Nutrition Journal
`
`BioMed Central
`
`Open Access
`Research
`Four-week short chain fructo-oligosaccharides ingestion leads to
`increasing fecal bifidobacteria and cholesterol excretion in healthy
`elderly volunteers
`Yoram Bouhnik*1, Lotfi Achour2, Damien Paineau3, Michel Riottot4,
`Alain Attar1 and Francis Bornet3
`
`Address: 1Pôle des Maladies de l'Appareil Digestif (PMAD), Service de Gastroentérologie et d'Assistance Nutritive, Hôpital Beaujon, 100 bd du
`Général Leclerc, 92110 Clichy Cedex, France, 2Institut Supérieur de Biotechnologie, avenue Taher El HADED, BP.74, 5000 Monastir, Tunisia ,
`3Nutri-Health SA, Immeuble Ampère, 8 rue Eugène et Armand Peugeot, 92566 Rueil-Malmaison Cedex, France and 4Université Paris Sud,
`Laboratoire de Physiologie de la Nutrition, Bat. 447, 91405 ORSAY Cedex, France
`
`Email: Yoram Bouhnik* - yoram.bouhnik@bjn.aphp.fr; Lotfi Achour - lotfiachour@yahoo.fr; Damien Paineau - d.paineau@nutri-health.eu;
`Michel Riottot - Michel.Riottot@ibaic.u-psud.fr; Alain Attar - alain.attar@bjn.aphp.fr; Francis Bornet - frj.bornet@nutri-health.eu
`* Corresponding author
`
`Published: 5 December 2007
`
`Nutrition Journal 2007, 6:42
`
`doi:10.1186/1475-2891-6-42
`
`This article is available from: http://www.nutritionj.com/content/6/1/42
`
`Received: 20 July 2007
`Accepted: 5 December 2007
`
`© 2007 Bouhnik et al; licensee BioMed Central Ltd.
`This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
`which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
`
`Abstract
`Background: Short-chain fructo-oligosaccharides (scFOS) are increasingly used in human diet for
`their prebiotic properties. We aimed at investigating the effects of scFOS ingestion on the colonic
`microflora and oro-fecal transit time in elderly healthy humans.
`Methods: Stools composition, oro-fecal transit time, and clinical tolerance were evaluated in 12
`healthy volunteers, aged 69 ± 2 yrs, in three consecutive periods: basal period (2 weeks), scFOS
`(Actilight®) ingestion period (8 g/d for 4 weeks) and follow-up period (4 weeks). Two-way
`ANOVA, with time and treatment as factors, was used to compare the main outcome measures
`between the three periods.
`Results: Fecal bifidobacteria counts were significantly increased during the scFOS period (9.17 ±
`0.17 log cfu/g vs 8.52 ± 0.26 log cfu/g during the basal period) and returned to their initial values at
`the end of follow-up (8.37 ± 0.21 log cfu/g; P < 0.05). Fecal cholesterol concentration increased
`during the scFOS period (8.18 ± 2.37 mg/g dry matter vs 2.81 ± 0.94 mg/g dry matter during the
`basal period) and returned to the baseline value at the end of follow-up (2.87 ± 0.44 mg/g dry
`matter; P < 0.05). Fecal pH tended to decrease during scFOS ingestion and follow-up periods
`compared to the basal period (P = 0.06). Fecal bile acids, stool weight, water percentage, and oro-
`fecal transit time did not change throughout the study. Excess flatus and bloating were significantly
`more frequent during scFOS ingestion when compared to the basal period (P < 0.05), but the
`intensity of these symptoms was very mild.
`Conclusion: Four-week 8 g/d scFOS ingestion is well tolerated and leads to a significant increase
`in fecal bifidobacteria in healthy elderly subjects. Whether the change in cholesterol metabolism
`found in our study could exert a beneficial action warrants further studies.
`
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`Background
`Short-chain fructo-oligosaccharides (scFOS) are a mixture
`of oligosaccharides consisting of glucose linked to fruc-
`tose units [1]. They are poorly absorbed in the human
`small intestine [2], but are fermented in the colon by the
`resident microflora [3]. It is now well established that
`scFOS meet criteria to be considered as prebiotic, defined
`as a non digestible food ingredient that beneficially affects
`the host by selectively stimulating growth and/or activity
`of one or a limited number of colonic bacteria, and thus
`improves host health [4]. We have shown in humans that
`dietary addition of 10 g/d scFOS led to increasing fecal
`counts of bifidobacteria [5]; moreover, the scFOS admin-
`istration dose-dependently increases fecal bifidobacteria
`in healthy volunteers, with an optimal and well-tolerated
`dose ranging from 2.5 to 10 g/d [6,7].
