Aliment Pharmacol Ther 2002; 16; 587-593.

Bifidobacterium animalis strain DN-173 010 shortens the colonictransit time in healthy women: a double-blind, randomized, controlled

study

P. MARTEAU*. E. CUILLERIER*, S. MEANCEt. M. F. GERHARDTt, A. MYARAt. M. BOUVIER§,C. BOULEYt. F. TONDUt, G. BOMMELAER~ & J. C. GRIMAUD§

*Gastroenterology Department, European Hospital Georges Pompidou, Paris. France; tDanone Vitapole. Nutrition ResearchGroup, Le Plessis Robinson, France; tBiochemistry Department, Saint-Joseph Hospital, Paris, France; §Gastroenterology

Department, Hôpital Nord, Marseille, France; and ~Gastroenterology Department, Centre Hospitalier et Universitqire,

Clermont Ferrand, France

Accepted

for publication 15 October 2001

SUMMARY

Background: A previous study bas suggested that BW-dobacterium animalis DN-173 010 shortens the colonictransit time in women.Aim: To confirm this efIect and to determine whethermodifications of the faecal bacterial mass and/or faecalsecondary bile salts may be the explanation.Methods: A double-blind. cross-over study was per-formed. Thirty-six healthy women were studied in fourconsecutive 10-day periods. During periods 2 and 4.they ingested three 125 g ClipS per clay of a fermentedmilk which was either a product containing B. animalisDN-173

010 or a control without bifidobacteria.

Periods 1 and 3 were run-in and washout periods;respectively. The total and segmentaI colonic transittimes were assessed using a pellet method.::lfi 12subjects, aIl stools were collected and analysed for pH.faecal weight, bacterial mass and bile acids.Results: The total and sigmoid transit times weresignificantly shorter during dosing with B. animaliscompared to the contrai period. The other transit times,faecal weight, pH, bacterial mass and bile acids wete notsignificantly affected.Conclusions: B. animalis DN-173010 shortens thecolonic transit time in healthy women. This effèct 'isnot explained by modifications of the faecal bacterialmass or secondary bile acids.

INTRODUCTION

There is a growing interest in functional food which can'beneficially affect one or more target functions in thebody in a way that is relevant either to promoting thestate of well-being and health and/or reducing the riskof disease'. Probiotics belong to these functional foodsand have been defined as 'microbial cell preparations or

components of microbial relis that have a beneikialeffect on the health and well~being of the hosf.1.2Bifidobacteria represent about 20% of the cultivablefaecal bacteria in adults and up to 80% ininfants. Saillestrains which are used in fermented milks have a highsurvival capacity in the gastrointestinal tract andexhibit probiotic properties in the colon.3-9Colonic transit disturbances (constipation and irritable

bowel syndrome) are frequent and represent an import-ant target for functional food. including probiotics.lProbiotics are believed to influence intestinal transit;however. the evidence is poor as randomized controlledstudies are lacking.2 An open study has suggested thatthe ingestion of propionibacteria may slow clown the

Correspondence ta: Dr P. Marteau. Service d'Hépato-Gastroentérologie,

Hôpital Européen Georges Pompidou. 20 rue Leblanc. 75908 Paris Cedex15. France.E-mail: philippe.marteau@egp.ap-hop-paris.fr

@ 2002 Blackwell Science Ltd 587

P. MARTEAU et al.588

similar colour, taste, texture and lactose content. TheBifidus product (Bifidus) was the commercial product'BIO', i.e. a semi-skimmed milk fermented by yoghurtcultures and B. animalis strain DN-173 010. Thecontrai product was the same fermented milk withoutbifidobacteria (i.e. fermented only with yoghurt cul-tures). The contrai and Bifidus products contained atleast 108 colony-forming units/g (CFU/g) of yoghurtcultures on the first day of each ingestion period. TheBifidus product contained additionally between 5 x 107and 108 CFU/g of B. animalis strain DN-173 010.

