International Journal of Food Microbiology 139 (2010) 168–176

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International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro

In vitro modulation of the human gastrointestinal microbial community byplant-derived polysaccharide-rich dietary supplements

M. Marzorati a,⁎, A. Verhelst b, G. Luta c, R. Sinnott c, W. Verstraete a, T. Van de Wiele a, S. Possemiers a,b

a Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, 9000 Gent, Belgiumb ProDigest, Technologiepark 3, 9052, Gent, Belgiumc Mannatech Inc, 600 S. Royal Lane, Suite 200 Coppell, Texas 75019, United States

⁎ Corresponding author. Tel.: +32 9 264 59 76; fax:E-mail address: massimo.marzorati@ugent.be (M. M

0168-1605/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.ijfoodmicro.2010.02.030

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 November 2009Received in revised form 30 January 2010Accepted 28 February 2010

Keywords:Gastrointestinal Resource ManagementBifidobacteriaColonic fermentationAmbrotoseGlyconutrientsPrebiotic

The use of prebiotics is a possible strategy to manage and steer the complex gut microbial communitytowards a health-promoting composition (Gastrointestinal Resource Management). In this study, theSimulator of the Human Intestinal Microbial Ecosystem was used to investigate the effects of twocommercially-available plant polysaccharide supplements on the structure, composition and metabolism ofan in vitro cultured colon microbial community.Microbial analyses showed both a bifidogenic (up to +1.3 log cfu/mL) and a lactobacillogenic (up to +0.9log cfu/mL) effect during treatment with the dietary supplements. Quantitative PCR confirmed that theincrease of Bifidobacteria spp. was statistically significant (Pb0.05) in all of the colon compartments andshowed a significant increase of the bacteroides–prevotella group concentration (+0.6 log cells/ml) in thecompartment simulating the proximal colon. Denaturant gradient gel electrophoresis analyses and a relativeecological interpretation, in combination with sugar and short-chain fatty acids quantification, providedevidence of a positive effect of both the tested products. Overall, the treatment period was associated with (i)good and selective fermentability of the polysaccharide supplements along the entire colon; (ii) positive andselective bifidogenic effect; (iii) the possibility of enhancing species belonging to Bacteroidetes, a phylumrecently associated with body weight management.

+32 9 264 62 48.arzorati).

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The human intestinal microbial ecosystem is responsible fornumerous processes involved in host health, such as extraction ofenergy from indigestible compounds, stimulation of the gut immunesystem and the synthesis of essential vitamins. On the other hand,specific microbial community assemblages may also be risk factorscontributing to disease states (e.g. allergies, bowel inflammation andobesity) (Turnbaugh et al., 2007; Leser and Mølbak, 2009; Possemierset al., 2009 and references within).

In recent years, many research efforts have focused onwhat has beenrecently definedasGastrointestinal ResourceManagement (GRM), i.e. themanagement of the complex gut microbiota and its metabolismwith theaim of improving the host health (Cummings and Macfarlane, 1991;Macfarlane andMacfarlane, 1997;ManningandGibson, 2004; Possemierset al., 2009). One approach to achieve this goal is the ingestion ofprebiotics to selectively influence the colonic microbial communitycomposition and activity (Gibson et al, 2004). The prebiotics currentlyused in the functional food industry are oligosaccharides such as lactulose,fructo-oligosaccharides (FOS), inulin, galacto-oligosaccharides (GOS) or

arabinoxylan-oligosaccharides (AXOS) (Manning and Gibson, 2004;Sanchez et al., 2009). Most of these compounds are predominantlyfermented in the proximal colon (with the exception of AXOS - Grootaertet al., 2009), where the microbiota is known to have a saccharolyticmetabolism (Macfarlane et al., 1992; Macfarlane and Macfarlane, 1997;Smith and Macfarlane, 1997). Consequently, there is a great interest infinding different prebiotics or mixtures of prebiotics that could exert abiological activity throughout the entire colon and reduce the occurrenceof chronic colonic diseases (for example, Crohn's disease and colorectalcancer) (Baumgart and Sandborn, 2007; Inoue et al., 2007).With this aim,specific studies have been conducted to identify potential key ingredientsin numerous medicinal plants known for their ancient uses as healingremedies (Eshun and He 2004). Among them, Aloe vera, larch andseaweed have been correlated with several positive effects in the humanbody (i.e. decreased ammonia absorption, immune modulation, possibleanti-metastatic activity, and increased short-chain fatty acids (SCFAs)production), and it has been suggested that the carbohydrates found inthese plants are indeed the biologically active components (Table 1)(Kelly, 1999; Robinson et al., 2001; Kim et al., 2002; Pogribna et al., 2008).

The aim of the present study was to evaluate the effect of twocommercially-available plant polysaccharide supplements (glyco-nutrients) on the structure, composition and metabolism of the residentmicrobial community in the different areas of the colon (i.e. ascending,

Table 1Protein and carbohydrate concentrations (expressed as molar percentage) in Larch extract, Aloe vera gel extract and gel powder, tragacanth gum, ghatti gum, rice starch and U.pinnatifida fucoidans (partially adapted from Luta et al., 2009).