`
`Bifidobacteria are considered beneficial to health [8], even
`if sound evidence of such effect is not available yet [4]. In
`mice, in vivo administered bifidobacteria along with
`fructo-oligosaccharides reduced 1,2-dimethylhydrazine
`induced carcinogenesis [9]. In rats, Bifidobacterium longum,
`administered alone or in association with non-digestible
`oligosaccharides, exerts strong antitumour activity [10,11]
`This effect could be due to colon acidification that inhibits
`bacterial degradation of primary to carcinogenic second-
`ary bile acids [12] and/or to increasing bifidobacteria pop-
`ulation. Indeed, bifidobacteria per se could have an anti-
`tumorigenic activity. Bifidobacteria reduce nitrosamine
`mutagenicity, and Bifidobacterium bifidum administered
`along with Lactobacillus acidophilus to healthy humans
`decreases nitroreductase activity in stools [13]. Lastly, oli-
`gosaccharide ingestion could result in increasing colonic
`contents and decreasing transit time [14], both factors
`may affect colonic carcinogen concentration and mucosal
`contact time [15]. Thus, taking into account the intrinsic
`anti-tumoral properties of bifidobacteria and the effects
`on colonic pH, fecal mass and transit time, a potential
`benefit of scFOS ingestion could be colon cancer preven-
`tion, in particular in the elderly, who are particularly at
`risk of developing colon cancer [16].
`
`Although colonic microbiota is relatively stable through-
`out adult life, age-related changes in the gastrointestinal
`tract inevitably affect its composition [17]. Bifidobacteria
`are numerically important colonic species that can be
`found in adults [18], and the decline in bifidobacteria
`numbers is one of the most marked changes in the elderly
`gut [19]. These changes, along with general reduction in
`species diversity in most bacterial groups, as well as
`changes in diet and digestive physiology, such as intesti-
`nal transit time, may result in increased putrefaction in
`the colon, and greater susceptibility to disease. Dietary
`supplements containing prebiotics have been suggested to
`counteract these changes in the elderly [20-22].
`
`In that context, the aim of our study was to assess in
`healthy elderly the effects of four-week scFOS ingestion
`on colonic microflora and oro-fecal transit time (OFTT).
`
`Methods
`Subjects
`Twelve elderly healthy volunteers, six men and six
`women, aged 69 ± 2 years, participated in the study. None
`of them had any gastrointestinal disease history. No anti-
`biotics or laxatives had been taken during the 3 months
`before the study. No other medication was allowed during
`the investigation period. The subjects signed a written
`informed consent to the protocol, which was approved by
`Lariboisière – Saint-Louis Hospital Ethics Committee.
`
`Study Design
`The study was conducted in Saint-Lazare Hospital, Paris,
`France. It was divided into three periods: basal (weeks
`1–2), scFOS (weeks 3–6), and follow-up (weeks 7–10)
`periods. Throughout the study, volunteers took their
`usual diet. Neither fermented dairy products containing
`viable bifidobacteria and FOS (onions, asparagus, rye,
`and Jerusalem artichoke) were allowed, nor food known
`to induce abdominal symptoms (beans, cabbage, raisin,
`banana, and wheat bran). During scFOS period, subjects
`received 8 g/d scFOS in two oral doses at the end of break-
`fast and diner. This dose was defined as a good compro-
`mise between efficacy and tolerance. We used scFOS from
`Actilight® (Beghin Meiji, Marckolsheim, France), which
`consist of 44% 1-ketose (GF2), 46% nystose (GF3), and
`10% 1F-β-fructofuranosyl nystose (GF4).
`
`To measure the mean oro-fecal transit time (OFTT), the
`subjects ingested, with their breakfast, 20 radio-opaque
`pellets of different shapes for three consecutive days [23].
`The first stools passed after the fourth day were collected,
`and their marker content analysed. Stools had been col-
`lected for three consecutive days before the end of weeks
`2 (basal period), 6 (ingestion period), and 10 (follow-up
`period), that is to say at the end of each feeding period.
