Yacon flour: Fructans: Calcium and magnesium balance: Bone mass and biomechanics: Caecum morphometry

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1 British Journl of Nutrition (27), 97, q The Authors 27 DOI: 1.117/S Effets of frutns-ontining yon (Smllnthus sonhifolius Poepp & Endl.) flour on eum muosl morphometry, lium nd mgnesium lne, nd one lium retention in growing rts Alexndre R. Loo 1,Céli Colli 1, Elin P. Alvres 2 nd Tulli M. C. C. Filisetti 1 * 1 Deprtment of Food nd Experimentl Nutrition, Fulty of Phrmeutil Sienes, University of São Pulo, São Pulo, Brzil, Av. Prof. Lineu Prestes, 58, Bloo , São Pulo, Brzil 2 Deprtment of Cell nd Developmentl Biology, Institute of Biomedil Sienes, University of São Pulo, São Pulo, Brzil, Av. Prof. Lineu Prestes, 1524, 558-9, São Pulo, Brzil (Reeived 19 My 26 Revised 2 Otoer 26 Aepted 1 Otoer 26) Yon roots hve een onsidered funtionl food due to the high levels of frutns they ontins. In the present study, C nd Mg lne, one mss nd strength, nd eum muosl morphometry were evluted. Growing mle Wistr rts (n 24) were fed d liitum ontrol diets or diets supplemented with yon flour (5 or 7 5 % frutooligoshrides) for 27 d. Minerl lne ws evluted in three periods of 5 d (strting on the 4th, 1th nd 16th dys). After the rts were killled, the ones were removed nd one minerl density ws mesured. C nlyses were performed on left femurs nd tiis nd iomehnil testing on right femurs. The eum ws removed nd tissue smples were olleted for histologil nlysis. Cel histology hnged notiely in rts fed yon flour: there ws n inrese in the depth nd numer of totl nd ifurted rypts s well. Yon flour onsumption signifintly (P, 5) resulted in positive C nd Mg lne, leding to higher vlues of one minerl retention nd iomehnil properties (pek lod nd stiffness) when ompred to the ontrol group. The positive effets on minerl intestinl sorption, one mss nd iomehnil properties showed n importnt role of yon roots in the mintenne of helthy ones. The inresed numer of ifurting rypts might e relted to the higher minerl sorption used y the enlrgement of the soring surfe in the lrge intestine of the nimls. Yon flour: Frutns: Clium nd mgnesium lne: Bone mss nd iomehnis: Ceum morphometry Frutns re reserve rohydrtes omprising frutose moleules linked or not to terminl surose moleule. They my hve liner or rnhed struture, with different onds etween frutose monomers: (1) (2,6) onds found in levn-type frutns; nd (2) (2,1) onds found in inulin-type frutns (Roerfroid & Delzenne, 1998). Inulintype frutns re ommonly found in lmost ll speies in the Asteree fmily, mny of whih re of eonomi importne, suh s Chiorium intyus nd Helinthus tuerosus (Crvlho & Figueiredo-Rieiro, 21). Yon (Smllnthus sonhifolius [Poepp & Endl.] H. Roinson, Asteree) is perennil plnt originlly from the Andes Mountins. It grows suessfully etween 8 nd 35 m ove se level, lthough it is lso grown t se level in New Zelnd nd in the USA. There re lso reports of its ultivtion in other ountries in South Ameri (Argentin, Colomi, Peru nd Venezuel) nd Europe (Germny, Itly, Frne nd Czeh Repuli) (Vlentová & Ulrihová, 23). Yon hs een ultivted in south-estern Brzil s rop sine 1991 from August to Septemer, yielding up to 1 t/h (Vilhen et l. 2). Its tuerous roots re sweet nd rispy nd n e eten either rw or ooked (Zrdini, 1991) nd re widely used, together with its leves, to rew mediinl te. More reently, its physiohemil properties hve ontriuted to its use in the development of everges nd kery produts (Quinteros, 21; Mostto et l. 26). Its underground system onsists of two different types of edile reserve orgns: the tuerous roots, the tul ommerilized rop; nd the rhizophores, the orgns of vegettive reprodution. Both umulte high mounts of frutns nd other solule rohydrtes, suh s frutose, gluose nd surose. Nevertheless, vritions in the onentrtion of suh rohydrtes nd in the hin length of frutns my our during plnt growth nd fter hrvesting (Ity et l. 22). Studies hve shown tht the est period to hrvest yon in tropil regions is etween the 31st nd the 35th week fter ultivtion, regrding the onentrtion of frutns nd their proportion in reltion to mono- nd dishrides (Oliveir & Nishimoto, 24). Furthermore, yon plnts present high hydrolyti tivity t mturtion phse of the tuerous roots, ontriuting to the predominne of low degree of polymeriztion (DP, 1; frutooligoshrides (FOS)) over high DP frutns (inulin) (Ity et l. 22). Arevitions: BMD, one minerl density; DF, dietry fire; DP, degree of polymeriztion; FOS, frutooligoshrides. * Corresponding uthor: Dr Tulli M. C. C. Filisetti, fx þ , emil tulli@usp.r Downloded from IP ddress: , on 2 Sep 218 t 17:39:1, sujet to the Cmridge Core terms of use, ville t