Composition

Larch extract Protein b1%Carbohydrate 93% of which galactose 90%, arabinose 9%, mannose 1% and glucose 0.5%

Aloe vera gel extract Protein 2%Carbohydrate 38% comprising galacturonic acid 41%, mannose 39%, galactose 6%, xylose 6%, glucose 4%, arabinose 1%, rhamnose 1% and fucose 1%

Aloe vera gel powder Protein 1.7%Carbohydrate 23% comprising glucose 52%, mannose 40%, galacturonic acid 6%, galactose 1%, arabinose 1%, rhamnose 1%, xylose 1%

U. pinnatifida fucoidans Protein 2%Carbohydrate 51% comprising fucose 63%, galactose 34%, mannose 1% and traces of glucose

Tragacanth gum Protein 1%Carbohydrate 29% comprising arabinose 34.7%, xylose 27.7%, glucose 12.5%, galactose 11.3%, fucose 10.3,%, rhamnose 3.6%

Ghatti gum Protein 1.5%Carbohydrate 49% comprising arabinose 53.5%, galactose 34.2%, mannose 4.6%, glucose 3.6%, rhamnose 3.6%, xylose 0.5%

Rice starch Glucose 97%

169M. Marzorati et al. / International Journal of Food Microbiology 139 (2010) 168–176

transverse and descending colon). The two products contained acombination of larch arabinogalactan (Larix occidentalis), rice starch,Aloe vera gel extract or gel powder and wakame algae extract (Undariapinnatifida fucoidans) and twodifferent gums (ghatti and tragacanth). Forthis purpose, the Simulator of Human Intestinal Microbial Ecosystem(SHIME) was used. This in vitro system has already been shown to be auseful model for nutrition studies investigating intestinal microbialcomposition and activity (De Boever et al., 2000; Van de Wiele et al.,2004; Decroos et al., 2006; Van de Wiele et al., 2007; Marzorati et al.,2009).

2. Materials and methods

2.1. Mixed-polysaccharide dietary supplements

The compositions of the two dietary supplements investigated inthis study are shown in Table 1. Product A (Ambrotose® complex)contained larch arabinogalactan, rice starch, Aloe vera gel extract,ghatti gum, glucosamine HCl, and tragacanth gum; product B(Advanced Ambrotose® powder) had a similar composition but Aloevera extract was substituted with Aloe vera gel powder and wakamealgae extract was added. Both products have been provided byMannatech, Incorporated (Coppell, Texas, USA).

2.2. SHIME study

The SHIME is a dynamic model of the human gastrointestinal tract.It consists of five double-jacketed vessels maintained at a temperatureof 37 °C, simulating the stomach, small intestine, ascending, trans-verse and descending colon, respectively, for a total time period(72 h) that approximates the typical retention time for ingested foodin developed countries (Macfarlane and Macfarlane 1997). The threecolon vessels of two SHIME systems were inoculated with bacteriafrom the same fecal sample of a healthy young adult volunteer whohad no history of antibiotic treatment in the six months prior to thestudy. The experimental setup of the SHIME run was performed aspreviously described (Van de Wiele et al., 2007). This included a two-week control period, a three-week treatment period, and a two-weekwashout period. The composition of the SHIME feed used during thebasal and the washout periods was starch (4.0 g/L), arabinogalactan(1.0 g/L), pectin (2.0 g/L), xylan (1.0 g/L), glucose (0.4 g/L), yeastextract (3.0 g/L), peptone (1.0 g/L), mucin (4.0 g/L), and cysteine(0.5 g/L) (Sigma-Aldrich, Schnelldorf, Germany). During the treat-ment period, 3.0 g/L of the starch was replaced with 3.0 g/L of theinvestigated supplement. Samples for plate counts and DNA analyseswere collected once a week; those for SCFA and ammonium analyseswere collected three times per week.

2.3. Analytical techniques

Short-chain fatty acid (SCFA) and ammonium levels weredetermined as described by Van de Wiele et al. (2004). D-lactic acidand L-lactic acid quantification was conducted using the UV-methodfor the determination of D- and L-lactic acid in foodstuffs and othermaterials (R-Biopharm Enzymatic BioAnalysis, Brussels, Belgium)according to the manufacturer's instructions. The molecular weight(MW) profiles of polysaccharides in the residual content wereanalyzed by size exclusion chromatography (SEC). Of each sample,10 mg was dissolved in 50 mM ammonium acetate buffer and filteredthrough a 0.45 µm Ultrafree-MC HV centrifugal filter, and 200 μL wasinjected into an Agilent 1100 HPLC system. SEC was performed on asuperose 12 column, using 50 mM ammonium acetate as eluent witha flow rate of 0.4 mL/min.

The total carbohydrate content of the polysaccharide ingredientswas determined by hydrolysis with acetylation, followed by the GC–MS of the alditol acetates; the total protein was quantified using aBradford assay. Both analyses were conducted as previously reported(Luta et al., 2009).

2.4. Cultured-based microbiological analysis

Decimal dilutions of the samples in physiological solution (NaCl8.5%) were plated and incubated at 37 °C. Plates specific for anaerobicspecies were incubated in anaerobic jars. Counts were performed onMcConkey-agar (Oxoid, Basingstoke, UK) for coliforms, LAMVAB forlactobacilli (Hartemink et al., 1997), Enterococcus-agar (Difco, Sparks,MD, USA) for enterococci, TSC-agar (Merck, Darmstadt, Germany) forclostridia, RB-agar (Hartemink et al., 1996) for bifidobacteria, MSA-agar(Oxoid) for staphylococci and BHI-agar (Oxoid) for facultative anae-robes and anaerobes.

