Faculty of Kinesiology and Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, AB T2N 1N4, Canada 2

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1 Nutrients 214, 6, ; doi:1.339/nu Article OPEN ACCESS nutrients ISSN Effect of the Novel Polyscchride PolyGlycopleX on Short-Chin Ftty Acid Production in Computer-Controlled in Vitro Model of the Humn Lrge Intestine Rylene A. Reimer 1, *, Annet J. H. Mthuis 2, Koen Venem 2, Michel R. Lyon 3,4, Rolnd J. Ghler 5 nd Simon Wood 4,6,7 1 Fculty of Kinesiology nd Deprtment of Biochemistry & Moleculr Biology, University of Clgry, Clgry, AB T2N 1N4, Cnd 2 TNO, Helthy Living, P.O. Box 36, Zeist, AJ 37, The Netherlnds; E-Mils: nnet.mthuis@tno.nl (A.J.H.M.); koen.venem@outlook.com (K.V.) 3 Cndin Centre for Functionl Medicine, 1552 United Boulevrd, Coquitlm, BC V3K 6Y2, Cnd; E-Mil: doctorlyon@shw.c 4 University of British Columi, Food, Nutrition nd Helth Progrm, Vncouver, BC V6P 2G9, Cnd; E-Mil: simonwood@shw.c Fctors Group R & D, 3655 Bonneville Plce, Burny, BC V3N 3S9, Cnd; E-Mil: rghler@nturlfctors.com InovoBiologic Inc., Kensington Rod NW, Suite 49, Clgry, AB T2N 4X7, Cnd School of Pulic Helth, Fculty of Helth Sciences, Curtin University, Perth, WA 6845, Austrli * Author to whom correspondence should e ddressed; E-Mil: reimer@uclgry.c; Tel.: ; Fx: Received: 9 Jnury 214; in revised form: 11 Ferury 214 / Accepted: 27 Ferury 214 / Pulished: 14 Mrch 214 Astrct: Mny of the helth enefits ssocited with dietry fier re ttriuted to their fermenttion y microiot nd production of short chin ftty cids (SCFA). The im of this study ws to investigte the fermentility of the functionl fier PolyGlyopleX ( ) in vitro. A vlidted dynmic, computer-controlled in vitro system simulting the conditions in the proximl lrge intestine (TIM-2) ws used. Sodium hydroxide (NOH) consumption in the system ws used s n indictor of fermentility nd SCFA nd rnched chin ftty cids (BCFA) production ws determined. NOH consumption ws significntly higher for Fructooligoscchride () thn, which ws higher thn cellulose (p =.2). At 32, 48 nd 72 h, cette nd utyrte production were higher for nd versus cellulose. Propionte production ws higher for thn cellulose

2 Nutrients 214, t 32, 48, 56 nd 72 h nd higher thn t 72 h (p =.14). Totl BCFA production ws lower for compred to cellulose, wheres production with ws lower thn for cellulose t 72 h. In conclusion, is fermented y the colonic microiot which ppered to dpt to the sustrte over time. The greter propionte production for my explin prt of the cholesterol-lowering properties of seen in rodents nd humns. Keywords: dietry fier; functionl fier; microil fermenttion; polyscchride; propionte 1. Introduction Dietry fier nd resistnt strches re sustrtes for cteril fermenttion in the humn gstrointestinl trct tht result in production of short chin ftty cids (SCFA), predominntly cette, propionte nd utyrte [1]. The interest in SCFA hs grown given the mounting evidence for their role in colonic helth nd metolic diseses, including irritle owel syndrome, inflmmtory owel disese, oesity, crdiovsculr disese nd cncer [1]. Beyond the non-digestile crohydrtes nturlly present in foods, there is lso potentil for novel fiers, those tht re extrcted nd modified through physicl, chemicl or enzymtic mens [2], to influence the production of SCFA. PolyGlycopleX ( ) is novel polyscchride tht is mnufctured y complexing three solule viscous polyscchrides. While it is elieved tht this new complex should e fermented y the intestinl microiot, this hypothesis ws tested experimentlly in this study. We hve previously demonstrted tht ws ssocited with higher fecl totl SCFA concentrtions in helthy dults consuming versus plceo for three weeks [3]. One drwck of mesuring fecl SCFA, however, is difficulty in determining their exct production due to the sustntil (up to 95%) sorption nd metolism of SCFA in the gut [4]. In contrst, SCFA production cn e ccurtely mesured in computer controlled model of the proximl lrge intestine [5 7]. The in vitro TIM-2 system is multi-comprtmentl nd dynmic lortory system, which simultes to high degree the colon, including complex, high density, metoliclly ctive, neroic microiot of humn origin. This model hs een vlidted with regrds to the numer nd rtio of the vrious micro-orgnisms which re similr in composition nd metolic ctivity with tht of the humn colon s well s the production of metolites, such s SCFA, rnched short chin ftty cids (BCFA; iso-utyrte nd iso-vlerte), mmoni nd phenolic compounds [5 7]. The ility to mesure BCFA in ddition to SCFA is importnt given the unique origin nd fte of these fermenttive products. While crohydrtes re fermented y scchrolytic cteri nd produce SCFA, H 2 nd CO 2 [8], proteins nd mino cids cn lso e fermented in the gut y proteolytic cteri to yield BCFA, H 2, CO 2, CH 4, phenols nd mines [9]. Mny products of protein fermenttion hve toxic effects [1]. Given the link etween colonic helth nd reduced risk of metolic disese nd the role of SCFA in providing tht protection, our ojective ws to determine the fermenttion potentil of the novel viscous polyscchride,, using the in vitro TIM-2 system [5]. Fructooligoscchride (), highly fermentle preiotic fier [9], ws used s the positive control nd cellulose, n insolule fier [11] s the negtive control.

