Implication of fermentable carbohydrates targeting the gut microbiota on conjugated linoleic acid production in high-fat-fed mice

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1 British Journl of Nutrition, pge 1 of 14 q The Authors 213 doi:1.117/s Impliction of fermentle crohydrtes trgeting the gut microiot on conjugted linoleic cid production in high-ft-fed mice Céline Drurt 1, Audrey M. Neyrinck 1, Evelyne M. Dewulf 1, Fienne C. De Bcker 1, Sm Possemiers 2, Tom Vn de Wiele 2, Frédéric Moens 3, Luc De Vuyst 3, Ptrice D. Cni 1, Yvn Lrondelle 4 nd Nthlie M. Delzenne 1 * 1 Metolism nd Nutrition Reserch Group, Louvin Drug Reserch Institute, Université ctholique de Louvin, Brussels, Belgium 2 Lortory of Microil Ecology nd Technology (LMET), Ghent University, Ghent, Belgium 3 Reserch Group of Industril Microiology nd Food Biotechnology (IMDO), Deprtment of Bioengineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium 4 Institut des Sciences de l Vie, Université ctholique de Louvin, Louvin-l-Neuve, Belgium British Journl of Nutrition (Sumitted 11 July 212 Finl revision received 19 Decemer 212 Accepted 19 Decemer 212) Astrct In vitro experiments hve shown tht isolted humn gut cteri re le to metolise PUFA into conjugted PUFA like conjugted linoleic cids (CLA). The hypothesis of the present pper ws tht high-ft (HF) diet feeding nd supplementtion with fermentle crohydrtes tht hve preiotic properties modulte the in vivo production of CLA y the mouse gut microiot. Mice were treted for 4 weeks s follows: control (CT) groups were fed stndrd diet; HF groups were fed HF diet rich in linoleic cid (18 : 2n-6); the third groups were fed with the HF diet supplemented with either inulin-type fructns (HF-ITF) or rinoxylns (HF-Ax). HF diet feeding incresed rumenic cid (cis-9,trns : 2 CLA) content oth in the cecl nd liver tissues compred with the CT groups. ITF supplementtion hd no mjor effect compred with the HF diet wheres Ax supplementtion incresed further rumenic cid (cis-9,trns : 2 CLA) in the cecl tissue. These differences etween oth preiotics my e linked to the high ft-inding cpcity of Ax tht provides more sustrtes for cteril metolism nd to differentil modultion of the gut microiot (specific increse in Roseuri spp. in HF-Ax v. HF). In conclusion, these experiments supply the proof of concept tht the mouse gut microiot produces CLA in vivo, with consequences on the level of CLA in the cecl nd liver tissues. We postulte tht the CLA-producing cteri could e meditor to consider in the metolic effects of oth HF diet feeding nd preiotic supplementtion. Key words: Conjugted linoleic cids: Gut microiot: High-ft diet: Preiotic supplementtion Conjugted linoleic cids (CLA) re group of positionl nd geometric isomers of linoleic cid (LA; cis-9,cis : 2) chrcterised y the presence of conjugted doule ounds with cis or trns configurtion. Some CLA isomers hve demonstrted interesting iologicl effects such s nticrcinogenic, nti-dipogenic, nti-therogenic or nti-oesity properties (1,2). In contrst, in certin conditions, some CLA isomers cn lso exert potentilly negtive effects such s insulin resistnce nd stetosis (3 5). In humns, the min dietry sources of CLA re diry products nd met derived from ruminnt nimls. The min CLA isomer found in these products is the isomer cis-9,trns : 2, clled rumenic cid (representing 75 to 9 % of totl CLA found in diry products) (6). CLA isomers re intermedites of the iohydrogention pthwy of LA crried out y rumen cteri, leding finlly to SFA, steric cid (18 : ) (7 9). It hs een proposed tht cteril reduction of PUFA is mechnism of protection ginst the toxicity of PUFA. Indeed, some PUFA prevent cteril growth (1,11). The iohydrogention pthwy lso leds to the production of other intermedites mong which vccenic cid (trns : 1) is the most importnt. All iohydrogention intermedites cn prtly escpe from the rumen nd e sored lter in the ruminnt intestine. Once in the niml tissues, they cn e stored or further metolised. Vccenic cid (trns : 1) cn notly e desturted into rumenic cid (cis-9,trns : 2 CLA) Arevitions: -LnA, -linolenic cid; Ax, rinoxylns; CLA, conjugted linoleic cid; CT, control; DPA, docospentenoic cid; FAME, ftty cid methyl esters; FBC, ft-inding cpcity; HF, high-ft; ITF, inulin-type fructns; LA, linoleic cid; qpcr, quntittive PCR; SCD-1, steroyl-coa desturse-1. * Corresponding uthor: Professor Nthlie M. Delzenne, fx þ , emil nthlie.delzenne@uclouvin.e

2 2 C. Drurt et l. British Journl of Nutrition through the ctivity of D-9 desturse enzyme (lso clled steroyl-coa desturse-1; SCD-1) (12 14). The vst numer of micro-orgnisms (1 14 cteri) in the humn gut cretes lrge metolic potentil to tke into ccount in host physiology (15). Bsed on the iohydrogention pthwy oserved in the rumen, some uthors hve proposed similr pthwy occurring in mmmlin gut microiot (16,17). Furthermore, severl rguments support role of the gut microiot in the endogenous production of CLA isomers. First, in vitro experiments hve shown tht severl gut cteri isolted from rodents or humns re le to metolise LA into CLA isomers nd vccenic cid (18). The three mjor gener including cteri le to produce CLA nd vccenic cid in mice gut re Bifidocteri spp. (1,19), Lctocillus spp. (2) nd Roseuri spp. (17). Second, the enzyme le to trnsform LA into CLA isomers, clled linolete isomerse, ws sequenced in some Lctocillus (21) nd Bifidocterium strins (22), two cteril gener found in the gut microiot. Third, Wll et l. (23) hve shown tht the ftty cid composition in host tissues ws modified (including n increse of rumenic cid; cis-9, trns : 2 CLA) fter co-dministrtion of proiotic strin (Bifidocterium reve) nd LA in mice. Finlly, nother interesting study showed tht incresing the mount of LA in the diet incresed the