2.5. Molecular microbiological analyses

Denaturant gradient gel electrophoresis (DGGE) and quantitativepolymerase chain reaction (qPCR) on total bacteria, bifidobacteria,lactobacilli andbacteroideswereperformed to study theeffect of productsA and B on the structure and composition of the colonic microbialcommunity. Metagenomic DNA was extracted from samples collectedevery week for a total of seven weeks from each vessel using the CTABprotocol (Boon et al., 2003). Samples were marked as SxVyWz, where Sstands for SHIME and x can be 1 or 2 (respectively for products A or B), yrepresents the number of the vessel and z the week of the sampling.

qPCRs for total bacteria, bifidobacteria, and lactobacilli wereperformed as reported by Possemiers et al. (2006). The qPCR for thebacteroides–prevotella group was conducted according to Rinttiläet al. (2004), that for the whole Bacteroidetes phylum according toGuo et al. (2008).

Table 2SEC results of the residual carbohydrates in the SHIME suspension during the differentexperimental periods. Data are listed per retention time in the chromatography column(Rt) molecular weight (MW) and percent of the total of the retrieved sugars.

Product A Product B

Period Rt(min)

MW % Rt(min)

MW %

Ascending colon Control 12.8 N200000 0.5 18.5 150000 8.722.2 80000 11.9 44.1 b2000 65.834.1 10000 8 46.5 b2000 25.537.1 10000 79.7

Treatment 17.7 180000 44.8 18.1 160000 1.629.5 20000 36.2 32.1 10000 14.644.0 b2000 6.2 44.1 b2000 54.146.7 b2000 12.9 46.5 b2000 29.8

Washout 18.4 160000 5.9 5.9 N200000 1.630.2 20000 8.4 18.5 150000 6.343.9 b2000 62.5 30.2 20000 7.746.7 b2000 23.2 44.0 b2000 62.5

46.5 b2000 21.8Transverse colon Control 9.8 N200000 41.5 44.1 b2000 68.7

44.1 b2000 38.7 46.5 b2000 31.346.5 b2000 19.8

Treatment 44.1 b2000 65.4 18.4 160000 12.946.3 b2000 34.6 29.8 20000 7.9

b2000 55.8b2000 23.4

Washout 7.2 N200000 64.8 44.1 b2000 63.444.1 b2000 21.9 46.6 b2000 36.646.5 b2000 13.3

Descending colon Control 44.1 b2000 59.8 44.1 b2000 73.446.5 b2000 40.2 46.3 b2000 26.6

Treatment 44.1 b2000 64.8 44.1 b2000 70.146.4 b2000 35.2 46.2 b2000 29.9

Washout 27.9 30000 40.6 44.1 b2000 64.529.6 20000 23.6 46.3 b2000 35.535.5 10000 35.8

170 M. Marzorati et al. / International Journal of Food Microbiology 139 (2010) 168–176

DGGEwith a 45–60% denaturant gradient was used to separate thePCR products obtainedwith a nested approach for the 16S rRNA genesof bifidobacteria (primers BIF164f–BIF662r — Satokari et al., 2001),lactobacilli (SGLAB0159f-SGLAB0667 — Heilig et al., 2002), andbacteroides–prevotella group (fD1-rBacPre — Possemiers et al.,2004). The first PCR round was followed by a second amplificationwith primers 338F-GC and 518R (Ovreas et al., 1997). The latterprimers were also used to amplify the 16S rRNA gene of total bacteriaon metagenomic DNA. The most prominent bands were cut, the DNAeluted, re-amplified and sequenced (Van de Wiele et al., 2004).Sequence analysis was performed using the BLAST server of theNational Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov).

2.6. Ecological interpretation of molecular fingerprints and statistics

The obtained DGGE patterns were subsequently analyzed using theBioNumerics software v.2.0 (Applied Maths, Sint-Martens-Latem,Belgium). A matrix of similarities for the densiometric curves of theband patterns was calculated based on the Pearson product–momentcorrelation coefficient and dendrograms were created by using UPGMAlinkage. Fingerprinting ecological interpretation based on a three-parameter analysis (range-weighted richness, dynamics and functionalorganization) was conducted as described by Marzorati et al. (2008).Briefly, range-weighted richness (Rr) valueswere calculated based on thetotal number of bands (N) and the denaturing gradient comprisedbetween the first and the last band of the pattern (Dg), according to theequation Rr=N2×Dg. The dynamics parameter (Dy) was based on thematrix of similarities for the densitometric curves of the band patternscalculated based on the Pearson product-moment correlation betweenday X and day X−7 (Wittebolle et al., 2005). In order to graphicallyrepresent the evenness of the bacterial communities, the Pareto–Lorenz(PL) distribution curves (Lorenz, 1905) were set up, based on the DGGEprofiles as previously described (Wittebolle et al., 2008). The curves, interms of functional organization (Fo), were evaluated by a horizontaly-axis projection of their intercepts with the vertical 20% x-axis line (F20value) and by the GINI coefficient (Wittebolle et al., 2009).

Significance of qPCR results was investigated using Matlab v.7.4with one-way ANOVA, and individual means were compared usingthe Tukey's test.

3. Results

Two independent SHIME systems were assembled to evaluate thelong-term effects of two mixed-polysaccharide dietary supplements onthe colon's microbial community. SEC analysis on product A showed thefollowing percent peak distribution of the total polysaccharides present:170,000 Da=7.6%; 10,000 Da=56.7%; and b2000 Da=35.7%. For prod-uct B: 160,000 Da=10.7%; 10,000 Da=23.2%; and b2000 Da=66.1%.