3 Nutrients 214, Experimentl Section 2.1. Test nd Control Fier The experimentl fiers were: (1) Positive control: Fructo-oligoscchride from chicory (Sigm Aldrich Chemie BV, Zwijndrecht, The Netherlnds); (2) Negtive control: (VWR Interntionl BV, Roden, The Netherlnds); (3) Test fier: PolyGlycopleX ( ), solule high viscosity polyscchride (HVP) mnufctured y proprietry process (EnviroSimplex ) from three dietry fiers; konjc (glucomnnn), sodium lginte nd xnthn gum ( : α-d-glucurono-α-dmnno-β-d-mnno-β-d-gluco, α-l-gulurono-β-d-mnnurono, β-d-gluco-β-d-mnnn; InovoBiologic Inc., Clgry, Cnd). The crohydrte properties of hve een previously descried [12 14] nd show tht n interction etween konjc glucomnnn xnthn gum complex nd sodium lginte forms new, ternry complex Intestinl Conditions of TIM-2 The conditions of the lrge intestine were reproduced in the TNO dynmic computer-controlled in vitro intestinl model, TIM-2. Detils of the in vitro model hve een previously pulished [5,7,15]. The following stndrdized conditions were simulted in the TIM-2 system: ody temperture, ph in the lumen, composition nd rte of secretion, delivery of pre-digested sustrte from the ileum (SIEM; Stndrd Ilel Efflux Medium), mixing nd trnsport of the intestinl contents, sorption of wter, presence of complex, high density, metoliclly ctive, neroic microiot of humn origin nd sorption of microil metolites vi semi permele memrne inside the colon model. The SIEM simultes mteril reching the colon in humns. For experiments in the TIM-2, SIEM ws modified slightly from the medium descried y Gison et l. [16], minly concerning the concentrtion of crohydrtes, pepton, csein nd Tween 8 [5,7]. SIEM contined the following components (g/l): 9. pectin, 9. xyln, 9. rinoglctn, 9. mylopectin, 43.7 csein, 74.6 strch, 31.5 Tween 8, 43.7 ctopepton,.7 ox-ile, 4.7 g K 2 HPO 4 3H2O, 8.4 g NCl,.9 g FeSO 4 7H 2 O,.7 g MgSO 4 7H 2 O,.8 g CCl 2 2H 2 O,.2 g hemin nd.3 g cysteine HCl, plus 1.5 ml of vitmin mixture contining (per litre): 1 mg mendione, 2 mg D-iotin,.5 mg vitmin B12, 1 mg pntothente, 5 mg nicotinmide, 5 mg p-minoenzoic cid nd 4 mg thimine. The ph ws djusted to 5.8. Prior to the performnce of ech experiment, the secretion fluids nd dilysis solutions were prepred fresh, the ph electrodes clirted, new memrne units instlled nd the system inoculted (one dy efore the strt of the test period) with microiot of humn origin (dults on Western type of diet s descried in detil in [15]). After n overnight dpttion period in which the microiot dpted to the model conditions, the test period strted Addition of Test nd Control Fiers During the test period the TIM-2 units were continuously fed with SIEM, ut without the stndrd crohydrtes. The test- nd control products were dded to TIM-2 during the dy on ll three test-dys in n 8 h period (Tle 1). The dded dose ws normlized on the mximum dose of which could e dded to TIM-2 during the dy (4. g, 2.5 g nd 3.5 g on ech of the

