers
should be investigated in greater detail. Recently,
Rousseaux and colleagues57 reported that certain
Lactobacillus strains can induce the expression of
pain-sensing l-opioid and cannabinoid receptors in
the intestinal epithelial cells. The mechanism underly-
ing this discovery remains unknown, but direct contact
of the lactobacilli with the epithelia was shown to
result in the induction of the receptors via the NF-jB
pathway. A study in rats demonstrated that Lactobacil-
lus reuteri can inhibit visceral pain through effects on
enteric nerves.58 Moreover, Verdu et al.59, 60 have
shown in two murine models that Lactobacillus
paracasei normalizes visceral hypersensitivity and
postinfective muscle hypercontractility. Lactobacillus
reuteri is, nonetheless, the only strain in these mech-
anistic studies that has been investigated in a clinical
trial, as well and in this case without success.61
Taken together, our findings demonstrate that a pro-
biotic combination of L. rhamnosus GG, L. rhamnosus
Lc705, P. freudenreichii ssp. shermanii JS and B. breve
Bb99 can be detected in the GI tract of adult IBS
patients. The microbiota and its metabolism remained
rather stable during the probiotic supplementation,
and thus other mechanisms besides those studied are
likely to contribute to the reduction in IBS symptoms
reported earlier. Carefully designed and conducted
experiments examining the modes of action behind
clinically proven probiotic strains and combinations
are warranted.
ACKNOWLEDGEMENTS
The authors wish to express their deepest gratitude to
Ms Sinikka Ahonen for her skilful assistance in the
microbial analyses, to Ms Anne-Maria Riihimaki for
carrying out the SCFA and enzyme analytics and to
Tuija Poussa, MSc for her statistical expertise.
Declaration of personal interests: K. Kajander and
R. Korpela are employees of Valio Ltd, Finland.
Declaration of funding interests: This study was funded
in full by Valio Ltd and the Finnish Funding Agency
for Technology and Innovation (TEKES). The prepar-
ation of this paper was funded in part by the Finnish
Academy. Initial data analyses were undertaken by
Tuija Poussa, who is an employee of StatConsulting,
Finland.
MICROBIOLOGICAL EFFECTS OF A PROBIOT IC 471
ª 2007 The Authors, Aliment Pharmacol Ther 26, 463–473
Journal compilation ª 2007 Blackwell Publishing Ltd
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