SEC analyses of supernatant samples suggest that the mixture ofplant-derived polysaccharides of product A was almost completelydegraded in the transverse colon where only fragments b2000 Da(MW) could be retrieved during the treatment period. A high MW(160,000 Da) fraction of product B was still available in the transversecolon and was fermented in the descending colon (Table 2).

3.1. Effect on microbial metabolism

Treatmentwithproduct A had noeffect on total SCFAproduction butled to an increase in acetate production in the compartments simulatingascending and transverse colon and led to a small, but significant,increase in the propionate concentration in the transverse colon.Furthermore, following the treatment the butyrate concentrationdecreased in all of the colon compartments (Table 3). In the washoutperiod, two different trends were observed. In the ascending colon, thecombined production of acetate and propionate decreased more than

40% and was compensated by a strong production of lactate(17.68 mmol/L). In thedistal colon, a decrease in acetate and propionatecorresponded to a significant increase in butyrate production, ascompared to the treatment period. Treatment with product B did notinduce any significant variation in the tested organic acids (Table 4). Theslight fluctuations in total SCFA production mainly corresponded tovariations in acetate production. Also, a sharp decrease in acetate andpropionate production in the ascending colon during the washoutperiod was compensated by a 6-fold increase in the lactate concentra-tion. A significant increase in butyrate production was also observed inthe descending colon during the washout period.

For both tested products and in all colon regions, a transientincrease in ammonium concentrations was observed during thetreatment periods (Tables 3, 4). In the washout period, the valuesreturned to baseline, with the exception of the ascending colon ofSHIME 1 where they remained significantly lower and the descendingcolon of SHIME 2, where they remained higher.

3.2. Effect on the structure and composition of the microbial community

Weekly analysis of coliforms, lactobacilli, enterococci, clostridia,bifidobacteria, staphylococci, and facultative anaerobes and anaerobeswas performed (Table S1). Both treatments induced a significantincrease of both bifidobacteria and lactobacilli counts (Table S1; Fig. 1).For the bifidobacteria, the average value of the last two weeks oftreatment was significantly higher (Pb0.05) than the average of thecontrol period in the distal part of the colon (transverse anddescending colon) for product A and only in the descending colonfor product B. In both cases, a general increasing trend in all of thecolon compartments could be observed following the treatment.Lactobacilli increased in all colon compartments following adminis-tration of product A (Pb0.1), and in the transverse colon during the

Table 3Concentrations of organic acids and ammonium ions in the three vessels of the SHIMEduring the control (n≥6), treatment with Product A (n≥9) and washout (n≥6)a

periods.

Control Treatment Washout

Ascending colonOrganic acids(mmol/L)

Acetate 22.85±2.30 26.60±1.37* 13.89±8.46*Propionate 21.11±0.14 20.56±1.50 13.50±6.39*Butyrate 9.33±0.29 6.44±0.67* 6.26±1.74Lactate 5.73±1.56 3.67±0.28 17.68±12.50*Other SCFAb 2.32±0.16 2.34±0.14 1.59±0.50Total SCFA 61.35±0.43 59.59±1.90 52.91±29.58

Proteolytic markers(mg/L)

Ammonium 240.9±41.5 284.1±28.1* 172.9±110*

Transverse colonOrganic acids(mmol/L)

Acetate 33.19±1.40 37.02±2.54* 30.63±6.10Propionate 25.73±0.35 27.31±0.69* 25.35±2.75Butyrate 13.79±1.45 9.65±0.64* 14.52±3.27*Lactate 4.67±0.58 4.67±0.36 6.58±2.09Other SCFAb 2.94±0.14 2.91±0.18 2.99±0.05Total SCFA 80.32±0.41 81.55±3.74 80.06±7.72

Proteolytic markers(mg/L)

Ammonium 370.1±43 401.3±14.6 399.8±34.1

Descending colonOrganic acids(mmol/L)

Acetate 37.50±1.80 39.19±2.54 36.35±7.18Propionate 25.40±1.34 26.36±1.72 26.37±2.64Butyrate 13.01±0.48 9.58±0.94* 14.72±3.61*Lactate 2.66±0.32 3.43±0.42 3.44±0.53Other SCFAb 6.56±0.81 5.48±0.34 5.38±0.29Total SCFA 85.14±1.52 84.04±4.10 86.25±7.04

Proteolytic markers(mg/L)

Ammonium 434.4±47.5 452.4±17.5 452.9±26.3

⁎ Significantly different from the previous period (CT vs TR; TR vs WO) according to aStudent's two-tailed t test (Pb0.05).

a Data are means±SD.b Sum of valerate, isovalerate and isobutyrate.

during the control (n≥6), treatment with Product B (n≥9) and washout (n≥6)periods.