tissue content of CLA isomers in conventionl rts (with norml gut microiot) ut not in germ-free rts (lcking gut microiot) (24). Altertions in the composition nd/or ctivity of gut microiot known s dysiosis hs een proposed to contriute to the development of oesity nd ssocited metolic disorders including low-grde inflmmtion, dietes nd dyslipidemi (25). We hve previously shown tht high-ft (HF) diet leding to oesity nd dietes chnges gut cteril composition (26 29). The interctions etween gut microiot nd host re, t lest in prt, medited y metolites produced y gut microes. For exmple, metolites such s SCFA minly produced during the fermenttion of non-digestile crohydrtes y scchrolytic cteri re le to e used oth s metolic sustrtes nd regultors in host tissues (3,31). In the present studies, we postulted tht CLA isomers endogenously produced upon metolic coopertion etween the gut microiot nd host intestinl nd liver tissues cn e new kind of metolites susceptile to regulte host metolism. Preiotics re non-digestile crohydrtes llowing the selective stimultion of growth nd/or ctivity(ies) of one or limited numer of microil genus(er)/species in the gut microiot tht confer(s) helth enefits to the host (32). Severl studies hve repetedly shown tht inulin-type fructns (ITF), non-digestile crohydrtes extrcted from chicory root, re le to counterct oesity (diet or genetic induced) in rodents (33 37). On the other hnd, rinoxylns (Ax) re the most importnt non-digestile crohydrtes present in whet nd represent new clss of potentil preiotics (38). We hve recently shown tht the supplementtion of either ITF or concentrte of wter-extrctle high-moleculr-weight Ax in the diet countercted HF-induced gut dysiosis together with n improvement in oesity (28,29). We decided to tret mice with HF diet nd preiotics (ITF or Ax) in order to evlute the potentil impct of these nutritionl modultions of gut cteril composition on ftty cid profile nd in prticulr CLA isomer profile in the trget host tissues (intestines nd liver). Experimentl methods Animls nd diets We performed two experiments using the sme conditions. Mle C57l6/J mice (Chrles River Lortories) ged 9 weeks t the eginning of the experiments were housed in controlled environment (12 h dylight cycle, lights off t 18. hours) with free ccess to diet nd wter. After 1 week of cclimtistion, the mice were seprted into three groups (eight mice per group; four mice per cge). In the first experiment, mice were fed either with control (CT) diet (AO4; SAFE) or HF diet (D12492; Reserch Diets) or the HF diet nd supplementtion of inulin-type fructns (ITF; 2 g/d per mouse of oligofructose provided y Orfti). In the second experiment, mice were fed either with the CT diet (AO4; SAFE), or the HF diet (D12492; Reserch Diets) or the HF diet nd supplementtion of rinoxylns (Ax; 2 g/d per mouse of Nxus provided y BioActor). Dietry tretments lsted for 4 weeks. Food intke ws recorded tking into ccount spillge twice per week. The HF diet contined 6 % lipids, 2 % proteins nd 2 % crohydrte s energy content. The HF diet provided (per 1 g): 35 g ft (soyen oil nd lrd), 9 g sucrose, 16 g mltodextrin, 26 g proteins nd 6 5 g cellulose (ccording to the mnufcturer s informtion). We nlysed the ftty cid profile of the CT diet nd HF diet y GC flme ionistion detector (see Tle 1). All mouse experiments were pproved y nd performed in ccordnce with the guidelines of the locl ethics committee. Housing conditions were specified y the Belgin Lw of 6 April 21 regrding the protection of lortory nimls (greement no. LA ). Tissue smples After 4 weeks of tretment nd 6 h period of fsting, mice were nesthetised y intrperitonel injection of ketmine (1 mg/kg; Anesketin; Eurovet) nd xylzine (1 mg/kg; Rompun; Byer Belgium). The liver, colon nd cecum were crefully collected nd frozen in liquid N 2 efore storge t 288C. The cecl content ws collected nd frozen for further microil nlysis. Dt relted to ody weight, ody weight gin nd tissue weights re presented in Supplementl Tle 1. Ft-inding cpcity nlysis The ft-inding cpcity (FBC) of ITF nd Ax ws nlysed ccording to n in vitro method of lipid dsorption descried y No et l. (39). We tested in prllel strch s negtive control nd chitosn (KiOnutrime-Cse; KitoZyme S.A.) s positive control. Gut microiot nlysis To study the gut microiot composition, the QIAmp DNA stool mini kit (Qigen) ws used ccording to the

3 Gut microiot nd conjugted linoleic cid 3 British Journl of Nutrition Tle 1. Ftty cid profile of the stndrd AO4 (control; CT) diet nd the high-ft (HF) diet (g ftty cid/kg diet) mnufcturer s instructions to extrct the metgenomic DNA from the cecl content of ll mice. To nlyse the quntittive effect of the different tretments on the composition of the gut microiot, quntittive PCR (qpcr) ws performed. qpcr for totl cteri nd specific qpcr for Bifidocterium spp., Lctocillus spp. nd Roseuri spp. were performed s reported previously (28,29,4). All qpcr were performed with n ABI PRISM SDS 7 Sequence Detection System (Applied Biosystems). The sttisticl nlysis ws performed on logrithmic vlues. Ftty cid profile nlysis CT diet HF diet 14 : cis-9-14 : 1 n.d : : cis-9-16 : : : trns-9-18 : trns : 1 n.d. 647 cis-9-18 : cis : : 2n : : 3n cis-9,trns : 2 n.d. 387 trns-1,cis : 2 n.d : 3n : 4n : 5n : 5n : 6n Totl n.d., Not detectle, elow the level of detection. To determine the ftty cid profile in tissues or diet, 1 mg of liver tissue or diet or 75 mg of cecl tissue were homogenised in methnol chloroform mixture (1:2, v/v). Homogentes were filtered with Whtmn filters no. 1 (porosity 1 mm). The filters were rinsed with 2 ml chloroform nd 1 ml methnol. Homogentes were purified successively with KCl ( 88 %) nd KCl ( 88 %) methnol (1:1, v/v). After centrifugtion (15 g, 5 min), the chloroform