Experimental design

The study comprised four consecutive periods, i.e. aID-clay run-in period and two ID-clay ingestion periodswith an interval of 10 days between them. Subjectswere asked to retain their normal stable diet, except thatmilk, sauces and fermented dairy products, includingyoghurt and soft cheeses, were excluded throughout thestudy. During the ingestion periods, the subjects weregiven, in a randomized, double-blind, cross-over fash-ion, three 125 g ClipS per clay of either contrai orBifidus. Half of the subjects received the contrai beforeBifidus (and were referred to as group A), and halfreceived the Bifidus before the contrai (group B). Thesubjects recorded the number of bowel movements inthe last week of each period. ln the last 3 days of therun-inand each test period, the subjects ingested everyclay 20 identical radio-opaque pellets at 09.00 h, andan abdominal X-ray was taken on the last day. Totaland segmentaI colonic transit times were calculatedaccording to the distribution of the markers in thedifferent segments of the bowel, using the formula:

transit time in the left colon in healthy men. ID We

observed. in a previous study, that a milk fermented byBifidobacterium animalis strain DN-173 010 shortenedth~ colonic transit time fi women, particularly thosewith a long transit time.ll The effect on faecal weightand stool frequency was not assessed and the mechan-ism is unknown. We hypothesized that an increase inthe faecal bacterial mass and/or in the secondary bilesalts in the colon may be responsible for this effect. Wespeculated tnat the probiotic bifidobacteria whichsurvive to the faecal stage may significantly increasethe faecal bacterial mass and/or stimulate colonicmotility by enhancing colonic concentrations of secon-clary bile salts. Indeed, some bifidobacteria may directlymetabolize bile acids12. 13 and some probiotics influence

bile salt metabolism by endogenous flora.14This study aimed to confirm, in a double-blind,

randomized, controlled trial, that B. animalis strainDN-173 010 shortens the colonic transit time inwomen, aQd to establish whether this effect is due toan incr~ased faecal bacterial mass or an increase insecondary faecal bile acids.

SUBJECTS AND METHODS

Subjects

This randomized, double-blind. cross-over study wasconducted in three centres in France (Clermont-Fer-rand, Marseille, Paris). Inclusion criteria were: healthyfemale volunteers. aged 18-45 years; absence of preg-nancy; body mass index < 25; absence of a past historyof digestive disease; absence of constipation; andabsence of symptoms of irritable bowel syndrome. Eachvolunteer provided written consent to the protocolwhich had been approved by the Ethics Committee ofthe Factulté de Médecine. Université Marseille Il. Thirty-six womenwere included (12 in each centre). Four wereexcluded because the study endpoint could not beassessed (errors in the timing of pellet ingestion). The 32volunteers in whom the study endpoints were measuredhad a mean age of 27 ::!: 7 years, a body weight of55 ::!: 5 kg and a height of 164 ::!: 6 cm.

1.2 x 2::nitransit time

where ni is the number of pellets present in eachsegment (right, left and sigmoid colon),lS

Stool analysis

A staal analysis was performed in the 12 volunteersfollowed in Paris. AlI stools in the last 3 days of eachstudy period were collected, and brought to themetabolic ward within 12 h in closed boxes carried ina larger box which was filled with ice. Faecal pH wasmeasured using a pH-meter (Radiometer, Copenhagen.Denmark). Samples were then frozen and kept at

Test products

The two test products were prepared by Danone(Danone Vitapole, Le Plessis Robinson. France) 15 daysbefore the beginning of the ingestion periods. They had

@ 2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16. 587-593

BIFIDOBACTERIA AND COLONIC TRANSIT TIME 589

-80 °C until analysis. Faecal bacterial mass wasdetermined on an aliquot according to the fractionationprocedure of Stephen and Cummings.I6 To determinethe faecal bile acids. 70% isopropanol was added to afaecal aliquot to inhibit bacterial degradation of bileacids. These samples were immediately freeze-dried. andthe wet and dry weights were measured before and afterfreeze-drying, respectively. Further steps included heat-ing with ethanol (three reflux steps during 2 h at90 °C), centrifugation, purification of bile acids withethyl acetate. extraction through a Bond Elut cartridge,methylation and sialylation. Separation of bile acids wasperformed by gas chromatography (Hewlett Packard5890 series II gas chromatograph equipped with an HP-5 MS capillary column) , and identification and quan-tification by mass spectrometry (HP 5971A massspectrometer). The analysis conditions have been des-cribed previously by Setchell et al.I?

include 36. Results are expressed as means with theirs.d.

Comparisons. The homogeneity of groups A and B waschecked with the non-parametric Mann-Whitney test.The analysis of product effect and period effect wasperformed using analysis of variance (ANOVA) or Krusk-alI-Wallis test. Comparisons between products werealso performed using Student's test for paired data.

RESULTS

Groups A and B did not differ significantly at the end ofthe run-in period for any transit time or faecalparameter, except for pH (Table 1).