Control Treatment Washout

Ascending colonOrganic acids(mmol/L)

Acetate 23.93±1.57 24.54±2.82 12.52±7.88*Propionate 18.69±1.82 19.80±1.70 9.78±9.56*Butyrate 8.60±1.17 8.07±0.37 6.26±5.91Lactate 8.30±0.23 4.39±1.21* 28.65±12.50*Other SCFAb 2.24±0.01 2.51±0.18 1.40±1.57Total SCFA 61.76±1.66 59.31±3.42 58.60±12.42

Proteolytic markers(mg/L)

Ammonium 217.7±55.0 237.2±93.8 154.1±87.1

Transverse colonOrganic acids(mmol/L)

Acetate 37.46±0.53 39.79±2.98 34.93±2.26Propionate 23.18±1.66 23.54±1.26 20.80±0.68Butyrate 11.57±1.17 8.98±0.84* 10.41±0.02Lactate 4.93±1.28 4.26±0.61 8.43±1.06*Other SCFAb 5.17±1.59 5.28±0.83 4.62±0.73Total SCFA 82.32±0.57 81.85±3.97 79.20±4.75

Proteolytic markers(mg/L)

Ammonium 377.7±28.7 399.5±34.4 364.9±39.9

Descending colonOrganic acids(mmol/L)

Acetate 55.45±5.33 55.99±5.36 48.65±3.80*Propionate 21.90±2.04 23.02±1.12 21.79±1.87Butyrate 4.44±3.45 3.89±2.17 7.14±0.05*Lactate 5.23±0.91 5.40±0.75 6.83±1.33Other SCFAb 5.69±0.98 6.12±0.43 6.13±0.76Total SCFA 92.70±1.72 94.42±5.43 90.54±7.71

Proteolytic markers(mg/L)

Ammonium 388.7±21.8 432.1±22.5* 437.9±19.7

⁎ Significantly different from the previous period (CT vs TR; TR vs WO) according to aStudent's two-tailed t test (Pb0.05).

a Data are means±SD.b Sum of valerate, isovalerate and isobutyrate.

171M. Marzorati et al. / International Journal of Food Microbiology 139 (2010) 168–176

treatment with product B. As shown in Fig. 1, the increase inlactobacilli also lasted during the washout period (more than 1 loghigher compared to control). Regarding the other bacterial groups, aslight decreasing trend of clostridia concentrations could be observedfollowing treatment with product A in all of the colon compartments(with the exception of the transverse colon) and a decrease ofstaphylococci concentrations was noted following treatment withproduct B in the transverse colon. Finally, an increasing trend inenterococci concentrations was observed following the treatmentonly with product A.

It is estimated that at least 80% of the gut microbiota consists ofmicroorganisms that cannot be cultured (Eckburg et al., 2005). Forthis reason, a molecular microbial ecology survey was applied, basedon qPCR and the molecular fingerprinting technique DGGE on totalbacteria, bifidobacteria, lactobacilli, the bacteroides–prevotella groupand the whole phylum of Bacteroidetes.

qPCR analyses showed a bifidogenic effect in all compartmentsimulating ascending, transverse and descending colon only for productB, whereas in product A the bifidogenic effect was significant only inascending colon (Table 5). During the control period, bifidobacteriawere0.3% of the total microbial community in all colon vessels, while theyaccounted for, respectively 1, 0.6 and 0.6% in the ascending, transverseand descending colon following the treatments. qPCR also showed thatboth products A and B induced a significant increase both of thebacteroides–prevotella group and of the Bacteroidetes concentration inthe compartment simulating the proximal colon (Table 5). Here,members of the bacteroides–prevotella group increased from about30% during control periods up to 59% and 77% of the total microbialcommunity following the treatment with, respectively, products A andB. Finally, in contrast with the plate counts, no statistically significantlactobacillogenic effect could be observed for the treatments, even

though the slight increase in lactobacilli lasted in the washout period inmost of the colon vessels (Table 5).Table 4Concentrations of organic acids and ammonium ions in the three vessels of the SHIME

a

The results of the DGGE analyses on bifidobacteria, total bacteria,the bacteroides–prevotella group and lactobacilli are shown, respec-tively, in Fig. 2 and Figs. S1–S3. Clustering of the DGGE lanefingerprints for total bacteria and the bacteroides–prevotella groupshowed the formation of two main clusters corresponding to thedifferent SHIME systems. Within each cluster, samples of W3, 4 and 5(treatment period) andW1, 2, 6 and 7 (control and washout periods)tended to sub-cluster together: more than 65% similarity for totalbacteria and 90% similarity for bacteroides–prevotella group (Figs. S1and S2). DGGE patterns from lactobacilli only clustered according tothe SHIME system of origin (Fig. S3), while those specific forbifidobacteria formed specific clusters, combining samples from thetreatment and washout period together (Fig. 2). This indicates thatproducts A and B affected the composition of the bifidobacteriacommunity in a way that remained stable after the treatment. Themost prominent bands were cut out of each gel and sequenced.Sequencing results are shown in Tables 6 and S2. All of the identifiedmicroorganisms were similar to species previously identified inhuman fecal samples. Following treatment with both products, twodistinct bands appeared and remained stable even in the washoutperiod in the DGGE profiles specific for bifidobacteria (black square inFig. 2), i.e. a species with 96% similarity to an uncultured Bifidobacter-ium sp. (FJ346169) and another one with 96% similarity toBifidobacterium bifidum (FJ169942) (Table 6).

3.3. Ecological interpretation of the DGGE analyses

To provide an ecological interpretation to the DGGE fingerprints,an approach based on a 3-level analysis (Range richness — Rr,Dynamics — Dy and Functional organization — Fo) has been applied(Marzorati et al., 2008). Table S3 reports the obtained values.

Fig. 1. Results from the plate counts for bifidobacteria (A and B for SHIME 1 and 2, respectively) and lactobacilli (C and D for SHIME 1 and 2, respectively). Samples were analyzed 1×/week from the ascending (AC), transverse (TC) and descending (DC) colon of the two SHIME systems. The data are presented for the control (2 weeks=black bars), treatment(3 weeks=dark grey bars) and washout (2 weeks=light grey bars) periods. Asterisks indicate samples significantly different from the control period (Pb0.05).