2 Yon flour ffets one 777 FOS hve een lssified s preioti ingredients sine they re seletively fermented y the miroflor in the lrge intestine, leding to modultion in the omposition of the nturl eosystem (Roerfroid et l. 1998). Studies rried out in nimls nd mn hve demonstrted tht frutns, through their gstrointestinl effets, my indiretly ffet the immunologil funtion nd the metolism of rohydrtes, lipids nd minerls (Roerfroid & Delzenne, 1998; Vn Der Heuvel et l. 1999; Buddington et l. 22). Although the smll intestine is the mjor site for minerl sorption, the lrge intestine plys n importnt role in minerl sorption s well, sine some dietry fires (DF; inluding frutns) re fermented y teri in the olon. SCFA, produed s result of the inrese in the metoli tivity of the intestinl miroflor, lower the luminl ph nd therey inrese minerl soluility. Both low ph nd SCFA result in the hypertrophy of the muosl ells, leding to enlrgement of the surfe re in the intestine nd enhned minerl sorption (Roerfroid & Delzenne, 1998). Moreover, diret oupling of SCFA to the minerls my led to n inrese in their uptke y the intestinl ells (Lutz & Shrrer, 1991). Finlly, n inrese in the expression of lindin-d9k (Oht et l. 1998; Rshk & Dniel, 25; Nzeusseu et l. 26) nd other genes involved in C trnsport in the lrge intestine (Rshk & Dniel, 25) hs een reported s result of frutns ingestion. These effets re refleted in inreses in one minerliztion s well (Kruger et l. 23; Zfr et l. 24; Nzeusseu et l. 26), possily relted to derese in osteolsti resorption (Kruger et l. 23; Zfr et l. 24; Nzeusseu et l. 26). The potentil preioti effet of frutns nd the presene of potentilly ntioxidnt phytohemils (suh s hlorogeni, ffei nd feruli ids) from yon tuerous roots hve een reported (Pedreshi et l. 23; Vlentová & Ulrihová, 23). Reently, study evluted the toxil effets in rts fed yon root flour offered 34 nd 68 mg FOS/ody weight per d over 4 months, nd no dverse effets were oserved (Gent et l. 25). Sine yon roots re rih soure of FOS, they my potentilly e used s novel soure of preioti omponents in diet supplements. Moreover, yon roots my e n lterntive to more trditionl frutn soures suh s Chiorium intyus nd Helinthus tuerosus. In the present study, we hve evluted the effets of supplementing diet with frutn-rih yon flour on C nd Mg intestinl sorption nd lne s result of its eum fermenttion in rts. We then ssessed its effets on one minerl density (BMD) nd ontent nd femur iomehnil properties s mesurement of minerl iovilility. To estlish the possile reltion of minerl sorption nd eum enlrgement, morphometry of the el muos (numer of rypts, rypt depth nd ifurted rypts) ws investigted. Mterils nd methods Plnt mteril Approximtely 7 kg yon tuerous roots from São Sestião Frm, in the ity of Iiún (São Pulo, Brzil) were hrvested 8 months fter ultivtion nd stored for 2 d t room temperture nd humidity, under dim light. They were then wshed with flushing wter, kept in otton-fire gs nd utolved (121 8C, 2 min), for intivtion of frutn hydrolses nd phenol oxidses present in Asteree speies (Ity et l. 22). The utolved smples were lended, freeze-dried (Lioténi Ind. Com. Ltd, Emu, São Pulo, Brzil) nd powdered to otin the flour. Animls nd diets Twenty-four 5-week-old mle Wistr rts, initilly weighing 65 7 g, were otined from the olony of the Fulty of Phrmeutil Sienes, University of São Pulo. The experimentl protool ws pproved y the Commission on Ethis in Animl Experiments of the Fulty of Phrmeutil Sienes of the University of São Pulo, ording to the guidelines of the Brzilin College on Animl Experimenttion. All rts were housed in individul stinless-steel wire-mesh ges under ontrolled temperture (22 ^ 2 8C) with 12 h light drk yle (lights on 7 19 hours). The experimentl diets supplemented with yon flour were modified from AIN-93G (Reeves et l. 1993; Tle 1) nd presented finl FOS onentrtions of 5 % (5 g/kg diet) nd 7 5 % (75 g/kg diet). The mounts of FOS nd solule sugrs (gluose, frutose, surose) present