Colonic transit time and number of bowel movements

The total colonic transit time and the sigmoid transit timewere significantly shorter after Bifidus than after contraiconsumption (Table 2). They did not differ significantlyfrom those measured at the end of the run-in period withany of the treatments (Table 2). Individual results areshawn in Figures 1 and 2. ln the sub-group of subjectswith an initial transit time above 40 h (n = 21), asignificant decrease in the total colonic transit time wasobserved during the Bifidus consumption period incomparison with the run-in period and the "controlperiod (Table 2). ln this sub-group, a significant decreasein the sigmoid transit time was also observed during theBifidus consumption period in comparison with the run-in period and the contrai period (Table 2). The number ofstools per week did not significantly differ between theBifidus,controlandrun-inperiods(7.9:t

2.7,7.6:t 2.8and 7.0 :t 1.6 stools/week, respectively).

Statistical analysis

Study end points. The major endpoint was the compar-ison of the total colonic transit time after consumptionof Bifidus vs. contrai. Secondary endpoints were thesegmentaI colonic transit times (the right. left andsigmoid colon). staal frequency. faecal weight. faecalbacterial mass. faecal pH and faecal bile salts (individualbile salts. pooled primary and pooled secondary bilesalts). Comparisons between each product period andrun-in period were also performed. ln our previousstudy using the saille method. the mean total colonictransit time in healthy volunteers was 36:t 16 h(mean :t s.d.). To test the hypothesis of a difference of12 h with an IX value of 0.05 and a f3 value of 0.15. wecalculated that 32 subjects were needed. and decided to

Table 1. Comparison between groups Aand B at the end of the run-in period(mean :t s.d.)

Group A(n = 17)

Group B(n = 15) P value*

57.2 :!17.8 :!15.0 :!24.4 :!

7.2 :!12.8 :!1468

:!

52.9 :!: 28.812.6 :!: 9.114.2 :!: 12.426.1 :!: 23.56.7 :!: 0.49.3 :!: 4.61256:!:

1012

0.5840.3440.7910.7190.0450.584O~R4

Total colonic transit time (h)Right colonic transit time (h)Left colonic transit time (h)Sigmoid colonic transit time (h)Paecal pHtPaecal bacterial mass (g/day)tPaecal secondary bile salts

(mmol/g dry weight)t

Group A. consumption of control then Bifidus. Group B. consumption of Bifidus then control..Mann-Whitney test.t n = 6 for both groups A and B.

@ 2002 Blackwell Science Ltd. Aliment Pharmacol Ther 16. 587-59:

:

28.0:

13.6:

Il.0:

14.2:

0.3:

5.7:

1370

P. MARTEAU et al.590

Group A (Control-Bifidus)

80.0 1

70.0

60.0

50.0

40.

30.

20.

10.00!

0.00,'1

;:s:Q)

:§-"00c

~c0

""60

"(ij"01-

BifidusControlRun-in

Figure 1. Total colonic transit time for each individu al volunteerin both groups during the run-in, Bifidus and control periods.

Figure 2. Transit time in the distal part of the colon'Ïor eachindividual volunteer in bath groups during the run-in. Bifidus and

contrai periods.

products

had no significant effect on the faecalbacterial mass (treatment effect: P = 0.938), but asignificant increase in the faecal bacterial mass wasobserved during the second consumption period inbath groups A and B (period effect: P = 0.042)

(Table 3).

Staal analysis

ln the 12 subjects analysed, faecai weight did notdiffer significantIy between the Bifidus and contraiperiods (96 :t 44 vs. 85 :t 36 g/day). Paecai pH wasnot different (6.89:t 0.44 vs. 6.83:t 0.28). The

Table 2. Total and segmentai coionic transit times (CTT) at the run-in period and after consumption of either Bifidus or contrai product

(mean 1: s.d.)

Subjects with total CTT > 40 h in run-in period (n = 21)Whole population (n = 32)

Run-inTransit tilDe (h)

TotalRight colonLeft colonSigmoid colon

Two values having different letters within the same row in the whole population and in the subjects with a long CTT are statistically different.

P < 0.05.

@ 2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16, 587-593

:!: 21.Sa:!: 12.7:!: II.9:!: IS.3a

BIFIDOBACTERIA AND COLONIC TRANSIT TIME 591

Table 3. Faecal bacterial mass (g/day) in 12 healthy women afterconsuming contrai or Bifidus product (mean :t s.d.)

~

Group AGroup B

13.3 :!: 6.014.9 :!: 4.8

Group A. consumption of control then Bifidus. Group B. consumptionof Bifidus then control.Treatment efTect: P = 0.938. Period efTect: P = 0.042.