`
`Tolerance to administered scFOS was evaluated using a
`daily chart in which the symptoms (excessive flatus, bor-
`borygmi, bloating, and abdominal pain) were rated from
`zero (no symptom) to three (severe symptom). Stool fre-
`quency and consistency were also graded by the volun-
`teers. Diarrhoea was defined as one or more watery stools,
`or more than three stools per day.
`
`Stool collection
`Stools were collected three times, for 48 h at the end of
`each period (weeks 2, 6, and 10). Samples were collected
`in plastic containers rendered anaerobic (Anaerocult A;
`Merck, Darmstadt, Germany), immediately transferred to
`the laboratory, and then analysed for bacterial counts and
`
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`pH within 2 hours. Stools were then frozen at -20°C for
`further analysis.
`
`Bacterial counts and pH
`Fresh faecal samples (1 g) were introduced in the first pre-
`weighed tube of the dilution series and thoroughly mixed,
`then further tenfold dilutions were made up to -9 in a
`reduced diluent (1/4 strength cysteinated Ringer diluent).
`0.1 ml of each dilution was spread on plates with different
`selective media to distinguish several bacterial genera:
`total anaerobic counts (Wilkins-Chalgren agar), Bifido-
`bacterium (Beerens' medium), Clostridium spp. (TNS
`medium), and enterobacteria (McConkey agar). The tests
`were duplicated for the first two media. Plates of the first
`three media were anaerobically incubated for 5 to 7 d, and
`McConkey agar was aerobically incubated for 48 hours.
`Colony counts were obtained and expressed as a log of the
`colony-forming units (CFU) per gram of fresh faeces.
`Extemporarily, the fresh stool pH was measured by pH
`meter (Bioblock, Illkirch, France).
`
`Bile acids
`For bile acid and neutral sterol analysis, frozen stools had
`been lyophilised and lipids had been extracted with etha-
`nol for 24 hours in a Soxhlet apparatus. Lipid fractions
`had been saponified in boiling ethanolic 2 m potassium
`hydroxide for 1 h. Sterols were extracted with hexane, and
`bile acids were deconjugated. [24] Total bile acids were
`measured by 3-hydroxy-steroid-dehydrogenase, according
`to slight modification of Stempfel and Sidbury technique
`[25]. Prior to enzyme determination, bile acids were dis-
`solved in 2-propanol. Free bile acids were methylated
`with
`diazomethane,
`silylated with Deriva-sil
`(Chrompack, Middelburg, The Netherlands), and assayed
`on Carlo Erba (Milan, Italy) HGRC 5160 gas chromato-
`graph equipped with standard fused silica WCOT capil-
`lary column cross-linked with OV1701 (Spiral, Dijon,
`France) (length, 25 m; film thickness, 0.2 lm; oven tem-
`perature, 240°C; hydrogen carrier gas flow rate, 2 mL/
`min). Faecal sterols were silylated with bis(trimethylsi-
`lyl)tri-fluoroacetamide (BSTFA) + 1% trimethylchlorosi-
`lane (TMCS) (Pierce, Rockford, IL, USA), and quantified
`using gas chromatography described above, with the fol-
`lowing modifications: fused silica WCOT OV 101 capil-
`lary column (Spiral, Dijon, France) (length, 25 m; film
`thickness, 0.2 lm; oven temperature, 220°C).
`
`Data analysis
`Faecal bacteria concentrations were expressed as log col-
`ony forming unit (cfu)/g wet weight. The results were
`expressed as means ± SEM for each period. Two-way
`ANOVA, with time and treatment as factors, was used to
`compare bacterial concentrations, pH, and faecal metabo-
`lites between the three periods. Following a significant F
`test (P < 0.05), Newman-Keuls test was used to identify
`
`differences between individual means. Symptoms experi-
`enced with scFOS were compared to those experienced
`with placebo using Wilcoxon signed rank test.