in the yon flour were sutrted from those of surose nd strh. The ontrol nd FOS diets were formulted in order to provide 7 5 g C/kg diet. The following ftors were used for energy lultions: 4, for rohydrtes nd proteins, 9 for lipids nd 1 for frutns (Roerfroid et l. 1993). Experimentl design All nimls were fed the sme ontrol diet for 5 d nd therefter either the ontrol or the FOS diets for 27 d. The food intke ws determined dily nd ody weight reorded every 2 d. Food nd deminerlized wter were offered d liitum. The feed effiieny ws determined s the weight gin/g food intke t the end of the experiment. Fees nd urine were olleted long three periods of 5 d (strting on the Tle 1. Composition (g/kg) of the experimentl diets Ingredients Control 5 % FOS 7 5 % FOS Csein* Fire Soyen oil l-cystine Coline itrtrte Vitmin mixture Minerl mixture Surose Corn strh Yon flour FOS, frutooligoshrides. * Csein protein ¼ % (N 6 25). AIN-93-VX vitmin mixture (Reeves et l. 1993). Modified from minerl mixture (AIN-93G-MX; Reeves et l. 1993) supplying (g/kg mixture): CCO 3, ; KH 2 PO 4, 196; NCl, 74; K 2 SO 4,46 6;K 3 C 6 H 5 O 7.H 2 O, 7 78; MgO, 24; ferri itrte, 6 6; ZnCO 3, 1 65; MnCO 3, 63; CuCO 3, 3; KIO 3, 1; N 2 SeO 4, 1 25; mmonium tetr-hydrted prmolydte, 795; N 2 SiO 3, 1 45; KCr(SO 4 ) 2.12H 2 O, 275; H 3 BO 3, 815; NF, 635; NiCO 3, 318; LiCl, 174; NH 4 VO 3, 66; surose, Downloded from IP ddress: , on 2 Sep 218 t 17:39:1, sujet to the Cmridge Core terms of use, ville t

3 778 A. R. Loo et l. 4th, 1th nd 16th dys of experiment), pooled ording to the olletion period nd stored t 22 8C. At the end of the experiment, the rts were fsted for 12 h efore eing killed. The nimls were nesthetized with 1:2 (v/v) mixture of ketmine (1 mg/kg) nd xylzine (25 mg/kg) nd their livers, spleens nd kidneys were removed, rinsed in sline solution ( 85 % NCl, w/v) nd weighed. The eum ws removed with its ontents, weighed, pled on ie (Lu et l. 2) nd ut open long the smll urvture. Aliquots of the ontents were frozen t 22 8C nd tissue smples were fixed in Bouin s regent (piri id formlin glil eti id, 75:2:15, y vol.) nd stored in 7 % ethnol. The hind lims were removed nd the ones were defleshed, weighed nd stored t 22 8C until nlysis. Chemil omposition of yon flour nd experimentl diets The following omponents were determined: moisture ontent, sh nd totl lipids (Instituto Adolfo Lutz, 1995); protein y miro-kjeldhl method (Assoition of Offiil Anlytil Chemists, 1995) (onversion ftor of 6 25) nd solule nd insolule DF y enzymti-grvimetri method, using 2-(4-morpholino)ethnesulphoni id Tris uffer (Lee et l. 1992). Frutns nd solule sugrs (gluose, frutose nd surose) were determined y n dpttion of the Hoeregs method (Hoeregs, 1997). In this method the smple is enzymtilly treted with lyophilized mylogluosidse (A-742; Sigm Chemil Co., St Louis, MO, USA) nd inulinse (Frutnse Mixture, Megzyme TM, Bry, Irelnd; t. E- FRMXLQ), nd the relesed sugrs re determined y nionexhnge hromtogrphy with pulsed mperometri detetion. The hydrolysis y mylogluosidse ws not performed due to the low strh ontents in the yon roots (Cpito, 21). C nd Mg were mesured y tomi sorption spetrophotometry (Polrized Zeemn AAS, Hithi Z-5; Hithi, Tokyo, Jpn), employing hollow thode lmp t nd 22 6 nm nd slits of 7 nd 1 3 nm, respetively, fter wet digestion (HNO 3 H 2 O 2, 5:1, v/v) nd ddition of 1 % (w/v) lnthnum s L 2 O 3. The working stndrd solutions were prepred y diluting CCl 2 (Titrisol; Merk) nd MgCl 2 (Tritisol; Merk, Drmstdt, Germny). Minerl lne Dry fees were milled nd the powdered smples, s well s urine smples, were utilized for C nd Mg nlyses. Apprent sorption nd lne were lulted y the following equtions: Apprent sorption ð%þ ¼1 ðingestion 2 fel exretionþ=ingestion; Minerl lne ðmmol=d or mmol=dþ¼ ingestion 2 ðfel exretion þ urinry exretionþ: Cel ph nd histology Cel ontents were entrifuged t 11 g in two 3 min yles nd the ph mesured with ph test strip (Merk) in the superntnt; moisture ws determined in n liquot of the remining el ontents. For histologil exmintion, the tissue fixed frgments were inluded in prffin nd pproximtely 5 mm-thik uts were otined nd stined with hemtoxylin nd eosin. Only rypts ut lengthwise were used to mesure the depth with the rypts divided into ritrry zones ording to the lines of n integrting oulr (Zeiss No. 