The faecal concentrations of total secondary bileacids, deoxycholic acid and lithocholic acid were notsignificantly affected during any period (Table 4).There was an increase in stool concentration ofprimary bile acids during the Bifidus period incomparison with the control period which approachedbut did not achieve statistical significance (Table 4;P = 0.079).

efIect of probiotics on intestinal transit. bas been poorlystudied. Bouglé et al. reported that the administration ofpropionibacteria may slow clown tra~sit in the left colonin healthy men; however, their study was open and didnot use a contrai group.10 The present study demon-strates, for the first time, that a probiotic strain maysignificantly shorten the colonic transit time. It is inaccordance with our previous trial, which suggestedthat B. animalis DN-173 010 shortened the sigmoidtransit time in women.11 This strain bas a high survivalin the small and large intestine when it is ingested in afermented dairy product.6. 7. 9 Although the clinical

relevance of our results is open to discussion, it bas beenshawn that modulation of the intestinal motility can beobtained with probiotics, and a rational mechanism forSaille of their efIects bas been provided.None of the mechanisms evaluated in the present

study (increase in faecal weight, especially in faecalbacterial mass, and modifications in secondary bile saltexcretion) can explain the efIect of the probiotic on thecolonic transit time. It is possible that saille efIects ofprobiotics on colonic bacteria may occur slowly andmay remain for more than 10 days, and this may be alimitation of our cross-over study design. Other studieshave shawn that modifications of faecal enzymesinduced by probiotic ingestion may indeed last for upto 3 weeks after cessation of ingestion. 9. 21 Future cross-

over studies aimed at assessing the efIects of prObioticson the colon ecosystem should include longer washoutperiods. ln the present study, an efIect of the strain onthe colonic transit time was observed, while the faecalbacterial mass and bile acids were not significantlyinfluenced.

DISCUSSION

This double-blind. randomized. controlled study dem-onstrates that healthy women have shorter total colonicand sigmoid transit times when they ingest 3 clips/clayof a fermented milk containing yoghurt cultures plusB. animalis DN-173 010 vs. the saille product withoutthe latter strain. The faecal weight. bacterial mass andfaecal excretion of secondary bile salts are not signifi-cantly influenced.Several studies have shawn that probiotics may

influence intestinal disturbances and. in particular.may prevent or shorten the duration of diarrhoea ininfants or adults with acute gastroenteritis.2. 18-21 The

Table 4. Individual and total secondary bile acid concentration and primary bile acidconcentration (mean :t s.d.; [extremevalues])in 12 healthy women (six in group A and six in group B) after consuming contrai or Bifidus

Bifidus

A

Bile acid (BA)concentration

(nmol/g dry weight)

Primary BAconcentrationSecondary BAconcentrationDeoxycholic acidconcentrationLithocholic acidconcentration

P value*

0.079

B

Control lliIJB::\\n1;\',A .., ,

\: 1:

67 :t 85 :..

[0; 226]1625:t 1794[200; 4956]904:t 1218[50; 3256]721:t 595[150; 1700]

188:t 280[9; 737]2413:t 1597[771; 5119]1170:t 942[218; 2906]1243:t 703[324; 2213]

22 :t28[0; 72]3260 :t 2540[1589; 8310]1606:t 1446[645; 4488]1654:t 1108[880; 3822]

B.

7:1: 10 j;t

[0; 25]2968:1: 1550

[1041; 5232]1349:1: 711

[540; 2322]1619 :1:878

[410; 2910]

0.660

Group A. consumption o[control then Bifidus. Group B. consumption of Bifidus then contrat.'Statistical significance of the treatment effect. i.e. contrai vs. Bifidus.

@2002 Blackwell Science Ltd,Aliment..PharmacoITher16. 587~593

\' "" "" :. .,

"" 1"WJ(J l Control(n'7 ~2) :." Blfidus«<'7:j.~)'~_.,~k~

P.