172 M. Marzorati et al. / International Journal of Food Microbiology 139 (2010) 168–176

According to Rr, the distribution of bands in the DGGE pattern can becorrelated with the percentage of denaturant gradient of the gelneeded to represent the sample's total diversity. Following thetreatment with product A, Rr values for bifidobacteria and totalbacteria increased, while those for lactobacilli decreased in the distalcolon. Both products induced a decrease in members of thebacteroides–prevotella group in the transverse colon and an increasein the descending tract. Product B also induced a decrease oflactobacilli and total bacteria in almost all of the colon compartments,but produced a 7-fold increase of bifidobacteria in the ascending colonand a 3-fold increase in the distal colon (Table S3). The Dy parametermeasures the number of species that, on average, come to significantdominance in a given habitat during a defined time interval(Marzorati et al., 2008). For both products, Dy values were medium/high for bifidobacteria and total bacteria, while almost no effect wasregistered for lactobacilli and the bacteroides–prevotella group, withDy values ≤10 in all colon compartments except for lactobacilli in theascending colon (Dy=25) (Table S3). Fo is a numerical value used todescribe the evenness distribution of a microbial community asdepicted by a Pareto–Lorenz curve (Marzorati et al., 2008). Overall thedata showed a decrease in evenness in almost all of the coloncompartments for bifidobacteria, the bacteroides–prevotella group

Table 5qPCR results for total bacteria, the phylum Bacteroidetes, bacteroides–prevotella group, bificolon compartments of SHIME 1 and 2. Data are reported as 16S rRNA gene copies/mL. SProducts A or B (n≥3) and washout period (n≥2) are significantly different (Pb0.05) acco

Total bacteria Bacteroidetes Bacteroide

Prod. A Prod. B Prod. A Prod. B Prod. A

AC Ctrl 1.86×1010 a 1.67×1010 a 8.37×109 a 7.26×109 a 5.72×109

Treat 2.42×1010 a 3.01×1010 b 1.34×1010 b 1.63×1010 b 1.10×101

Wash 9.35×109 b 4.37×109 a 2.25×109 c 1.84×108 c 1.29×109

TC Ctrl 1.28×1010 a 1.63×1010 a 5.40×109 a 7.11×109 a 3.98×109

Treat 1.26×1010 a 9.92×109 b 4.46×109 a 5.84×109 a 2.96×109

Wash 1.26×1010 a 1.38×1010 ab 8.60×109 b 7.33×109 a 5.31×109

DC Ctrl 7.19×109 a 1.24×1010 a 3.14×109 a 3.62×109 a 1.99×109

Treat 5.62×109 a 9.10×109 a 2.06×109 a 2.19×109 a 1.43×109

Wash 7.53×109 a 2.01×1010 b 3.66×109 a 8.14×109 b 3.08×109

and total bacteria. There were a few exceptions: following thetreatment with product B, Fo of bacteroides–prevotella and totalbacteria increased in the transverse colon (Table S3).

Finally, Fig. 3 shows a cluster analysis conducted on a compositedata set (DGGE analyses of total bacteria, bifidobacteria, bacteroides–prevotella and lactobacilli) using the unweighted pair group withmathematical averages (UPGMA) and distance matrices of eachDGGE, based on the Pearson correlation similarity coefficients. Bothproducts modulated the resident microbial communities in all coloncompartments. Fig. 3A shows that samples from the SHIME runningproduct A formed two separate clusters corresponding to the controlperiod for all of the colon vessels and W6 and 7 for the descendingcolon (cluster II) and to all the other samples (cluster I). Within eachcluster, samples sub-clustered according to the colon compartment oforigin. Product A had an effect on the structure and composition of themicrobial community of the three colon compartments and this effectwas stable in the ascending and transverse colon, while only transientin the descending colon. Product B was comparable to A, with themain difference being that it was still able to shape the residentmicrobial community during the washout period in all the coloncompartments (Fig. 3B). Three main clusters could be retrieved:cluster I corresponding to samples from the ascending colon; cluster

dobacteria and lactobacilli in the ascending (AC) transverse (TC) and descending (DC)amples with different code letters within each control period (n≥2), treatment withrding to one-way ANOVA.

s–prevotella Bifidobacteria Lactobacilli

Prod. B Prod. A Prod. B Prod. A Prod. B

a 5.34×109 a 4.10×107 a 6.44×107 a 1.89×105 a 2.53×105 ab0 b 1.44×1010 b 9.66×107 b 1.85×108 b 2.05×105 a 2.53×105 a

c 2.34×108 c 4.62×107 a 6.70×107 a 1.17×105 b 4.07×104 ba 5.69×109 a 6.81×107 a 6.77×107 a 9.30×104 a 1.85×105 aa 4.62×109 a 7.63×107 a 1.20×108 b 1.48×105 a 9.05×104 bb 5.49×109 a 6.56×107 a 8.34×107 ab 2.64×105 b 1.93×105 aa 3.00×109 a 9.48×107 a 5.91×107 a 6.41×104 a 1.15×105 ab 2.13×109 a 8.25×107 a 1.18×108 b 6.26×104 a 9.81×104 ac 6.01×109 b 8.01×107 a 5.79×107 a 1.54×105 b 4.19×105 b

Fig. 2. DGGE fingerprint patterns for bifidobacteria using a 45–60% denaturant gradient.Clustering analysis was based on the Pearson product–moment correlation coefficientand dendrograms were created by using UPGMA linkage. S1 refers to the SHIME1treated with product A; S2 to the SHIME2 treated with product B. V1 refers to ascendingcolon, V2 to transverse colon, and V3 to descending colon. Samples are named asfollows: SxVyWz, where x represents the number of the SHIME, y the vessel and z theweek of the sampling. Numbers 1–5 refer to the sequenced bands as reported in Table 6.