phse ws collected in new tues nd then evported under N 2 flux. The ftty cids were then sujected to n lkline hydrolysis. For tht purpose, solution of KOH in methnol ws dded nd incuted t 78C for 2 h (sponifiction of lipids). The ftty cids were methylted s follows: 4 ml HCl in methnol (1 2 M) ws dded in the tue nd incuted t 78C for 2 min. Ftty cid methyl esters (FAME) were then extrcted with hexne. Quntifiction of FAME ws mde y GLC (GC Trce ThermoQuest; Thermo-Finnign). The chromtogrph ws equipped with flme ionistion detector, utomtic injector nd fused silic cpillry column (length 1 m; inside dimeter 25 mm; film thickness 2 mm) coted with film of iscynopropyl polysiloxne (RT-256; Restek) using H 2 s the crrier gs t constnt pressure of 2 kp. The initil oven temperture ws 88C, incresed t 258C/min to 1758C (held for 25 min), then incresed t 18C/min to 258C (held for 4 min), then incresed t 18C/min to 2258C (held for 2 min) nd finlly decresed t 28C/min to 88C. The temperture of the flme ionistion detector ws mintined t 2558C. H 2 flow to the detector ws 35 ml/min, nd irflow ws 35 ml/min. The identifiction nd the quntifiction of ech pek were mde y comprison of retention times with pure FAME stndrds (Alltech Assocites; except CLA isomers from Nu-Chek Prep). Rel-time quntittive PCR Totl RNA ws extrcted from the liver nd colon using the TriPure isoltion regent (Roche Dignostics Belgium). cdna ws prepred y reverse trnscription from 1 mg of totl RNA using the Kit Reverse Trnscription System (Promeg). Rel-time PCR ws performed with the Setp OnePlus TM rel-time PCR system nd softwre (Applied Biosystems) using SYBR-Green (Mes Fst qpcre; Eurogentec) for detection. Riosoml protein L19 (RPL19) RNA ws chosen s the housekeeping gene to normlise the dt, nd further nlysed ccording to the 2 2DDCT method (41). Sttisticl nlysis Results re presented s men vlues with their stndrd errors. Sttisticl significnce of difference etween groups ws ssessed y one-wy ANOVA followed y Tukey s post hoc multiple comprison test (GrphPd Prism Softwre). P, ws considered s sttisticlly significnt. Vlues with unlike superscript letters re significntly different (P, ) ccording to Tukey s post hoc ANOVA sttisticl nlysis. Results Modultion of numer of conjugted linoleic cid-producing cteri in cecl content y high-ft diet nd dditionl preiotic tretments In oth experiments, the HF diet decresed the numer of CLA-producing cteri, clculted s the sum of the three most cited gener le to produce CLA (Bifidocterium spp., Roseuri spp., Lctocillus spp.) (Tle 2). Preiotic supplementtions countercted the HF diet-induced decrese of CLA-producing cteri: ITF supplementtion restored the numer of CLA-producing cteri to the CT level wheres following Ax supplementtion, the numer of CLA-producing cteri incresed ut did not rech the CT vlue (Tle 2). The cteril gener modulted y ech preiotic were different (Tle 2). ITF nd Ax oth incresed Bifidocterium spp. compred with the HF diet, ut the ifidogenic effect of ITF ws more importnt thn the one of Ax. ITF decresed Roseuri spp. nd Lctocillus spp. compred with the HF diet, wheres Ax incresed Roseuri spp. without

4 4 C. Drurt et l. Tle 2. Gut microiot nlysis in the cecl content of mice fter 4 weeks of dietry tretment* (Men vlues with their stndrd errors) Experiment 1 Experiment 2 Diet... Men SEM Men SEM Men SEM Men SEM Men SEM Men SEM Totl cteri CLA-producing c 119 cteri Bifidocterium spp Lctocillus spp Roseuri spp c 13 CT, control; HF, high-ft; HF-ITF, high-ft diet supplemented with inulin-type fructns; HF-Ax, high-ft diet supplemented with rinoxylns; CLA, conjugted linoleic cids.,,c Men vlues within row with unlike superscript letters were significntly different (P,; Tukey s post hoc ANOVA sttisticl nlysis). * Quntities of cteri re expressed s log 1 (DNA copies/totl cecl content). CLA-producing cteri represent the counts of cteril groups typiclly ssocited with CLA production. This numer of CLA-producing cteri ws clculted s follows (log 1 (sum of Bifidocterium spp., Lctocillus spp. nd Roseuri spp.)). British Journl of Nutrition modifiction of Lctocillus spp. compred with the HF diet. It is worth noting tht preiotic supplementtions did not chnge the numer of totl cecl cteri compred with HF groups (Tle 2). Modultion of profile of cteril derived n-6 PUFA in the liver y high-ft diet nd dditionl preiotic tretments In the cecl tissue nd in the liver, chnges in LA (cis- 9,cis : 2), rumenic cid (cis-9,trns : 2 CLA) nd vccenic cid (trns : 1), the three mjor ftty cids of the linoleic iohydrogention pthwy, occurred upon dietry mnipultion. The mount of totl ftty cids, LA (cis- 9,cis : 2) nd n-6 PUFA metolites (including cteril derived metolites) re presented in Fig. 1 (liver tissue) nd Fig. 2 (cecl tissue). In the liver, we oserved (in oth experiments) the presence of rumenic cid (cis-9,trns : 2 CLA) nd vccenic cid (trns : 1) in the CT groups, despite the fct tht these ftty cids were undetectle in the CT diet (Tle 1). The heptic totl ftty cids nd LA (cis-9,cis : 2) contents were not significntly modified in the HF groups compred with the CT groups, wheres HF diet feeding incresed rchidonic cid (2 : 4n-6) nd tended to increse rumenic cid (cis-9,trns : 2 CLA) (Fig. 1 nd Supplementl Tle 2). Regrding vccenic cid (trns : 1), its content ws not modified y the HF diet in the first experiment wheres it decresed in the second experiment (Fig. 1 nd Supplementl Tle 2). Concerning the n-3 PUFA profile, the HF diet did not significntly chnge -linolenic cid (-LnA; 18 : 3n-3), docospentenoic cid (DPA; 22 : 5n-3) nd DHA (22 : 6n-3) contents ut decresed EPA (2 : 5n-3) (Fig. 3). Preiotic supplementtions (oth ITF nd Ax) hd no significnt effect on totl ftty cids nd the n-6 PUFA profile compred with the HF diet ut