2). These lines limited spes or zones whih enled us to lulte the numer of spes within eh rypt. Eh spe ws then tken s stndrd mesurement. At lest thirty rypts per niml were used. For the determintion of the numer of ifurting rypts, the rypts with n indenttion t the se or presenting longitudinl fission (one rypt mouth nd two ses) were onsidered (Mskens, 1978). The lultion ws rried out y determining the numer of the ifurting rypts per mirosopi field, onsidering the sme riteri used in the mesure of the depth of the rypts. To estimte the totl numer of rypts per mirosopi field, lso the oliquely setioned rypts were onsidered. At lest twenty mirosopi fields per niml were nlysed. Bone minerl ontent, density nd iomehnis C nd Mg onentrtions in previously dried tii nd femur (15 8C, 12 h) of the left lim were determined. BMD of the right femur nd tii ws mesured y dul-energy X-ry sorptiometry (Freits et l. 23), using pdexa Sre Densitometer nd the pdexa Sre softwre, version (Norlnd Medil Systems, Fort Atkinson, WI, USA), oth designed for smll nimls. The regionl differene ws estimted y evlution of the regionl BMD (25 % proximl, 5 % middle (midshft) nd 25 % distl of eh one). After nlyses, the ones were gin stored t 22 8C until iomehnil strength tests were rried out. The storge t 22 8C does not lter the mehnil properties of the ones (Sedlin & Hirsh, 1966). The mehnil properties were evluted in the mid-diphysel region of the right femurs using three-point ending test (Shimno et l. 22) nd loding ontinued to filure, through TA-TX2 texture nlyser oupled to the Texture Expert softwre, version 1.2 (Stle Miro Systems, Hslemere, UK). For the ssy, the ones were thwed t room temperture nd rehydrted in sline solution. The one length (mesured from the femur hed to the ondilus) nd dimeter (mesured in the mid-diphysel region) were mesured y liper. The entre of the ones ws frtured under the following onditions: 16 mm smple spe, 2 mm/ s plunger speed nd 25 kg lod rnge. Sttistil nlysis Results re expressed s mens with their stndrd devitions nd nlysed onsidering 5 % signifine level. The sttistil model used ws the ANOVA (Neter et l. 1996) under ompletely rossed plnning, where the tretments were defined y the omintion of period nd group ftors nd ll mesurements of period ftor repeted. As the effets etween group nd period ftor were not signifint (P. 5), new models without intertions were djusted. Downloded from IP ddress: , on 2 Sep 218 t 17:39:1, sujet to the Cmridge Core terms of use, ville t

4 Yon flour ffets one 779 In order to study the vriles mesured t the end of the experiment, ANOVA with two levels in the group ftor were rried out. All sttistil nlyses were rried out using SPSS for Windows, version 11.5 (SPSS, Chigo, IL, USA). Results Chemil omposition of yon flour nd experimentl diets Yon flour ontins FOS (55 3 %) s the min storge sugr nd lso fetures lower gluose, frutose nd surose ontents (8 9, 13 5 nd 13 4 %, respetively), s well s solule nd insolule DF (3 nd 7 %, respetively) (Tle 2). The gluose, frutose, surose nd frutns onentrtions in yon flour were used to lulte the mounts of ingredients in the experimentl diets. As onsiderle mounts of free sugrs were oserved in the yon flour, they were sutrted from the totl mount of surose (5 nd 7 5 % FOS groups) or strh (7 5 % FOS group) to e dded to the experimentl diets. Low ontents of C nd Mg were found in yon flour (Tle 2) nd thus the ddition of the flour did not signifintly lter the ontent of these minerls in the diets. Chemil nlysis of the diets showed C nd Mg onentrtions of 12 17, nd 12 7 mg C/kg, nd 48, 52 nd 59 mg Mg/kg, in the ontrol, 5 % FOS nd 7 5 % FOS diets, respetively. The energy vlues (kj/g) of the diets were 16 5, 15 4 nd 14 9, in the ontrol, 5 % FOS nd 7 5 % FOS diets, respetively. The nimls fed diets supplemented with yon flour showed no gstrointestinl disturnes. Effets on fel output nd el prmeters Tle 3 shows the results for dry weight nd wter ontent of fees olleted within three experimentl periods. A signifint inrese (P, 1) in the fees men dry weight ws oserved during the three experimentl periods in nimls fed yon flour, when ompred to the ontrol group, lthough the results hve not shown ny differenes etween the FOS groups. Yon flour-supplemented diet intke resulted in n inrese (P, 5) in the perentge of wter ontent in