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II.1 corresponding to the control period of the transverse anddescending colon; and cluster II.2 corresponding to the treatmentand washout period of the transverse and descending colon. Withincluster II, the samples sub-clustered according to the period and thecolon region.

4. Discussion

A healthy gut is the key to a healthy body and certain prebioticsshow promise in supporting gut health. In this work, it was shown

that two mixtures composed of mainly Aloe vera gel and larcharabinogalactans and seaweed fucoidans (only in product B) possessstrong characteristics in terms of potential prebiotic activity. ProductAwas the basic formulation of the supplements; for product B the Aloevera gel extract was replaced with Aloe vera gel powder, and awakame algae extract was added. These formulation changes resultedin a lower concentration of mannose and glucose and a relativeincrease in fucose and galacturonic acid in product B (data notshown). Both products have already been shown to have beneficialeffects in an animal colitis model, by providing a good source ofdietary fiber and by stimulating the immune-enhancing functions,mainly associatedwith larch arabinogalactans and Aloe vera (Patankaret al., 1993; Kelly, 1999; Langmead et al., 2004; Koetzner et al., 2009).No study has ever been conducted up to now to evaluate the effect ofsuch complex polysaccharide supplements in the gastrointestinaltract. Such an effect, considering the huge number of cross-feedingsoccurring in the gutmicrobial community, cannotbe reduced to the sumof the effects of the single ingredients. This warrants the evaluation ofcomplex mixtures rather than the single constituents to elucidate thepotential final health effect.

At the metabolic level, the substitution of an easily fermentablecarbon source, such as starch, with a complex mixture of polysacchar-ides did not induce significant changes in SCFA concentrations (even aslight increase in acetate and propionate production) (Tables 3 and 4).This was indicative of a good but still selective (seemolecularmicrobialdata) fermentation of both products. Arabinogalactan have beenassociated to significant increases of total fecal anaerobes andLactobacillus spp. with no other changes in pH or SCFA production(Robinson et al., 2001). During the washout period in the ascendingcolon of both the SHIME systems, increased lactate production wasobserved in combination with decrease of the other main SCFAs.Bacteria in the gut regulate the production of SCFAs in response toseveral factors, and among them carbon availability plays a key role.Lactate is normally produced under conditions of carbon excess (toaffect redox balance during fermentation) and starch is the mainprecursor of this organic acid (Macfarlane andMacfarlane, 2003). In thissituation, the lactate producing bacteria possibly grew on the residualstarch or other components of the diet during the treatment period.When the products were again substituted with starch (C-excess)during thewashout period, themicrobiota promptlymetabolized it intolactate. Lactate was thus an intermediate for butyrate production in thedistal colon (Duncanet al., 2004). The temporary increase inammoniumproduction may be seen as a marker for limited carbohydrate substrateavailability for the bacteria during the treatment period. The bacteriathat were not selectively affected by the treatment and thus could notuse the administered products as efficiently as they were able to utilizestarch as an energy source, may have shifted to more proteolyticmetabolism, resulting in increased ammonium concentrations. Finally,SEC analyses showed that high MW fractions of the mixture of plant-derived polysaccharides in product B were still available in thetransverse colon and could reach the distal colon, thus potentiallyexerting a prebiotic effect throughout the colon (Table 2).

Plate counts showed that the concentrations of cultivablebifidobacteria and lactobacilli were positively influenced by treatmentwith both products A and B (Fig. 1). As both genera are commonlyassociated with health benefits for the human host, an indication ofbeneficial effect of the treatment, based on the plate counts, could bededuced. qPCR analyses confirmed the bifidogenic effect for product Ain the ascending colon, while for product B in all of the colon com-partments. Previous in vivo and in vitro works already showed thatseveral Lactobacillus spp. and Bifidobacterium spp. possess themetaboliccapabilities to utilize complex carbohydrates containing fucose, man-nose, arabinose and galactose (Crociani et al., 1994; Degnan andMacfarlane, 1995; Robinson et al., 2001; Topping and Clifton, 2001).

Bacteroidetes have been recently positively correlated with bodyweight control. The relative abundance of two dominant bacterial

Table 6Sequencing results of themost prominent bands in the DGGE patterns. Trend column indicates if the relative importance of the species in the community increased (↑), decreased (↓)or remained unaffected (=) by the treatment in the ascending (AC), transverse (TR) or descending (DC) colon of SHIME 1 (SH1) or 2 (SH2). If no specific code is present, the effect isgeneralized for all colon compartments. N.A. = not applicable

Band* DGGE Most related species Strain-clone Similarity (%) Ref. number Trend SH1 Trend SH2

1 Bifidobacteria Uncult. Bifidobacterium sp. Clone G04_5 96 FJ346169 ↑ ↑2 Bifidobacteria Bifidobacterium bifidum Strain FHHMB 96 FJ169942 ↑ ↑3 Bifidobacteria B. adolescentis BBMN23 97 GQ380694 N.A.a N.A.a

4 Bifidobacteria B. adolescentis BBMN23 99 GQ380694 ↓ ↓5 Bifidobacteria Uncult. Bifidobacterium sp. clone 6PL157 99 EU422543 = =

⁎ Band numbers are reported in Fig. 2.a The band appears only in the washout period.