induced chnges in the n-3 profile. The level of EPA (2 : 5n-3), DPA (22 : 5n-3) nd DHA (22 : 6n-3) ws not modified y ITF supplementtion wheres Ax supplementtion decresed EPA (2 : 5n-3) nd DPA (22 : 5n-3) nd tended to decrese DHA (22 : 6n-3) compred with HF diet feeding (Fig. 3). Modultion of profile of cteril derived n-6 PUFA in the cecum y high-ft diet nd dditionl preiotic tretments In the cecl tissue, s oserved in the liver, we detected nd quntified rumenic cid (cis-9,trns : 2 CLA) nd vccenic cid (trns : 1) in the CT groups (Fig. 2). Regrding the modifictions of the n-6 PUFA profile y HF diet feeding nd preiotic supplementtions, we oserved different effects etween experiments 1 nd 2. In experiment 1, HF diet feeding did not significntly chnge totl ftty cids, LA (cis-9,cis : 2) nd rumenic cid (cis-9,trns : 2 CLA) content, ut HF diet feeding incresed the rchidonic cid (2 : 4n-6) content (produced in host tissue from LA) nd the vccenic cid (trns : 1) content ( cteril metolite of LA) (Fig. 2). Furthermore, ITF supplementtion hd no dditionl effects compred with the ones oserved following HF diet feeding, except tht the reltive proportion of rumenic cid (cis-9,trns : 2 CLA) (expressed s percentge of the totl ftty cids identified) ws incresed y ITF compred with the HF diet (P¼; t test) (Supplementl Tle S3). In experiment 2, HF diet feeding tended to increse totl ftty cids nd LA (cis-9,cis : 2) contents compred with the CT diet (Fig. 2). Both contents in the HF group were 1 4-fold higher thn the contents mesured in the CT group. HF diet feeding significntly incresed vccenic cid (trns : 1) content compred with the CT diet (Fig. 2). HF diet feeding did not significntly chnge the solute content of rumenic cid (cis-9,trns : 2 CLA) ut significntly incresed the reltive proportion of rumenic cid (cis-9,trns : 2 CLA) (results expressed s percentge of the totl ftty cids identified) (Supplementl Tle S3). Totl ftty cids nd LA (cis-9,cis : 2) content incresed upon Ax supplementtion (Fig. 2). The level in the HF-Ax group ws 1 5 times higher thn the mount mesured in the HF group nd two times higher thn the mount mesured in the CT group. Ax supplementtion incresed the rumenic cid (cis-9,trns : 2 CLA) content (solute content nd reltive proportion) compred with oth the CT nd HF groups (Fig. 2 nd Supplementl Tle S3). Ax supplementtion

5 Gut microiot nd conjugted linoleic cid 5 hd no dditionl effect on the vccenic cid (trns : 1) content compred with the effect oserved upon HF diet feeding. In ddition to the chnges oserved in the n-6 PUFA profile, the n-3 PUFA profile ws lso modified fter the different dietry tretments (Fig. 4). -LnA (18 : 3n-3) content tended to e incresed fter HF diet feeding. The level in the HF group ws out 2-fold higher thn the mount mesured in the CT group in oth experiments. We lso oserved chnges in the level of long- nd very-long chin n-3 PUFA, consisting of decresed EPA (2 : 5n-3) content nd n incresed DPA (22 : 5n-3) content following HF diet feeding wheres (A) Totl FA (µg/mg liver) CT HF HF-ITF Totl FA (µg/mg liver) CT HF HF-Ax British Journl of Nutrition (B) LA (µg/mg liver) (C) AA (µg/mg liver) CT HF HF-ITF CT HF HF-Ax 6 5, AA (µg/mg liver) LA (µg/mg liver) (D) 6 6 RA (µg/mg liver) RA (µg/mg liver) (E) VA (µg/mg liver) VA (µg/mg liver) Fig. 1. Totl ftty cids (FA) (A), linoleic cid (LA; 18 : 2n-6) (B) nd n-6 PUFA metolites rchidonic cid (AA; 2 : 4n-6) (C), rumenic cid (RA; cis-9,trns : 2) (D) nd vccenic cid (VA; trns : 1) (E) in the liver tissue of mice fter 4 weeks of dietry tretment: control (CT) diet; high-ft (HF) diet; or HF diet supplemented with inulin-type fructns (HF-ITF) or rinoxylns (HF-Ax). Vlues re mens, with their stndrd errors represented y verticl rs., Men vlues with unlike letters were significntly different (P,; Tukey s post hoc ANOVA sttisticl nlysis).

6 6 C. Drurt et l. DHA (22 : 6n-3) content ws not modified upon HF diet feeding (Fig. 4). ITF supplementtion hd no dditionl effect on the n-3 PUFA profile (-LnA, EPA, DPA nd DHA) compred with HF diet feeding (Fig. 4). Ax supplementtion further incresed -LnA (18 : 3n-3) cid content, this increse ppering significnt s compred with the CT group (Fig. 4). Ax supplementtion hd no dditionl effect on EPA (2 : 5n-3) content ut it decresed the reltive proportion of DPA (22 : 5n-3) nd DHA (22 : 6n-3) compred with HF diet feeding (Supplementl Tle 3). Effect of high-ft diet feeding nd dditionl preiotic tretments supplementtion on steroyl-coa desturse-1 mrna expression nd ctivity in the liver nd cecl tissue We oserved chnges in the rumenic cid (cis-9,trns : 2 CLA) content in the cecl tissue nd in the liver fter HF diet feeding. Rumenic cid (cis-9,trns : 2 CLA) my e produced directly y cteri, or indirectly in host tissues from vccenic cid (trns : 1). In order to evlute the contriution of host metolism to the chnges in rumenic (A) British Journl of Nutrition Totl FA (µg/mg cecl tissue) (B) LA (µg/mg cecl tissue) (C) AA (µg/mg cecl tissue) (D) 125 RA (µg/mg cecl tissue) (E) VA (µg/mg cecl tissue) CT HF HF-ITF Totl FA (µg/mg cecl tissue) CT HF HF-ITF CT LA (µg/mg cecl tissue) AA (µg/mg cecl tissue) RA (µg/mg cecl tissue) VA (µg/mg cecl tissue) , 1 CT, HF, HF-Ax HF, HF-Ax Fig. 2. Totl ftty cids (FA) (A), linoleic cid (LA; 18 : 2n-6) (B) nd n-6 PUFA metolites rchidonic cid (AA; 2 : 4n-6) (C), rumenic cid (RA; cis-9,trns : 2) (D) nd vccenic cid (VA; trns : 1) (E) in the cecl tissue of mice fter 4 weeks of dietry tretment: control (CT) diet; high-ft (HF) diet; or HF diet supplemented with inulin-type fructns (HF-ITF) or rinoxylns (HF-Ax). Vlues re mens, with their stndrd errors represented y verticl rs., Men vlues with unlike letters were significntly different (P,; Tukey s post hoc ANOVA sttisticl nlysis).