the fees reltive to the nimls fed the ontrol diet in the three predetermined periods long the experiment. Yon flour-fed nimls hd lower el wll nd el ontent weights (wet weight in g/1 g ody weight) thn those fed ontrol diet (P, 1). The inrese in the wet weight of el ontents ws ompnied y inreses in moisture nd residue weight otined fter entrifugtion of the el ontents (þ57 nd þ68 %, for the 5 nd 7 5 % FOS groups, respetively, P, 5) ompred to the ontrol group (Tle 3). No signifint differenes in the ph vlues were oserved in the superntnt of the el ontents mong the groups. Diet supplementtion with 7 5 % FOS signifintly inresed (P, 5) the numer nd depth of el rypts ompred to the ontrol group (Fig. 1). Furthermore, these effets were onomitnt with greter numer of ifurting rypts in nimls fed yon flour (5 nd 7 5 % FOS groups) when ompred to the ontrol group (Fig. 2). Body weight gin, feed intke nd effiieny, nd weight of orgns The onsumption of the yon flour-supplemented diets did not ffet the growth nd development (P. 1) of the nimls during the experimentl period (27 d), s the men feed effiienies were similr (Tle 3). Sttistilly signifint differenes (P, 5) in the totl feed intke nd in spleen, liver nd kidney reltive men weights (g/1 g ody weight) etween the experimentl groups were not oserved. Tle 2. Composition (g/1 g) of the yon flour* (Men vlues nd stndrd devitions) Components Men SD Ash Protein (N 6 25) Ether extrt 61 2 Insolule fire Solule fire Frutns Gluose Frutose Surose Clium (mg/g) 83 1 Mgnesium (mg/g) 62 9 * n 3 exept for insolule nd solule fire, n 4; frutns, gluose, frutose nd surose, n 6. Intestinl sorption nd lne of lium nd mgnesium The pprent C sorption in the yon flour groups ws sttistilly higher (P, 5) thn tht from the ontrol group in ll experimentl periods, nd this differene inresed from pproximtely 51 % in the first period to 56 % in the lst (Fig. 3(A)). The C lne of the FOS groups ws sttistilly different (P, 5) from tht of the ontrol group (Fig. 3(B)). No signifint differenes were oserved in the Mg pprent sorption mong the experimentl groups (Fig. 3(C)). However, the 7 5 % FOS group showed signifintly higher Mg lne when ompred to the ontrol group (Fig. 3(D)). Effets on one prmeters No signifint differenes were oserved in the dry weight, length nd dimeter of femur (Tle 4) mong the experimentl groups. On the other hnd, yon flour orresponding to 7 5 % FOS in the diet used sttistilly signifint inrese (P, 5) in C onentrtion in femur nd tii, when ompred to the ontrol group (Fig. 4). No signifint differenes were oserved in Mg onentrtions in femur nd tii etween the experimentl groups (117 (SD 23), 16 (SD 7) nd 19 (SD 8) mmol/g, in dry femur, nd 12 (SD 16), 17 (SD 11) nd 111 (SD 8) mmol/g, in dry tii, for the ontrol, 5 % FOS nd 7 5 % FOS groups, respetively). BMD ws evluted ex vivo using dul-energy X-ry sorptiometry. The nimls whih onsumed FOS presented no sttistilly signifint differene in BMD (P, 5) when ompred to Downloded from IP ddress: , on 2 Sep 218 t 17:39:1, sujet to the Cmridge Core terms of use, ville t

5 78 A. R. Loo et l. Tle 3. Body weight gin, food intke, fel output nd el prmeters in rts fed ontrol diet nd yon flour-supplemented diets* (Men vlues nd stndrd devitions, n 8) Dietry tretment Control 5 % FOS 7 5 % FOS Anlyses Men SD Men SD Men SD Body weight gin (g) Food intke (g/27 d) Food effiieny Fel output (dry g/5 d) First period (dys 4 8) Seond period (dys 1 14) Third period (dys 16 2) Fel wter ontent (%) First period (dys 4 8) Seond period (dys 1 14) Third period (dys 16 2) Cel wll (wet g/1 g rt) Cel ontents (wet g/1 g rt) Ceum ontents Superntnt ph Moisture (%) FOS, frutooligoshrides.,, Men vlues within row with unlike supersript letters were signifintly different (P, 5). * For detils of proedures, see p those from the ontrol group. Tle 4 presents results onerning the mehnil properties evluted in the midshft region of the femur. An inrese in ll studied prmeter vlues ws oserved (pek lod, yield lod, stiffness, resiliene nd sored energy) in the FOS groups in omprison to the ontrol group, ut only vlues regrding pek lod nd stiffness presented sttistilly signifint differenes (P, 5) etween the experimentl groups. Disussion The present results showed tht the onsumption of yon flour ontining FOS stimulted pprent C intestinl sorption s well s C