`
`Results
`Bacterial counts and pH
`Table 1 summarises bacterial counts and pH during basal,
`scFOS, and follow-up periods. Faecal bifidobacteria
`counts were significantly increased during the scFOS
`period (9.17 ± 0.17 log cfu/g vs 8.52 ± 0.26 log cfu/g dur-
`ing the basal period; P < 0.05), and returned to their base-
`line values during the follow-up period (8.37 ± 0.21 log
`cfu/g). Total anaerobe counts did not change during
`scFOS period compared to the basal period, but decreased
`in the follow-up period compared to the ingestion period
`(P < 0.05). Faecal Clostridium counts were significantly
`increased during the follow-up period compared to the
`basal and scFOS periods (P < 0.05). Faecal enterobacteria
`counts did not change during the three periods. Faecal pH
`tended to decrease during scFOS and follow-up periods
`compared to the basal period (P = 0.06).
`
`Faecal neutral sterols and bile acids
`Faecal cholesterol concentration increased during the
`scFOS period (8.18 ± 2.37 mg/g dry matter vs 2.81 ± 0.94
`mg/g dry matter during the basal period; P < 0.05)), and
`returned to the baseline value during the follow-up period
`(2.87 ± 0.44 mg/g dry matter (figure 1). However, no sta-
`tistical differences were
`reported
`for coprostanol,
`cholestanol, and ketones for the three periods (Table 2).
`Total neutral sterol concentrations and outputs did not
`change, but tended to increase (p = 0.08) during the
`scFOS period.
`
`Total bile acid concentrations and outputs were similar in
`the three periods. Concentrations of secondary (litho-
`cholic and deoxycholic acids) and primary bile acids
`(cholic and chenodeoxycholic acids) did not change for
`the three periods (Table 2).
`
`Table 1: Faecal bacterial counts (log cfu/g wet weight) and pH in
`elderly healthy volunteers during basal (2 wks), scFOS (4 wks)
`and follow-up (4 wks) periods (n = 12, mean ± SEM)
`
`Basal period scFOS period
`
`Bifidobacteria
`Total anaerobes
`Clostridium
`Enterobacteria
`PH
`
`8.52 ± 0.26
`10.09 ± 0.07
`3.25 ± 0.25
`7.69 ± 0.21
`6.57 ± 0.10
`
`9.17 ± 0.17a
`10.22 ± 0.06b
`3.45 ± 0.26b
`7.45 ± 0.28
`6.32 ± 0.10
`
`a different from basal and follow-up periods (P < 0.05)
`b different from follow-up period (P < 0.05)
`c different from basal period (P < 0.05)
`
`Follow-up
`period
`
`8.37 ± 0.21
`9.94 ± 0.09
`4.29 ± 0.30c
`7.48 ± 0.24
`6.26 ± 0.07
`
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`*
`
`12
`
`10
`
`8
`
`6
`
`4
`
`2
`
`0
`
`mg/g dry matter
`
`Coprostanol
`
`Cholestanol
`
`Cholesterol
`
`Ketones
`
`Neutral sterols
`
`Basal period
`
`scFOS period
`
`Follow-up period
`
`Effect of 4-wk scFOS ingestion (8 g/d) on faecal cholesterol in healthy volunteers (means ± SEM, n = 12)Figure 1
`
`Effect of 4-wk scFOS ingestion (8 g/d) on faecal cholesterol in healthy volunteers (means ± SEM, n = 12). *P <
`0.05 between scFOS period and both basal and follow-up period
`
`Stool weight and oro-fecal transit time
`Stool wet weight, dry matter, and faecal water did not
`change throughout the study. Oro-fecal transit time was
`
`Table 2: Faecal neutral sterols and bile acids (mg/g dry matter) in
`elderly healthy volunteers during the basal (2 wks), scFOS (4 wks)
`and follow-up (4 wks) periods (n = 12, mean ± SEM)
`
`Neutral sterols Basal period scFOS period
`
`Coprostanol
`Cholestanol
`Cholesterol
`Ketones
`Total
`
`9.00 ± 1.40
`0.29 ± 0.11
`2.81 ± 0.94a
`0.27 ± 0.05
`12.38 ± 1.15
`
`8.29 ± 2.09
`0,13 ± 0.05
`8.18 ± 2.37b
`0.16 ± 0.08
`16.75 ± 1.94
`
`Bile acids
`
`Basal period scFOS period
`
`Lithocholic
`Deoxycholic
`Cholic
`Chenodeoxycholic
`Ketones
`Total
`
`2.00 ± 0.43
`1.80 ± 0.35
`0.46 ± 0.21
`0.24 ± 0.05
`1.26 ± 0.30
`5.77 ± 0.66
`
`1.29 ± 0.29
`2.58 ± 0.50
`0.58 ± 0.19
`0.20 ± 0.04
`1.52 ± 068
`6.17 ± 1.25
`
`a ≠ b : P < 0.05
`
`Follow-up
`period
`
`7.99 ± 1.62
`0.23 ± 0.17
`2.87 ± 0.44a
`0.21 ± 0.06
`11.30 ± 1.57
`
`Follow-up
`period
`
`1.26 ± 0.17
`2.61 ± 0.63
`0.87 ± 0.24
`0.30 ± 0.07
`1.33 ± 0.19
`6.37 ± 1.02
`
`not significantly modified by scFOS ingestion compared
`to the basal and follow-up periods (Table 3).