7 Gut microiot nd conjugted linoleic cid 7 (A) α-lna (µg/mg liver) CT HF HF-ITF α-lna (µg/mg liver) CT HF HF-Ax (B) 2 2 EPA (µg/mg liver) 15 1 EPA (µg/mg liver) 15 1 c British Journl of Nutrition (C) DPA (µg/mg liver) (D) DHA (µg/mg liver) CT HF HF-ITF DPA (µg/mg liver) DHA (µg/mg liver) , CT HF HF-Ax 3 5, Fig. 3. -Linolenic cid (-LnA; 18 : 3n-3) (A) nd n-3 PUFA metolites EPA (2 : 5n-3) (B), docospentenoic cid (DPA; 22 : 5n-3) (C) nd DHA (22 : 6n-3) (D) in the liver tissue of mice fter 4 weeks of dietry tretment: control (CT) diet; high-ft (HF) diet; or HF diet supplemented with inulin-type fructns (HF-ITF) or rinoxylns (HF-Ax). Vlues re mens, with their stndrd errors represented y verticl rs.,,c Men vlues with unlike letters were significntly different (P,; Tukey s post hoc ANOVA sttisticl nlysis). cid (cis-9,trns : 2 CLA) content oserved in the cecl tissue nd in the liver, we mesured SCD-1 mrna expression nd estimted its ctivity in intestinl segments nd in the liver. The expression of SCD-1 mrna ws t lest 2-fold higher in the liver thn in the different intestinl segments (dt not shown). As shown in Tle 3, SCD-1 mrna expression ws decresed y the HF diet in the liver nd the colon tissue in oth experiments. Both preiotic supplementtions hd no mjor effect on SCD-1 mrna expression in the liver nd in the colon compred with the HF diet, except tht Ax further decresed mrna expression in the liver compred with the HF diet. In order to strengthen the SCD-1 mrna expression results, we lso estimted the SCD-1 ctivity y clculting desturtion rtios. In oth experiments, HF diet feeding decresed the cis-9-14 : 1/14 : nd cis-9-16 : 1/16 : rtios in the liver nd in the cecl tissue wheres the cis-9-18 : 1/18 : rtio ws unchnged in these tissues (Tle 3). Preiotic supplementtions hd no dditionl effect compred with the effects of the HF diet (Tle 3). Ft-inding cpcity of rinoxylns nd inulin-type fructns As shown in Fig. 5, we otined FBC of 15 % for strch used s the negtive control nd FBC of 245 % for chitosn used s the positive control. The FBC of ITF ws wek (18 %) wheres Ax hd much higher FBC (43 %). This result shows the high FBC of Ax. Discussion Gut microiot composition/ctivity cn influence host physiology (25). The gut microiot intercts with the host in

8 8 C. Drurt et l. (A) α-lna (µg/mg cecl tissue) CT HF HF-ITF α-lna (µg/mg cecl tissue) CT, HF HF-Ax British Journl of Nutrition (B) EPA (µg/mg cecl tissue) (C) DPA (µg/mg cecl tissue) (D) DHA (µg/mg cecl tissue) CT HF HF-ITF CT HF HF-Ax ,, CT HF HF-ITF DPA (µg/mg cecl tissue) EPA (µg/mg cecl tissue) DHA (µg/mg cecl tissue) , CT HF HF-Ax Fig. 4. -Linolenic cid (-LnA; 18 : 3n-3) (A) nd n-3 PUFA metolites EPA (2 : 5n-3) (B), docospentenoic cid (DPA; 22 : 5n-3) (C) nd DHA (22 : 6n-3) (D) in the cecl tissue of mice fter 4 weeks of dietry tretment: control (CT) diet; high-ft (HF) diet; or HF diet supplemented with inulin-type fructns (HF-ITF) or rinoxylns (HF-Ax). Vlues re mens, with their stndrd errors represented y verticl rs., Men vlues with unlike letters were significntly different (P,; Tukey s post hoc ANOVA sttisticl nlysis). prt through the production of metolites, such s SCFA or ile cid metolites (25). Bcteri cn lso produce lipophilic metolites, in prticulr conjugted isomers of LA (CLA) (1,16,18,42 44) nd conjugted isomers of -LnA (44 47). The min ojective of our experiments ws to study if the gut microiot modultes the production of CLA in vivo upon HF diet feeding nd to evlute the potentil effect of preiotic supplementtions in chnging the mount of CLA produced y modultion of the gut microiot composition/ctivity. Although rumenic cid (cis-9,trns : 2 CLA) nd vccenic cid (trns : 1) were undetectle in the CT diet, these ftty cids were present oth in the liver nd the cecl tissue of control mice, strongly supporting their endogenous production y the gut microiot through the iohydrogention pthwy of LA. The gut microiot composition of HF-fed mice ws different from CT mice: for instnce, the counts of cteril groups typiclly ssocited with CLA production were decresed nd this ws mostly due to decrese in Lctocillus spp. However, the quntity of the sustrte (LA; 18 : 2n-6) ville for cteril metolism is much higher in the HF diet compred with the CT diet (the HF diet contins twenty times more LA thn the CT diet). Interestingly, lthough the counts of CLA-producing cteri were decresed upon HF diet feeding, we oserved n increse in rumenic cid (cis-9,trns : 2 CLA) nd in vccenic cid (trns : 1) in the cecl tissue. This oservtion suggests tht the quntity of sustrte ville is the rte-limiting fctor for CLA synthesis y gut microiot in control conditions. One explntion my e tht nerly ll the ftty cids re sored in the jejunum nd ileum (proximl prt) in the control conditions (CT diet), leding to very smll quntities of lipids reching the cecum. In this wy, we cn ssume tht the mount of LA (18 : 2n-6) ville for CLA synthesis in the cecum, where cteri re the

9 Gut microiot nd conjugted linoleic cid 9 British Journl of Nutrition Tle 3. Steroyl-CoA desturse-1 (SCD-1) mrna expression nd ctivity in the liver nd intestinl tissues of mice fter 4 weeks of dietry tretment* (Men vlues with their stndrd errors) Experiment 1 Experiment 2 Diet... Men SEM Men SEM Men SEM Men SEM Men SEM Men SEM Liver SCD-1 mrna c 271 Colon SCD-1 mrna Liver 14 : 1/14 : Liver 16 : 1/16 : Liver 18 : 1/18 : Cecl tissue 14 : 1/14 : , Cecl tissue 16 : 1/16 : Cecl tissue 18 : 1/18 : CT, control; HF, high-ft; HF-ITF, high-ft diet supplemented with inulin-type fructns; HF-Ax, high-ft diet supplemented with rinoxylns.,,c Men vlues within row with unlike superscript letters were significntly different (P,; Tukey s post hoc ANOVA sttisticl nlysis). * SCD-1 ctivity ws estimted y clcultion of the desturtion rtio, the rtio etween the MUFA produced y the SCD-1 nd their respective SFA, the sustrte of the SCD-1. most undnt, could e influenced y the dietry ft intke nd the sorption cpcity of the jejunum ileum. It is importnt to consider tht the dietry intke of LA (18 : 2n-6) nd -LnA (18 : 3n-3) in humns rnges respectively from 6 8 to 21 6 g/d nd from 5 to 2 2 g/d ccording to country nd sex (48). The sorption efficiency of LA (18 : 2n-6) nd -LnA (18 : 3n-3) is estimted t 96 % of dietry intke, mening tht quntity rnging from 272 to 864 mg for LA (18 : 2n-6) nd from 2 to 88 mg for -LnA (18 : 3n-3) reches the cecum ech dy (48). Therefore, non-negligile quntity of LA (18 : 2n-6) is present in the cecl lumen nd is ville for cteril metolism. In the present studies, we hve regrouped the CLA-producing cteri, tking