nd Mg lne, leding to higher one C ontent nd greter mehnil resistne of the femur t the mid-diphysis, s evluted y the pek lod nd stiffness vriles, in omprison to the ontrol group. In ddition, n enlrgement of the eum ws oserved, s demonstrted y inreses in the numer nd depth of el rypts, s well s in the numer of ifurted rypts, suggesting n inrese in the sorptive surfe re of the eum nd proly ontriuting to minerl sorption. In this ontext, the positive effets of frutns (FOS nd inulin) onsumption on minerl sorption in the lrge intestine hve een rodly studied using different experimentl protools (Oht et l. 1994,, 1995, 1998; Kruger et l. 23; Zfr et l. 24; Rshk & Dniel, 25). However, to our knowledge, this is the first report out the effets of yon FOS on minerl (C nd Mg) sorption nd one C retention. The presene of food in the intestine is the most importnt determinnt for the stimultion of ell prolifertion, onsidering tht the lk of nutrients in the intestinl lumen (s seen in totl prenterl nutrition ses) or the ingestion of DF-free diets leds to trophy of the muos (Wong & Wright, 1999). This trophy n e redued y the presene of some type of DF in the diet (Goodld & Wright, 1983). Frutns shre some properties with DF suh s non-digestiility in the smll intestine nd fermentility in the lrge intestine (Shneemn, 1999) nd prtiulr ttention hs een given to their ility to stimulte the seletive growth of some teril speies, onsidered enefiil to the host (Roerfroid et l. 1998). Pedreshi et l. (23) demonstrted in vitro tht yon FOS hve the potentil to e fermented y ifidoteri nd ltoilli speies nd they lssified it s novel soure of preiotis. In the present study, feeding rts with yon flour ontining FOS indued signifint inrese in the el wll nd ontents (Tle 3), proly s result of the inrese in iomss due to greter fermenttion in the eum. Furthermore, this trophi effet hs een elieved to e medited y SCFA produed s result of the inrese in the metoli tivity of the intestinl miroflor (Rémésy et l. 1993; Cmpell et l. 1997). Among these SCFA, utyrte hs een reognized s the min soure of energy for the olonoytes, promoting the helth nd integrity of the el/ oloni muos (Le Bly et l. 1999). Feeding rts with 7 5 % FOS diet resulted in signifint inrese (P, 5) in the numer nd depth of el rypts (Fig. 1). Moreover, 5 % FOS in the diet ws enough to provide very high inidene of ifurting rypts in the eum (Fig. 2). Other studies lso hve shown hnge in the length nd density of rypts fter onsumption of fermentle rohydrtes (Rémésy et l. 1993; Howrd et l. 1995). MCullough et l. (1998) demonstrted tht fermentle fires inresed oth the numer of rypts per irumferene nd the numer of rnhed rypts in the proximl olon of rts. It hs een proposed tht inresed rypt size is the mjor stimulus for rypts to divide (Totfurno et l. 1987). Smll indenttions re oserved in the se of Downloded from IP ddress: , on 2 Sep 218 t 17:39:1, sujet to the Cmridge Core terms of use, ville t

6 Yon flour ffets one 781 (A) 12 No rypts per field Control 5% FOS 7 5% FOS (B) Crypt depth (ritrry units) Control 5% FOS 7 5% FOS Fig. 1. The photogrphs re histologil setions of el muos from rts fed ontrol diet nd yon flour-supplemented diets (5 nd 7 5 % frutooligoshrides (FOS)) (hemtoxylin nd eosin; sle rs ¼ 14 mm). (A), Numer of rypts per mirosopi field; (B), rypt depth (ritrry units). For detils of proedures, see p Vlues re mens with their stndrd devitions depited y vertil rs (n 8)., Men vlues with unlike supersript letters were signifintly different (P, 5). rypt, whih then send longitudinlly nd proeed until there re two seprte rypts (Totfurno et l. 1987), proess thought to e relted to expnsion of rypt stem ell popultion (Bjerknes & Cheng, 25). This proess is inresed in postntl development nd during reovery of the intestine from injury (Mskens, 1978; Cheng et l. 2). In ddition, the prodution of new rypts y fission n provide n lterntive or dditionl mens of inresing tissue mss (MCullough et l. 1998). Bifurting rypt per field (%) % FOS Control 5% FOS7 5% FOS Fig. 2. The photogrph is histologil setion showing rypt in the ifurtion proess (rrow) (hemtoxylin nd eosin; sle r ¼ 14 mm). The grph shows ifurting rypts per mirosopi field (%) in the eum from rts fed ontrol diet nd yon flour-supplemented diets (5 nd 7 5 % frutooligoshrides (FOS)). For detils of proedures, see p Vlues re mens with their stndrd devitions depited y vertil rs (n 8)., Men vlues with unlike supersript letters were signifintly different (P, 5). Downloded from IP ddress: , on 2 Sep 218 t 17:39:1, sujet to the Cmridge Core terms of use, ville t