4 Nutrients 214, consecutive dys). The control products were dded in t these doses in similr timefrme s the test product. ws dded to the lumen of TIM-2 (12 ml) during the dy. Becuse of its viscosity, ws dded to lrge volume (3 ml) of dilyste efore ddition to TIM-2. The controls were dded to TIM-2 in the sme wy. A totl of 3 independent 72 h runs were mde with nd 2 runs ech for nd cellulose. Tle 1. Composition of medi with control nd test fiers. Medium Component Dy 1/Dy 2/Dy 3 Dy 1/Dy 2/Dy 3 Dy 1/Dy 2/Dy 3 Csein 2.6 g 2.6 g 2.6 g Tween g 1.9 g 1.9 g Bctopepton 2.6 g 2.6 g 2.6 g Ox-ile 42 mg 42 mg 42 mg K 2 HPO 4 3H 2 O 282 mg 282 mg 282 mg NCl.5 g.5 g.5 g FeSO 4 7H 2 O.54 mg.54 mg.54 mg MgSO 4 7H 2 O 42 mg 42 mg 42 mg CCl 2 2H 2 O 48 mg 48 mg 48 mg Hemin 1.2 mg 1.2 mg 1.2 mg Cysteine HCl 18 mg 18 mg 18 mg Vitmins * 9 mg 9 mg 9 mg 4. g/2.5 g/3.5 g 4. g/2.5 g/3.5 g 4. g/2.5 g/3.5 g * The vitmin formultion contined (mg/l): mendione, 1; iotin, 2; vitmin B 12,.5; pntothente, 1; nicotinmide, 5; pr-minoenzoic cid, 5; thimine, Smpling from TIM-2 Lumen smples were collected in duplicte t the strt of the test period nd fter 8, 24, 32, 48, 56 nd 72 h. Totl volumes of the dilysis liquid were mesured nd smples were collected t the strt of the test period nd fter 8, 24, 32, 48, 56 nd 72 h. Smples were stored t < 18 C Fermenttion nd ph Fermenttion of non-digestile crohydrtes y the microiot produces SCFA nd lctte which cuse the lumen to cidify. During the TIM-2 experiments ph ws regulted to ph 5.8 y sodium hydroxide (NOH) secretion. Sodium hydroxide usge during the experiments therefore indicted the ility of the microiot to ferment the crohydrtes dded to the TIM-2 system Short-Chin nd Brnched Chin Ftty Acid Anlysis SCFA (cette, propionte, n-utyrte) nd BCFA (iso-utyrte nd iso-vlerte) nlyses were performed s descried efore [7]. In rief, smples were centrifuged (~12, g, 5 min) nd mixture of formic cid (2%), methnol nd 2-ethyl utyric cid (internl stndrd, 2 mg/ml in methnol) ws dded to the cler superntnt. A.5 µl smple ws injected on GC-column

5 Nutrients 214, (Stilwx-DA, length 15 m, ID.53 mm, film thickness.1 mm; Vrin Chrompck, Bergen op Zoom, The Netherlnds) in Chrompck CP91 gs chromtogrph Sttisticl Anlyses Results re presented s men ± SEM. Repeted mesures ANOVA with Bonferroni correction ws used to ssess the effect of time nd fier on SCFA nd BCFA production. Finl NOH usge ws nlyzed with one-wy ANOVA with Tukey s post hoc test. Significnce ws p Results 3.1. Sodium Hydroxide Usge NOH usge reflects the production of SCFA which, when not corrected, would result in decrese in ph of the luminl contents s fermenttion occurs. The use of NOH y the TIM-2 system to mintin the ph t 5.8 is shown for, nd cellulose in Figure 1. There ws no NOH usge during the test runs with the negtive control cellulose indicting tht the fier ws not fermented y the microiot. The positive control showed the highest mount of NOH usge which reflects the gretest cid production from fermenttion of the sustrte. The test product lso showed NOH usge indicting fermenttion of the y the lrge-intestinl microiot present in TIM-2. One wy ANOVA nlysis of the finl NOH usge indicted significntly higher vlues for (37. ± 2.8 ml) thn for (19.5 ±.5 ml) which ws higher thn for cellulose (.3 ±.3 ml) (p =.2). When clculted ck to the mount of sustrte dded to TIM-2, NOH usge during fermenttion ws stle over the three dy test period. With, NOH consumption ws stle over the first 2 dys ut showed higher consumption on dy 3 in ll three seprte runs, indicting dpttion of the intestinl microiot to the sustrte nd incresed ility to ferment the sustrte. Figure 1. Sodium hydroxide usge during multiple TIM-2 runs over time for fructooligoscchride (), PolyGlycopleX () nd cellulose. Vlues t the strt of the test period were set to zero. Individul runs re shown where n = 2 for nd cellulose nd n = 3 for. 45 2M NOH (ml) Runtime (hours)