into ccount the dt ville from in vitro studies mostly. The three mjor gener including cteri le to produce CLA in the mouse gut re Bifidocteri spp., Lctocillus spp. nd Roseuri spp. (49). The clcultion of the numer of CLA-producing cteri should e cutiously interpreted. Even if our dt suggest tht Roseuri spp. could e prticulrly interesting, we hve insufficient knowledge to relly clim the link etween one group of cteri nd CLA production. In fct, only cultivle cteri hve een tested for their cpcity to metolise LA or -LnA in vitro. A limited numer of cteril strins hve een tested up to now nd found to e le to produce CLA s individul strins. Moreover, in complex microil ecosystems, interctions nd competition exist etween cteri, which re not studied upon simple culture conditions in vitro, nd would require nlysis in complex iosystems mimicking the in vivo sitution, or trnscriptomic nlysis to point out key cteril functions ssocited with CLA production. Wll et l. (23) hve lredy shown tht supplementtion of mice with LA (18 : 2n-6) together with Bifidocterium reve incresed rumenic cid (cis-9,trns : 2 CLA) content in host tissues compred with supplementtion with LA (18 : 2n-6) lone. In our studies, we hve shown tht higher content of LA (18 : 2n-6) in the diet without ny preor proiotic interventions is le to increse LA (18 : 2n-6) vilility for cteril metolism nd is sufficient to oserve n increse in rumenic cid (cis-9,trns : 2 CLA) content in host tissue (23). By using HF diet, in the present studies we showed for the first time in mice tht incresing the sustrte (LA; 18 : 2n-6) vilility for cteril metolism incresed the proportion of metolites such s rumenic cid (cis-9,trns : 2 CLA) nd vccenic cid (trns : 1) in host tissues. Unfortuntely, we cnnot exclude tht the presence of vccenic cid (trns : 1) nd rumenic cid (cis-9,trns : 2) in the HF diet, proly due to the presence of lrd used s n ingredient, contriuted to the incresed mount of these ftty cids oserved in host tissues fter HF diet feeding. It would e interesting to perform n nlysis of the ftty cid profile in the gut content, nmely in the cecl content, in order to evlute if we could oserve difference in PUFA, CLA nd vccenic cid content fter HF diet feeding nd/or preiotic supplementtion. Such nlysis would give informtion on the vilility of PUFA in the distl prt of the gut nd would e strong evidence of PUFA metolism nd CLA production y the gut microiot.

10 1 C. Drurt et l. British Journl of Nutrition FBC (% of compound weight) Strch In order to evlute the reltive contriution of cteril species in the production of cteril derived LA metolites, we dded two non-digestile crohydrtes (ITF nd Ax) prone to selectively modulte the gut microiot (28,29,32,38). Interestingly, we oserved tht oth preiotic supplementtions in the HF diet incresed the numer of CLA-producing cteri even if the cteril gener modulted y ech preiotic were different. Neither preiotic chnged the totl numer of cteri nd Lctocillus spp. compred with HF diet feeding. Both preiotics incresed the numer of Bifidocterium spp. compred with HF diet feeding ut ITF hd stronger ifidogenic effect thn Ax. ITF decresed Roseuri spp. wheres Ax incresed this cteril genus compred with HF diet feeding. Regrding the effects of preiotic supplementtions on the host tissue ftty cid profiles, we oserved tht Ax, ut not ITF, significntly incresed the rumenic cid (cis-9,trns : 2 CLA) content nd tended to increse the LA (18 : 2n-6) content nd vccenic cid (trns : 1) content in the cecl tissue. This difference etween the two preiotic supplementtions could e explined y the different modultion of the gut microiot. Indeed, Roseuri spp., tht were incresed upon Ax supplementtion, possess the highest linolete isomerse ctivity nd thus the highest cpcity to produce CLA nd other metolites of LA (16). In fct, this oservtion supports the ide tht the presence of Roseuri spp. is more importnt thn the presence of Bifidocterium spp. for CLA production. Another importnt difference etween the two preiotics used in our studies is their cpcity to ind dietry ft. In fct, in vitro dt showed tht ITF hve wek FBC wheres Ax hve strong FBC. Ax supplementtion could thus lso promote CLA production y incresing LA (18 : 2n-6) vilility in the lower prt of the gut. Indeed, we oserved tht Ax incresed totl ftty cid, LA (18 : 2n-6) nd -LnA (18 : 3n-3) contents in the cecl tissue compred with HF diet feeding wheres ITF supplementtion hd no effect on these ftty cid contents. Even if we oserved higher proportion of rumenic cid (cis-9,trns : 2 CLA) nd vccenic cid (trns : 1) c ITF Ax Chitosn Fig. 5. Ft-inding cpcity (FBC) of strch (negtive control), inulin-type fructns (ITF), rinoxylns (Ax) nd chitosn (positive control). Vlues re mens, with their stndrd errors represented y verticl rs.,,c,d Men vlues with unlike letters were significntly different (P,; Tukey s post hoc ANOVA sttisticl nlysis). d in the liver following HF diet feeding, the further increse of these two ftty cids upon preiotic supplementtions ws oserved in the cecum ut not in the liver. This oservtion supports the ide tht modultion of the cteril metolites of LA is restricted to intestinl tissues, the tissues situted ner the production site of CLA. However, we hve previously found n increse in rumenic cid (cis-9,trns : 2 CLA) in dipose tissue fter Ax supplementtion, suggesting tht the increse in rumenic cid (cis-9,trns : 2 CLA) fter Ax supplementtion is not restricted to intestinl tissue ut lso concerns tissues situted fr from the intestine (29). In view of this, we postulte tht rumenic cid (cis-9,trns : 2 CLA) produced y the gut microiot cn hve some systemic effects, in ddition to the locl effects in intestinl tissue. However, due to the rther low increse in vccenic cid nd rumenic cid content in host tissue, the size of the effect ttriuted to these chnges in the ftty cid profile should e interpreted cutiously. Even if the chnges in vccenic cid nd rumenic cid content in host tissue re reltively modest, metolic effect could e expected since in the cow, dose s smll s % of trns-1,cis : 2 CLA is le to inhiit the mmmry iosynthesis of milk ft y down-regultion of the enzymes implicted in mmmry lipid synthesis (5). The chllenging question is now to identify whether CLA re produced in the upper prt of the gstrointestinl trct nd sored y the clssicl route of intestinl ft sorption or if CLA re only produced y the dense gut microiot situted in the cecum nd sored y n unidentified mechnism of sorption. (7 9) In view of the metolic pthwys known in ruminnts s well s the dt otined in vitro (16) nd in our studies, we propose mechnism of CLA synthesis in which the gut microiot nd the host re involved in symiosis (Fig. 6). We know tht SCD-1 ctivity in host tissues cn influence the CLA content in these tissues. To identify the reltive contriution of gut microiot metolism s compred with the host metolism involved in the CLA content chnges, we mesured SCD-1 expression nd estimted its ctivity in host tissue. SCD-1 mrna expression ws decresed y the HF diet in the liver nd the colon tissue in oth experiments. A wy commonly used in the literture to estimte the SCD-1 ctivity is to clculte the desturtion rtio, which mens the rtio etween MUFA (myristoleic cid, plmitoleic cid, oleic cid) produced y the SCD-1 nd corresponding SFA sustrtes (myristic cid, plmitic cid nd steric cid, respectively) (51,52). In oth experiments, HF diet feeding decresed the cis-9-14 : 1/14 : nd cis-9-16 : 1/16 : rtios in the liver nd in the cecl tissue ut hd no effect on cis-9-18 : 1/18 : in these tissues. Preiotic supplementtions hd no dditionl effect compred with the effects of the HF diet. In generl, HF diet feeding decresed SCD-1 mrna expression nd ctivity in the liver nd in the cecl tissue without mjor effect of the preiotic supplementtion. Thus, the higher rumenic cid (cis-9,trns : 2 CLA) content in the cecl tissue results from the modultion of the cteril metolism of LA rther thn chnges in host desturse (SCD-1) expression nd/or ctivity. Although there is strong evidence for n effect of the gut microiot on the tissue

11 Gut microiot nd conjugted linoleic cid 11 Microiot in gut lumen Host tissues Isomerse Linoleic cid (cis-9,cis : 2) Isomerse nd reductses Reductses Rumenic cid (cis-9,trns : 2) Other CLA Reductses Vccenic cid (trns : 1) Reductses Rumenic cid (cis-9,trns : 2) Other CLA 9-Desturse (SCD-1) Vccenic cid (trns : 1) Isomerse α-linolenic cid (cis-9,cis 12,cis : 3) CLnA Steric cid (18 : ) CLnA British Journl of Nutrition Fig. 6. Proposed mechnism of conjugted linoleic cid (CLA) nd conjugted linolenic cid (CLnA) synthesis in mice. Cultivted gut cteri re le to metolise -linolenic cid (cis-9,cis-12,cis : 3) into CLnA nd linoleic cid (cis-9,cis : 2) into CLA through cteril isomerse ctivities. Some CLA nd CLnA re metolised into vccenic cid (trns : 1) y one or severl cteril reductses. Metolic pthwys of -linolenic cid nd linoleic cid go further to SFA (steric cid; 18 : ). In our experiments we hve shown tht rumenic cid (cis-9,trns : 2 CLA) nd vccenic cid (trns : 1) produced endogenously y the gut microiot cn ccumulte in host tissues. However, the sorption nd trnsport mechnisms of these lipophilic cteril metolites from the intestinl lumen to the host tissues re completely unknown. In host tissues, vccenic cid (trns : 1) cn e desturted y the D-9 desturse (steroyl-coa desturse-1; SCD-1) into rumenic cid (cis-9,trns : 2 CLA)., Bcteril ctivity;, host ctivity;, unknown pthwy. levels of rumenic cid (cis-9,trns : 2 CLA), contriution of the endogenous host desturse (SCD-1) ctivity cnnot e ruled out. In ruminnts, the mjority of rumenic cid (cis-9,trns : 2 CLA) found in tissues results from the desturtion of cteril metolite, vccenic cid (trns : 1), y SCD-1 in ruminnt tissues (12). We my propose tht the increse of rumenic cid (cis-9,trns : 2 CLA) oserved in cecl tissue is the result of the desturtion y host SCD-1 enzyme of the high mount of vccenic cid (trns : 1) produced y the gut microiot. It hs lredy een reported tht dministrtion of commensl gut cteri leds to chnges in ftty cid profile in different host tissues (for exmple, dipose tissue, liver nd rin) (53,54). Wll et l. reported tht supplementtion with CLA-producing cteri (Bifidocterium reve) (1,16) nd LA (18 : 2n-6) not only incresed rumenic cid (cis-9, trns : 2 CLA) content in murine tissues ut lso chnged the n-3 PUFA profile (23). They oserved n increse in oth EPA (2 : 5n-3) nd DHA (22 : 6n-3) contents in the lrge intestine nd dipose tissue (23). In our studies, we oserved tht HF diet feeding decresed EPA (2 : 5n-3) content in the liver nd cecl tissues nd Ax supplementtion further decresed EPA (2 : 5n-3) content in the liver. It is interesting to note tht, even if -LnA (18 : 3n-3) content ws incresed fter Ax supplementtion, EPA (2 : 5n-3) content did not increse fter Ax supplementtion lthough -LnA (18 : 3n-3) is the precursor of very-long-chin n-3 PUFA. This oservtion is in fvour of the requisitioning of the enzymes required for the synthesis of very-long-chin n-3 PUFA in nother pthwy such s the production of conjugted verylong-chin n-6 PUFA from CLA. Indeed, the production of conjugted very-long-chin n-6 PUFA from CLA (55) hs een descried, nd those metolites could led to interesting iologicl properties (55). In ddition, it hs lso een shown tht the ctivity of the enzymes implicted in very-long-chin PUFA synthesis (desturse nd elongse) is regulted y the nutritionl sttus nd y the composition of the dietry ft (56 58). Deficiency in essentil ftty cids increses enzyme ctivity wheres consumption of essentil ftty cids decreses enzyme ctivity. Therefore, the increse in -LnA oserved fter Ax supplementtion could decrese the ctivity of the enzymes implicted in the long-chin PUFA synthesis nd contriute to the decrese in EPA content. The nlyticl method used to nlyse the ftty cid profile in tissue llowed us to quntify only rumenic cid (cis-9,trns : 2 CLA). This is the mjor CLA isomer produced y Bifidocterium spp. (44,59) ; however, CLA re lrge group of isomers nd quntifiction of other isomers would e interesting since the literture indictes tht the physiologicl effects of CLA re isomer specific (2,4,8,6,61). Finlly, some cteri isolted from the mmmlin gut re lso le to produce conjugted LnA in vitro (44,47) nd some iologicl effects re now ttriuted to this other group of conjugted ftty cids (45,46,62,63). So, n nlysis of conjugted LnA nd even glol nlysis of conjugted PUFA in host tissues would e n interesting perspective of our studies. In conclusion, we showed for the first time tht the gut microiot of mice is le to produce CLA without ny supplementtion. We showed tht HF diet feeding incresed CLA content in host tissues y incresing sustrte vilility for the gut microiot. Preiotic supplementtions further incresed CLA content in the cecl tissue, y further incresing sustrte vilility nd modulting the gut microiot composition. Just s some ppers hve proposed tht CLA production is chrcteristic of proiotic cteri (49,64),we propose tht CLA production is chrcteristic to tke into ccount in the metolic effects of non-digestile crohydrtes with preiotic properties.