`
`Digestive tolerance
`During scFOS ingestion, excessive flatus and bloating
`were significantly more frequent when compared to the
`basal period (P < 0.05), but symptom intensity was very
`mild (Table 4). Borborygmi and abdominal pain were not
`significantly different in all periods.
`
`Discussion
`The present experiment showed that four-week scFOS
`ingestion, with a dose of 8 g/d, is well tolerated and leads
`
`Table 3: Mean oro-fecal transit time (OFTT) and mean 24-h
`faecal wet weight, dry weight and percentage of faecal water in
`elderly healthy volunteers during the basal (2 wks), scFOS (4
`wks) and follow-up (4 wks) periods (n = 12, mean ± SEM)
`
`Basal period scFOS period
`
`OFTT (h)
`Wet weight (g/d)
`Dry weight (g/d)
`Faecal water (%)
`
`37.2 ± 3.4
`155.4 ± 20.9
`32.8 ± 3.3
`77 ± 2
`
`39.9 ± 3.3
`137.7 ± 17.3
`28.8 ± 2.9
`77 ± 1
`
`Follow-up
`period
`
`37.8 ± 3.7
`174.8 ± 22.0
`35.2 ± 3.5
`76 ± 2
`
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`Table 4: Digestive symptom intensity (ranged from 0 to 3) in
`elderly health volunteers during the basal (2 wks), scFOS (4 wks)
`and follow-up (4 wks) periods (n = 12, mean ± SEM)
`
`Basal period
`
`scFOS period
`
`Excessive flatus
`Bloating
`Borborygmi
`Abdominal
`pain
`
`0
`0
`0
`0
`
`0,83 ± 0,3a
`0,67 ± 0,26a
`0
`0,42 ± 0,23
`
`Follow-up
`period
`
`0,25 ± 0,13
`0,33 ± 0,14
`0
`0,25 ± 0,13
`
`Symptom intensity was rated as follow: 0: no symptom ; 1: mild
`symptoms; 2: moderate symptoms; 3: severe symptoms
`a different from basal period (P < 0.05)
`
`to significant increase in faecal bifidobacteria and choles-
`terol excretion in healthy elderly. The sc-FOS bifidogenic
`effect has been extensively demonstrated in adults [6,26-
`28], but rarely in elderly [19,21,22]. Among the very few
`available studies about functional foods in elderly, one
`recent double-blind trial testing a symbiotic (B. lactis BL-
`01, B. bifidum BB-02 and an inulin-based prebiotic) also
`found promising results [29]. Significant increase in total
`bifidobacteria counts was indeed observed in the symbi-
`otic group compared with the placebo group.
`
`We also found increasing Clostridium spp. after sc-FOS
`ingestion discontinuation. Clostridium spp. is a major com-
`ponent of normal anaerobic microflora and it can not be
`considered as a deleterious or beneficial genus. Since
`some toxinogenic subspecies of Clostridium difficile are
`related to an increased risk of pseudo membranous colitis
`and/or infection in older people, it would have been inter-
`esting to measure the sc-FOS effects on these subspecies.
`However, we did not perform those analyses, for they
`were out of our study scope. Further studies may investi-
`gate this point, using adequate measurement methods for
`species concentrations and toxinogenic properties (cellu-
`lar cultures, biomolecular analysis). Culture-based enu-
`meration of microbiota does not usually allow for
`bacterial species measurement, but mainly bacterial
`genus.