6 Nutrients 214, SCFA Production The mount of SCFA t the strt of the test period ( h) ws rtificilly set to zero. Susequent vlues reflect the cumultive production of SCFA fter the ddition of the test products. There ws significnt effect of time (p =.1) nd the interction of time with the test fier (p =.1) for cette, propionte, utyrte nd totl SCFA production (Figure 2). At 32, 48 nd 72 h, cette production ws higher for nd compred to cellulose (p <.5; Figure 2). Propionte production ws higher for thn for cellulose t 32, 48, 56 nd 72 h (Figure 2). ws intermedite etween nd cellulose nd t 72 h ws significntly higher thn cellulose (p =.1) ut lower thn (p =.14). Butyrte production ws higher for thn for cellulose t ll time points during the test period (Figure 2c; p<.5). ws higher thn cellulose nd not different from, t 32, 56 nd 72 h. Totl SCFA production ws significntly higher for nd t ll time points from 24 to 72 h (Figure 2d; p <.5). The contriution of the individul SCFA to totl SCFA production t 72 h is depicted in Figure 3. Figure 2. The verge cumultive production of () cette, () propionte, (c) utyrte nd (d) totl short chin ftty cids (SCFA) in TIM-2, during 72 h on the fructooligoscchride (), PolyGlycopleX (), or cellulose sustrte. Vlues re men ± SEM (n = 2 for nd cellulose nd n = 3 for ). Leled mens without common letter t the sme time point differ, p <.5. Y-xis scles differ cross sufigures. 1 3 ) ) Time P =.1 Time Fier P = Acette (mmol) Butyrte (mmol) 2 Propionte (mmol) 5 Time P =.1 Time Fier P = c) d) 2 1 Time P =.1 15 Time Fier P =.1 Time P =.1 75 Time Fier P = Totl SCFA (mmol) c

7 Nutrients 214, Figure 3. Contriution of cette, propionte nd utyrte to totl SCFA production in TIM-2 t 72 h on the fructooligoscchride (), PolyGlycopleX () or cellulose sustrte. Dt re men vlues (n = 2 for nd cellulose nd n = 3 for ). SCFA (mmol) Butyrte Propionte Acette BCFA Production As with the SCFA, the mount of BCFA metolites ws set to zero t the strt of the test period ( h). There ws significnt effect of time for iso-utyrte, iso-vlerte nd totl BCFA (p =.1). There were no significnt differences in iso-utyrte production etween the test fiers (Figure 3A). Iso-vlerte production ws significntly lower for versus cellulose ut not t 56 h (p <.5). At 72 h, production of iso-vlerte ws lower for versus cellulose nd (p =.5) nd ws lower thn cellulose (p =.5). Totl BCFA production ws significntly lower for compred to cellulose t 48, 56 nd 72 h (p <.5) wheres ws lower thn cellulose t 72 h (p =.5). 4. Discussion The present study exmined the fermenttion potentil of the novel polyscchride,, using the in vitro TIM-2 system. SCFA hve een proposed s meditors of t lest some of the eneficil effects of dietry fier on colonic helth, crdiovsculr risk fctors, nd oesity nd type 2 dietes risk [1,11]. We report tht totl SCFA production from is similr to the positive control. Compred to, there ws lower cette (NS) nd higher propionte production for (p =.14) which my explin some of the metolic effects of including reducing hypercholesterolemi [17 2]. The first mjor finding of this study is tht totl SCFA production t the end of the 72 h test period ws similr etween nd nd significntly higher thn cellulose. While cette nd utyrte production ws similr etween nd, with eing slightly higher, the production of propionte with t the end of the fermenttion period ws significntly higher thn nd cellulose. Although the resons for the genertion of distinct SCFA production profiles etween nd re not entirely known, it is likely tht the glycosidic linkges nd monoscchride composition of the two fiers result in different microorgnisms tht ferment the sustrtes. For exmple, the genome of Bcteroides thetiotomicron contins numerous (>1) glycosylhydrolses [21] tht hve een hypothesized to e involved in the rekdown of severl host nd dietry glycns. While Bcteroides species hve long een known to produce propionte [22], it is