12 12 C. Drurt et l. British Journl of Nutrition Supplementry mteril To view supplementry mteril for this rticle, plese visit Acknowledgements Finncil support hs een provided y grnts from the Wlloon Region (Generl Directory of Agriculture, convention D31 117) nd Fonds de l Recherche Scientifique (FRS-FNRS; no F). P. D. C. is Reserch Associte from the FRS-FNRS, Belgium. F. M. nd L. D. V. enefit from finncil support y the Reserch Foundtion Flnders (Fonds Wetenschppelijk Onderzoeck (FWO) Vlnderen) nd the Reserch Council of the Vrije Universiteit Brussel. F. M. is the recipient of PhD fellowship of the Reserch Foundtion Flnders (FWO Vlnderen). S. P. nd T. V. D. W. re postdoctorl reserchers from the Reserch Foundtion Flnders (FWO Vlnderen). C. D. enefits from Dnone Institute grnt. The funders hd no role in study design, dt collection nd nlysis, decision to pulish, or preprtion of the mnuscript. The uthors thnk Christine Turu nd Eric Mignolet for their excellent technicl ssistnce in the ftty cid profile nlysis; nd Brr Pchikin, Lure Bindels, Amndine Everrd, Lucie Geurts nd Bouzz Es Sdi for helpful discussion nd technicl support. C. D. performed dt collection nd nlysis, interpreted the dt nd wrote the mnuscript. A. N. M. designed the experiments, performed dt collection nd nlysis, nd interpreted the dt. E. D. W. designed the experiments, nd performed dt collection nd nlysis. F. C. D. B. performed dt collection. S. P. nd T. V. D. W. performed nd interpreted gut microiot nlysis. F. M. nd L. D. V. prticipted to the gut microiot nlysis. P. D. C. performed the tissue smpling t the end of the experiment. N. M. D. plnned nd reviewed ll experiments nd mnuscript preprtion. A. M. N., P. D. C., N. M. D. nd Y. L. provided intellectul input on the pper nd reviewed the pper. The uthors declre no conflicts of interests. References 1. Benjmin S & Spener F (29) Conjugted linoleic cids s functionl food: n insight into their helth enefits. Nutr Met (Lond) 6, Churruc I, Fernndez-Quintel A & Portillo MP (29) Conjugted linoleic cid isomers: differences in metolism nd iologicl effects. Biofctors 35, Clément L, Poirier H, Niot I, et l. (22) Dietry trns-1, cis-12 conjugted linoleic cid induces hyperinsulinemi nd ftty liver in the mouse. J Lipid Res 43, Tylor CG & Zhrdk P (24) Dietry conjugted linoleic cid nd insulin sensitivity nd resistnce in rodent models. Am J Clin Nutr 79, 1164S 1168S. 5. Stout MB, Liu LF & Belury MA (211) Heptic stetosis y dietry-conjugted linoleic cid is ccompnied y ccumultion of dicylglycerol nd incresed memrne-ssocited protein kinse C epsilon in mice. Mol Nutr Food Res 55, Chin SF, Liu W, Storkson JM, et l. (1992) Dietry sources of conjugted dienoic isomers of linoleic cid, newly recognized clss of nticrcinogens. J Food Compos Anl 5, Jenkins TC, Wllce RJ, Mote PJ, et l. (28) Bord-invited review: recent dvnces in iohydrogention of unsturted ftty cids within the rumen microil ecosystem. J Anim Sci 86, Bumn DE, Bumgrd LH, Corl BA, et l. (2) Biosynthesis of conjugted linoleic cid in ruminnts. J Anim Sci 77, 1e 15e. 9. Chillird Y, Glsser F, Ferly A, et l. (27) Diet, rumen iohydrogention nd nutritionl qulity of cow nd got milk ft. Eur J Lipid Sci Technol 19, Cokley M, Ross RP, Nordgren M, et l. (23) Conjugted linoleic cid iosynthesis y humn-derived Bifidocterium species. J Appl Microiol 94, Mi MR, Chudhry LC, Bestwick CS, et l. (21) Toxicity of unsturted ftty cids to the iohydrogenting ruminl cterium, Butyrivirio firisolvens. BMC Microiol 1, Griinri JM, Corl BA, Lcy SH, et l. (2) Conjugted linoleic cid is synthesized endogenously in lctting diry cows y D9-desturse. J Nutr 13, Turpeinen AM, Mutnen M, Aro A, et l. (22) Bioconversion of vccenic cid to conjugted linoleic cid in humns. Am J Clin Nutr 76, Sntor JE, Plmquist DL & Roehrig KL (2) Trnsvccenic cid is desturted to conjugted linoleic cid in mice. J Nutr 13, Cni PD & Delzenne NM (211) The gut microiome s therpeutic trget. Phrmcol Ther 13, Devillrd E, McIntosh FM, Duncn SH, et l. (27) Metolism of linoleic cid y humn gut cteri: different routes for iosynthesis of conjugted linoleic cid. J Bcteriol 189, McIntosh FM, Shingfield KJ, Devillrd E, et l. (29) Mechnism of conjugted linoleic cid nd vccenic cid formtion in humn fecl suspensions nd pure cultures of intestinl cteri. Microiology 155, Brrett E, Ross RP, Fitzgerld GF, et l. (27) Rpid screening method for nlyzing the conjugted linoleic cid production cpilities of cteril cultures. Appl Environ Microiol 73, Roserg-Cody E, Ross RP, Hussey S, et l. (24) Mining the microiot of the neontl gstrointestinl trct for conjugted linoleic cid-producing ifidocteri. Appl Environ Microiol 7, Lee HY, Prk JH, Seok SH, et l. (26) Humn originted cteri, Lctocillus rhmnosus PL6, produce conjugted linoleic cid nd show nti-oesity effects in diet-induced oese mice. Biochim Biophys Act 1761, Mcouzet M, Roert N & Lee BH (21) Genetic nd functionl spects of linolete isomerse in Lctocillus cidophilus. Appl Microiol Biotechnol 87, Mcouzet M, Lee BH & Roert N (21) Genetic nd structurl comprison of linolete isomerses from selected food-grde cteri. J Appl Microiol 19, Wll R, Ross RP, Shnhn F, et l. (29) Metolic ctivity of the enteric microiot influences the ftty cid composition of murine nd porcine liver nd dipose tissues. Am J Clin Nutr 89, Chin SF, Storkson JM, Liu W, et l. (1994) Conjugted linoleic cid (9,11- nd 1,12-octdecdienoic cid) is produced in conventionl ut not germ-free rts fed linoleic cid. J Nutr 124,