`
`In our study, several parameters were assessed with the
`objective to better understand scFOS physiological effects
`in healthy elderly, such as transit time, stool characteris-
`tics, and colonic environment. We did not find scFOS
`ingestion changed faecal weight and oro-fecal transit time
`in elderly. Gibson et al. have shown that prebiotics can
`increase stool output: they studied 8 volunteers under
`controlled diet, and showed that with 15 g/d fructo-oli-
`gosaccharides, stool output significantly increased from
`136 to 154 g/d [27]. Other two human studies did not
`demonstrate increasing stool output [2,30]. but the diet
`was not controlled in none of these studies, which may
`
`have hidden any slight effect. In the study of Alles et al., 12
`healthy subjects were given 4.8–19.2 g/d oligomate (52%
`galacto-oligosaccharides), which did not result in any
`change in bowel habit. However, the subjects started with
`unusually high faecal weights under controlled diet, 272
`± 26 g/d On the other hand, studies using probiotics dem-
`onstrated bifidobacteria could reduce human colonic
`transit time, but not all bifidobacteria strains have the
`same effects [31]. This specific strain-dependent effect
`could explain the reason why our prebiotic, which stimu-
`lates global endogenous bifidobacteria, had no effect.
`
`In our study, the microbial transformation of cholesterol
`into coprostanol was not influenced by scFOS ingestion.
`Another study observed that sterol and fatty acid biohy-
`drogenation by intestinal microflora is altered by oli-
`gosaccharide fermentation [32]. Coprostanol production
`results from intestinal anaerobic bacteria action [33].
`Concerning bile acid metabolism, no differences were
`observed during the three periods. Furthermore, the use of
`poorly digestible carbohydrates in rats, hamsters and pigs
`demonstrated that prevention of microbial conversion of
`bile acids depended on the carbohydrate dose in the diet
`[23,34] This suggests that the low carbohydrate dose, 8 g/
`d scFOS, used in this experiment is unable to modify
`microbial conversion of bile acids.
`
`Endogenous or exogenous bile acids, as well as dietary
`cholesterol are carcinogenic factors involved in colon can-
`cer in laboratory animals [35,36] Various epidemiological
`studies suggest those steroids could also be involved in
`colon cancer in men [12,37]. According to these studies,
`low scFOS dose ingestion by humans, which prevented
`microbial conversion of cholesterol into cytotoxic mole-
`cule, (coprostanol, potentially carcinogenetic), could be
`interesting for humans. In our study, the intake of 8 g/d
`scFOS led to increasing faecal cholesterol. The mechanism
`of such increase could be related to decreasing cholesterol
`bacterial transformation, although we failed to find any
`significant sc-FOS effect on cholesterol bacterial metabo-
`lism. Moreover, the low scFOS dose used in our study was
`also probably not sufficient to significantly reduce micro-
`bial conversion of bile acids. However, in our previous
`study evaluating a higher scFOS dose (12.5 g/d), we also
`failed to show any significant effect in bile acids and neu-
`tral sterol [28]. These negative results could be explained
`by a questionable capacity of various bifidobacteria to
`take up cholesterol into their cellular membrane [38].
`
`Conclusion
`Overall, we showed that 8 g/d scFOS ingested are well tol-
`erated and led to significant increase in faecal bifidobacte-
`ria in healthy elderly. Under our experimental conditions,
`i.e. 8 g/d for 12 days, we failed to show any sc-FOS effects
`on OFTT, which is commonly increased in elderly living
`
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`in industrialised countries. We found significant change
`in cholesterol metabolism, which could potentially exert
`protective action against colon cancer; however, this find-
`ing warrants further studies.
`
`List of abbreviations used
`OFTT: Oro-fecal transit time; scFOS: Short-chain fructo-
`oligosaccharides
`
`Competing interests
`The author(s) declare that they have no competing inter-
`ests.
`
`Authors' contributions
`YB participated in the study design, data collection, data
`analysis, and manuscript writing. LA participated in data
`collection. MR carried out bile acids and neutral sterols
`analysis. DP participated in data analysis and manuscript
`writing. FB designed the study and participated in manu-
`script writing. All authors read and approved the final
`manuscript.
`
`Acknowledgements
`The study was supported by a grant from Beghin-Meiji, Marckolsheim,
`France.
`
`2.
`
`4.
`
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