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phyla, the Bacteroidetes and the Firmicutes, appears to play a role inthe ability of the microbiome to harvest energy from the diet: obesemice showed a decrease in the abundance of Bacteroidetes and aproportional increase in Firmicutes (Ley et al., 2005; Turnbaugh et al.,2006). Both products induced a significant increase in the concentra-tion of species belonging to the Bacteroidetes phylum in the proximalcolon.

DGGE analyses with specific primers confirmed that the treatmentwith both products had an effect in modulating the structure andcomposition of the microbial community belonging to bifidobacteriaand the bacteroides–prevotella group. This effect was transient for thebacteroides–prevotella group (Fig. S2) while, for bifidobacteria, thecluster analysis indicated that the composition of the community wasmodified in a way that remained stable after the treatment (Fig. 2). Inall three vessels, two common bands phylogenetically related to anuncultured Bifidobacterium sp. and to B. bifidum, appeared during andafter treatment with both products A and B. The B. bifidum level isnormally enhanced by human milk oligosaccharides (mainly galacto-oligosaccharides) and glycans (Newburg, 2009) and it has beenrelated to several positive effects for human health: immunomodula-tion, anti-inflammation, and effectiveness against neonatal necrotiz-

Fig. 3. Cluster analysis conducted on a composite data set (DGGE analyses of total bacteria,bifidobacteria, bacteroides–prevotella and lactobacilli) using the unweighted pair groupwith mathematical averages (UPGMA) and distance matrices of each DGGE based on thePearson correlation similarity coefficients. S1 refers to the SHIME1 treatedwith product A;S2 to the SHIME2 treated with product B. V1 refers to ascending colon (square), V2 totransverse colon (round), and V3 to descending colon (triangle). Samples are named asfollows: SxVyWz,where x represents thenumberof the SHIME,y thevessel and z theweekof the sampling.

ing enterocolitis, as well as with an improvement in the intestinalbarrier function in rat models (Ménard et al., 2008; Khailova et al.,2009).

The increasing effect of the treatment was not observed for thelactobacilli bothwith the qPCR— in contrast with the plate counts thatshowed that cultivable fraction of bacteria belonging to this genuswas enhanced by the treatment — and DGGE (Fig. S3). This specificgroup is normally present at low levels in the human gut and both invitro and in vivo studies have already shown that the abundance ofsmaller groups, like Lactobacillus sp., naturally fluctuates in human gut(Zoetendal et al., 1998; Possemiers et al., 2004). This normal variationappeared to be a more influencing factor than the treatmentsthemselves.

The cluster analysis conducted on a composite data set (Fig. 3) andthe ecological interpretation on bifidobacteria and bacteroides–prevotella group — based on Rr, Dy and Fo parameters (Marzoratiet al., 2008) - showed a possible mode of action for the two products.In both cases, PL curves showed in all the three vessels balancedmicrobial communities characterized by a medium ‘functionalorganization’, where some species were dominant and many othersresilient, thus normally preserving their functionality (Marzoratiet al., 2008). Product A induced a change on the structure of themicrobial community of the three colon compartments and this effectremained stable in the ascending and transverse colon. In contrast, inthe descending colon, where only small fractions of the polysacchar-ides mixture were present, the change was transient (Table 2). Theeffects of product A were comparable to B, with the difference beingthat the effect of the latter in the distal colon was more pronouncedand longer lasting. Again, this behavior could be induced by theslower fermentability of some fractions of the polysaccharidemix thatthus remained available throughout the colon for a longer time. In thiscontext, the bifidobacteria richness increase (Rr↑) was associated witha concomitant high rate of change per week (Dy↑) and this process ledto a more balanced microbial community (Fo↓) where the abundanceof the initial dominant strains decreased in favor of other species(bands 1 and 2 in Fig. 2) that can selectively use these products.Regarding the bacteroides–prevotella group, the DGGE showed astimulatory effect of the treatments specific for every coloncompartment. In the ascending colon, all of the bacteria could useboth products thus leading to an increase in the concentration(qPCR↑) and in the degree of evenness (Fo↓). In the transverse colon,it was registered a selective stimulatory effect on initially lowabundant species (Rr↓ and Fo↓) for product A and even a stronger effectfor product B that led to a microbial community more dominated (Fo↑)by those bacteroides species that could preferentially benefit from thetreatment (e.g. band 17, Table S2). An opposite trend was observed inthe descending colon for both products.

A prebiotic is a selectively fermented product that allows specificchanges in the composition and/or activity in the gastrointestinalmicrobiota, which confers benefit upon host health (Gibson et al.,2004). This work indicated that the prolonged supplementation of theSHIME system with mixtures of plant-derived polysaccharidesresulted in: (i) good and selective fermentability along the entire

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colon; (ii) a positive and selective bifidogenic effect; (iii) the potentialto enhance species belonging to the Bacteroidetes phylum. Inconclusion, in terms of GRM, both products are promising topositively impact the human gut microbiota with Advanced Ambro-tose® powder exerting a more pronounced microbial modulationthan Ambrotose® complex.

Acknowledgements

Tom Van deWiele and Sam Possemiers are Postdoctoral Fellows ofthe Fund for Scientific Research of Flanders (Belgium).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ijfoodmicro.2010.02.030.

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