7 782 A. R. Loo et l. (A) C sorption (%) 1st Period 2nd Period 3rd Period (C) Mg sorption (%) st Period 2nd Period 3rd Period (B) C lne (mmol/d) (D) Mg lne (µmol/d) st Period 2nd Period 3rd Period 1st Period 2nd Period 3rd Period Fig. 3. Clium (A, B) nd mgnesium (C, D) intestinl sorption nd lne of rts fed ontrol diet (A) nd yon flour-supplemented diets (B, 5 % frutooligoshrides (FOS); B, 7 5 % FOS). For detils of proedures, see p Vlues re mens with their stndrd devitions depited y vertil rs (n 8).,, Men vlues with unlike supersript letters were signifintly different (P, 5). Cel ph, whih is onsidered n indiret result of SCFA prodution, ws not ffeted y yon flour onsumption in the present experimentl onditions. Furthermore, the vlues were higher thn those reported in the literture s response to teril fermenttion of frutns nd other fermentle rohydrtes (Levrt et l. 1991; Cmpell et l. 1997; Lu et l. 2). Wolf et l. (1998), fter feeding growing rts with different dietry FOS onentrtions for 27 d, ttriuted the high el ph vlues t the end of their experiment to the high uffering ility of the AIN-93G diet. In the present study, nimls were deprived of food for 12 h efore eing killed. Aording to Le Bly et l. (23), SCFA my vry onsiderly depending on the period etween the lst mel nd the moment of smple olletion, whih might thus explin the more elevted ph vlues oserved in the present study. Finlly, entrifugtion of eum ontents my hve Tle 4. Biomehnil properties of femur in rts fed ontrol diet nd yon flour-supplemented diets* (Men vlues nd stndrd devitions, n 8) Dietry tretment Control 5 % FOS 7 5 % FOS Anlyses Men SD Men SD Men SD Wet weight (g) Length (mm) Dimeter (mm) Pek lod (N) Yield lod (N) Stiffness (N/mm) Resiliene (Nmm) Asored energy (Nmm) FOS, frutooligoshrides., Men vlues within row with unlike supersript letters were signifintly different (P, 5). * For detils of proedures, see p Downloded from IP ddress: , on 2 Sep 218 t 17:39:1, sujet to the Cmridge Core terms of use, ville t

8 Yon flour ffets one 783 Bone C (mmol/g dry weight) Femur ontriuted to the voltiliztion of SCFA present in the smples, thus ffeting the ph of the solule phse fter proessing (Lu et l. 2). The present results showed tht the intke of yon FOS inresed C pprent intestinl sorption nd lne in omprison to the ontrol group, s evluted in three periods of 5 d eh (Fig. 3). Studies hve demonstrted the effets of fermentle rohydrtes on C sorption, lthough some onditions like short-term or long-term onsumption of these rohydrtes, minerl nd rohydrte levels or their rtio in the diet my ffet their ility to promote minerl sorption. Levrt et l. (1991) evluted the effets of different inulin onentrtions (5, 1 nd 2 % in the diets) on pprent C sorption in rts nd oserved dose response pttern. In nother study, Coudry et l. (24) evluted the impt of dietry C levels ( 25, 5 or 75 %) on the stimultory effet of inulin on C sorption nd demonstrted tht C sorption effiieny ws inversely orrelted with dietry C intkes nd tht the highest effet of inulin ws oserved in the group reeiving the lowest dietry C level. Contrrily to the effet on C sorption, yon flour onsumption did not ffet Mg sorption, despite the signifint inrese in Mg lne oserved in the 7 5 % FOS group. In this respet, studies hve een oserved tht n elevted C ingestion my negtively ffet Mg sorption (Couzy et l. 1993). Miur et l. (1999) oserved signifint (P, 5) derese in the Mg pprent sorption nd serum nd femur onentrtions in growing rts fter ingestion of 1 5 % C diet. In the present study, Mg urinry exretion in the ontrol nimls ws numerilly higher (dt not shown) thn tht in the rts onsuming 7 5 % FOS diet, whih t lst led to higher Mg retention in the FOS group. Previous studies hve shown tht the onsumption of frutns not only stimultes minerl sorption in the lrge intestine, ut lso inreses one minerl retention (Kruger et l. 23; Zfr et l. 24; Nzeusseu et l. 26). The present results showed tht the onsumption of 7 5 % yon FOS used signifint inrese (P, 5) in C onentrtion Tii Fig. 4. Clium onentrtion in femur nd tii of