8 Nutrients 214, lso plusile tht other gener esides Bcteroides my e involved in fermenttion of. The higher production of propionte with is interesting in light of the proposed stiety-enhncing effects nd demonstrted cholesterol-lowering effects of propionte [23]. Propionte hs een shown to inhiit heptic cholesterol synthesis in oth humns nd niml models [23 25]. Propionte my lower serum cholesterol y reducing cholesterol synthesis in the liver, vi 3-hydroxy 3-methylglutryl CoA reductse [26], or enhncing secretion nd synthesis of ile cids [27]. These mechnisms my help explin prt of the ility of to lower serum cholesterol in rodents [17 19] nd humns [2]. In ddition, recent ex vivo experiments with dipose tissue hve shown tht propionte eneficilly ffects cytokine nd dipokine secretion y dipose tissue [28,29]. The second mjor finding is tht sodium hydroxide consumption, n indictor of fermenttion in the system, incresed for on dy 3 compred to the first two dys. This pttern of fermenttion suggests tht the intestinl microiot dpted to the sustrte mking them etter le to ferment the sustrte with incresing time. This period of dpttion is perhps not surprising in light of evidence in humn sujects where chnges in serum SCFA in response to dietry fier intke took mny months (i.e., >3 months) to occur [3]. It is likely tht t lest prt of this dpttion period cn e ttriuted to chnges in the complex interctions of the cteri community in the host [31]. Adpttion to occurred reltively quickly in our in vitro system (i.e., y dy 3), likely due to the sence of other fermentle crohydrtes. There my e importnt implictions for the period of dpttion in humns consuming the product in tht enefits ttriutle to fermenttion per se would ccumulte over time. The third mjor finding is tht the production of BCFA, mrker of protein fermenttion, ws highest for cellulose, lowest for nd intermedite for. While SCFA re the principl products of scchrolytic ctivity (i.e., rekdown of crohydrtes) in the gut y Bcteroides spp., Lctocillus spp. nd Bifidocterium spp., BCFA re the product of protein degrdtion crried out y dominnt proteolytic cteri including Bcteroides spp. nd Clostridium spp. [32]. BCFA, chiefly iso-utyrte nd iso-vlerte, re mrkers of cteril protein fermenttion nd their presence is ssocited with the production of toxic metolites such s mmoni nd phenols [1]. The production of BCFA in the TIM-2 system is reflective of the mount of crohydrte sustrte ville for the microiot. Indeed, one of the mjor mechnisms y which crohydrtes cn reduce the production of BCFA is vi vilility of fermentle crohydrte sustrte [33]. When crohydrte energy is low in the lumen, cteri will ferment protein or mino cids to otin energy [33]. In our system, cellulose is poorly or not fermented [34], s demonstrted y the lck of NOH consumption in Figure 1 nd therefore fils to provide sustrte to the gut microiot. As result, the fermenttion of proteins derived from the Stndrd Ilel Efflux Medium nd cteri secretions occurs. on the other hnd is highly fermentle sustrte, s shown y the gretest NOH consumption in Figure 1 nd therey supplies undnt crohydrte sustrte. While the lck of NOH consumption with cellulose (Figure 1) my seem contrdictory with the genertion of ftty cids seen in Figure 2, the SCFA produced in the experiments with cellulose re in fct exclusively from protein fermenttion. Previous work hs shown tht cellulose is poorly fermented [15] nd tht the SCFA produced stem from protein fermenttion, s lso indicted y the increse in mmoni in those experiments [15]. Given tht mmoni rises the ph, there is no need for the system to secrete NOH. Indeed, the ph in the runs with cellulose slowly incresed to 6.9 (not