MARTEAU et al.592

Saille bifidobacteria can deconjugate and dehydroxy-late bile salts. i.e. con vert conjugated primary bile saltsinto free secondary bile salts which can stimulatecolonic motility and secretion.12. 13 ln the present

study, B. anirnalis DN-173 010 ingestion did notinfluence the faecal excretion of secondary bile salts.We believe that this is a good thing, as secondary bilesalts may increase the risk of colon cancer.22-24 Theshortening of the colonic transit time was thereforenot due to significant bile salt modifications. Theconcentrations of primary bile acid tended to increaseafter consumption of the Bifidus product and thiscould be due to shortening of the colonic transittime.25 The mechanism of the efIect of the testedstrain on the colon motility remains unknown. Onemay hypothesize that a product of bacterial originmay decrease the sigmoid tonus and or stimulate thecolonic motility. One of these products might bephloroglucinol which can be produced by Saillecolonic bacteria and has strong efIects on colonicmotility.26. 27 Further studies should be performed to

evaluate this hypothesis.We conclude that the regular consumption of a Bifidus

fermented milk (BIO. Danone) influenced the colonicmotor function. This efIect is specific to the probioticstrain as it was not observed with a contrai fermentedmilk. Further studies are needed to determine themechanism. and also to assess the clinical efficacy ofthe BIO product in constipated patients.

through the human small intestine: an in vivo study usingintestinal perfusion. Am J Clin Nutr 1992; 55: 78-80.

7 Berrada N, Lemeland JF, Laroche G. et al. Bifidobacterium fromfermented milks: survival during gastric transit. J Dairy Sci1991; 74: 409-13.

8 Marteau P. Pochart P. Flourié B. et al. EfIect of chronicingestion of a fermented dairy product containing Lactobacil-lus acidophilus and Bijidobacterium bijidum on metabolicactivities of the colonic flora in man. Am J Clin Nutr 1990;52: 685-8.

9 Duez H. Pelletier C. Cools S. et al. A coIGny immunoblottingmethod for quantitative detection of a Bijidobacterium animalisprobiotic strain in human faeces. J Appl Microbiol 2000; 88:1019-27.

10 Bouglé D. Roland N, Lebeurrier F, Arhan P. ElTect of propi-onibacteria supplementation on fecal bifidobacteria and seg-mentaI colonic transit time in healthy human subjects. ScandJ Gastroenterol1999; 34: 144-8.

Il Bouvier M, Méance S. Bouley C. Berta JL. Grimaud JC. ElTectsof consumption of a milk fermented with the probiotic strainBifidobacterium animalis DN-173 010 on colonic transit timesin healthy humans. Biosci Microflora: in press.

12 Tanaka H. Doesburg K, Iwasaki T. Mierau 1. Screening oflactic acid bacteria for bile salt hydrolase activity. J Dairy Sci1999; 82: 2530-5.

13 Grill JP. Manginot-Dürr C. Schneider F. Ballongue J. Bifido-bacteria and probiotic elTects: action of Bifidobacterium specieson conjugated bile salts. Curr Microbiol1995; 31: 23-7.

14 Salvioli G. Salati R, Bondi M. et al. Bile acid transformationby the intestinal flora and cholesterol saturation in bile. Ef-fect of Streptococcus faecium administration. Digestion 1982;23: 80-8.

15 Chaussade S, Khyari A, Roche H. et al. Determin~tion of totaland segmentaI colonic transit time in constipated patients. DigDis Sci 1989; 34: 1168-72.

16 Stephen AM. Cummings JH. The microbial contribution tohuman faecal mass. J Med Microbiol1980; 13: 45-56.

17 Setchell KD. Lawson AM. Tanida N. Sjovall J. General methodsfor the analysis of metabolic profiles of bile acids and relatedcompounds in feces. J Lipid Res 1983; 24: 1085-100.

18 Isolauri E. Juntunen M, Rautanen T, et al. A human Lacto-bacillus strain (Lactobacillus casei sp strain GG) promotesrecovery from acute diarrhea in children. Pediatrics 1991; 88:90-7.

19 Shornikova A V. Casas lA, Mykkanen H, et al. Bacteriotherapywith Lactobacil/us reuteri in rotavirus gastroenteritis. PediatrInfect Dis J 1997; 16: 1103-7.

20 Saavedra JM. Bauman NA. Oung 1. et al. Feeding of Bijido-bacterium bijidum and Streptococcus thermophilus to infants inhospital for prevention of diarrhoea and shedding of rotavirus.Lancet 1994; 344: 1046-9.

21 Goldin BR. Gorbach SH. The elTect of milk and lactobacillusfeeding on human intestinal bacterial enzyme activity. Am JClin Nutr 1984; 39: 756-61.

22 Biasco G. Paganelli GM. Owen RW. et al. Faecal bile acids andcolorectal cell proliferation. The ECP Colon Cancer WorkingGroup. Eur J Cancer Prev 1991; l(Suppl. 2): 63-8.

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