Effects of multispecies probiotic supplementation on intestinal microbiota in irritable bowel syndrome

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


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



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.




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.




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




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

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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.




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.




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Effects of multispecies probiotic supplementation on intestinal microbiota in irritable bowel syndrome