9 Nutrients 214, shown), rther thn stying constnt t 5.8. This is due to the production of significnt mounts of mmoni which hs pk of Hence, no NOH secretion ws needed to mintin ph of 5.8, ut SCFA production ws due to the proteinceous sustrtes dded with SIEM (Figures 2 nd 3), reflected y incresed BCFA (Figure 4). Figure 4. The verge cumultive production of () iso-utyrte, () iso-vlerte nd (c) totl BCFA in TIM-2 during 72 h on the fructooligoscchride (), PolyGlycopleX () or cellulose sustrte. Vlues re men ± SEM (n = 2 for nd cellulose nd n = 3 for ). Leled mens without common letter t the sme time point differ, p <.5. Y-xis scles differ cross sufigures. A Iso-Butyrte (mmol) Time P =.1 B Iso-Vlerte (mmol) Time P =.1 Time Fier P =.1 c C Totl BCFA (mmol) Time P =.1 Time Fier P =.1 c The suppression of BCFA production y supports previous work in which inulin suppressed BCFA nd mmoni production in the TIM-2 system [7] nd similr results hve een otined with severl glucose-sed fiers [15]. ws shown to e intermedite in oth NOH consumption nd BCFA production. The stimultion of crohydrte fermenttion y the microiot is eneficil, not only for the production of SCFA s n energy source for the host nd prticulrly colonocytes ut the microiot lso provides for nitrogen sink. As scchrolytic cteri grow they incorporte putrefctive metolites into cellulr proteins nd therey remove proteins nd hrmful protein metolites from contct with host tissue [1,35]. In humns, resistnt strch nd other fermentle sustrtes such s solule mize fier nd polydextrose hve een shown to reduce the ccumultion of potentilly hrmful metolites of protein fermenttion in the colon [35,36]. ppers to ct similrly, leit to lesser extent thn would e seen with. Given the rpid sorption of SCFA nd the inherent limittions of using fecl concentrtions of SCFA to predict their in situ production, the TIM-2 system provides cler dvntge for ccurtely determining the SCFA production from. In future experiments it might e wise to lso mesure ny remining fter fermenttion, e.g., fter 24, 48 nd 72 h. In ddition, there would e dded enefit in mesuring other fermenttion products such s lctte nd putrefctive fctors such s mmoni which would provide n even etter indiction of the shift in lnce etween production of helthy nd toxic metolites. Lctte is produced y mny gut microiot including lctocilli,

10 Nutrients 214, ifidocteri nd enterococci [37]. In ddition to the cidifying nd nti-microil effect of lctte, certin lctte-utilizing cteri re le to convert lctte into utyrte, key gut-protective SCFA [37]. Although not mesured here, fermenttion of is known to produce lctic cid (lctte) [7] nd therefore it is highly likely tht the higher NOH consumption occurring with (Figure 1) is cused y production of lctte. 5. Conclusions In conclusion, this study demonstrted tht is fermented y the microiot in TIM-2, representtive in vitro model of the humn lrge intestine. In ddition, it ws shown tht when dministered dily, the microiot dpted (in composition nd/or metoliclly) over time with incresed ility to ferment the sustrte. The greter propionte production with my explin, t lest in prt, the consistent cholesterol-lowering properties of seen in oth rodents nd humns. Given emerging evidence for the role of gut microiot nd SCFA in regulting inflmmtion [28 4], it will e interesting for future work to exmine the effects of on inflmmtory mrkers, prticulrly in the context of metolic syndrome. Acknowledgments, PolyGlycopleX nd EnviroSimplex re registered trdemrks of InovoBiologic Inc., Clgry, Cnd. Funding for this work ws provided y InovoBiologic Inc. Conflicts of Interest All uthors hve finncil reltionship with the sponsor of the study, InovoBiologic, Inc., Clgry, Alert, Cnd. RAR received finncil support for the preprtion of this mnuscript. The TIM-2 studies performed t TNO were funded y InovoBiologic nd coordinted y AJHM nd KV. RJG owns the Fctors Group of Compnies, which retins n interest in. MRL receives consulting fees from the Fctors Group of Compnies nd SW receives consulting fees from InovoBiologic Inc. References 1. Wong, J.M.; de Souz, R.; Kendll, C.W.; Emm, A.; Jenkins, D.J. Colonic helth: Fermenttion nd short chin ftty cids. J. Clin. Gstroenterol. 26, 4, Bureu of Nutritionl Sciences; Helth Cnd. Proposed Policy: Definition nd Energy Vlue for Dietry Fire; Helth Cnd: Ottw, Cnd, Reimer, R.A.; Pelletier, X.; Crin, I.G.; Lyon, M.R.; Ghler, R.J.; Wood, S. Fecl short chin ftty cids in helthy sujects prticipting in rndomised controlled tril exmining solule highly viscous polyscchride versus control. J. Hum. Nutr. Diet. 212, 25, Topping, D.L.; Clifton, P.M. Short-chin ftty cids nd humn colonic function: Roles of resistnt strch nd nonstrch polyscchrides. Physiol. Rev. 21, 81,

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