rts fed ontrol diet (A) nd yon flour-supplemented diets (B, 5 % frutooligoshrides (FOS); B, 7 5 % FOS). For detils of proedures, see p Vlues re mens with their stndrd devitions depited y vertil rs (n 8)., Men vlues with unlike supersript letters were signifintly different (P, 5). oth in femur nd in tii (Fig. 4), s well s higher BMD when ompred to the ones from the ontrol group, lthough BMD vlues hve not rehed sttistil signifine. Using growing helthy rts, Kruger et l. (23) oserved signifint inrese (P, 5) in femur BMD fter inulin ingestion (DP. 23) for 4 weeks, lthough this effet hd not een oserved fter the onsumption of oligofrutose (DP etween 2 nd 8). Whole-ody BMD ws ltered only fter 22 weeks in helthy growing rts fed diets supplemented with 5 % nd 1 % inulin nd 2 %, 5 % or 1 % C (Roerfroid et l. 22). In reent study, Nzeusseu et l. (26) verified greter inrese in the re nd BMD of the nellous one of the proximl tii nd verter in growing rts fed inulin thn in those fed oligofrutose fter 3 months, s ssessed y peripherl quntittive omputerized tomogrphy mesurements. The mehnisms involved in the modultory effets of frutns on one metolism hve een disussed. Zfr et l. (24) oserved tht the signifint inrese in C intestinl sorption in rts onsuming frutns deresed the one turnover through suppression of osteolst resorption. In growing rts onsuming inulin for 4 weeks, Kruger et l. (23) oserved signifint derese in urinry exretion of frgments of type 1 ollgen, iohemil mrker for one resorption. The nti-resorptive effet of frutns ws lso onfirmed y Nzeusseu et l. (26), who demonstrted signifint derese in serum levels of C-telopeptide. The resistne of omposite mteril like one my e evluted through the onstrution of lod deformtion urve, where most of the lod pplied to the one is supported y the minerl phse (Shimno et l. 22). Bone reking strength mesures re simple to perform nd n diretly determine lods required to use frture (Shimno et l. 22). In the present study, three-point ending test ws utilized for evluting some mehnil properties in the mid-diphysel region of the femur, region essentilly omprised of ortil one. A trend towrds n inrese of ll properties studied ws deteted in yon FOS groups, lthough only the 7 5 % FOS group differed from the ontrol group regrding the pek lod nd stiffness vriles, providing positive influene of yon flour on resistene nd rigidity of femorl diphyses. Nevertheless, it should e kept in mind tht one strength lso depends on the rhiteturl disposition of one mteril (one geometry prmeters, e.g. rosssetionl re, moment of inerti, ortil thikness) nd other minerliztion unrelted ftors suh s rystl rrngement nd size, the degree of ollgen ross-linking, nd the mount nd distriution of tissue mirodmge (Burr, 22). In this view, the previous results of our lortory showed n inrese in the pek nd yield lod of femur in rts fed 5 % FOS-supplemented diets, suggesting n influene of FOS on orgni omponent (Loo et l. 26). In summry, the present study showed stimultory effet of yon FOS on C intestinl sorption nd lne, resulting in higher one C ontent nd stronger one struturl properties in the femorl midshft of growing rts. Moreover, we speulte whether the enlrgement of the el wll used y ifurting rypts might hve ontriuted to the inresed minerl sorption in the nimls fed frutns. This spet, of ourse, needs further reserh. More urte studies expnding the present findings on one rhiteture nd intestinl physiology open new perspetives for the use of yon flour s n ingredient in funtionl foods, s well s other frutn-ontining rops. Downloded from IP ddress: , on 2 Sep 218 t 17:39:1, sujet to the Cmridge Core terms of use, ville t

9 784 A. R. Loo et l. Clinil trils should e performed in order to verify the effet of yon onsumption on mn. Aknowledgements Prt of this work hs een presented in strt form: FASEB Journl (24) 18, A15. We thnk Anit Hgy (in memorim) for supplying of the yon tuerous roots; Dr Ceíli Gouvei for her ollortion in the BMD nlyses; Dr José Alfredo Gomes Arês, Dr Luiz Antonio Gioielli nd Dr Chiu Chi Ming for their ollortion in the one iomehnil ssys; Fundção de Ampro à Pesquis do Estdo de São Pulo (reserh projet 2/ ) for supporting the reserh, nd Coordenção de Aperfeiçomento de Pessol de Nível Superior for the mster s fellowship wrded to Alexndre Rodrigues Loo. We lso thnk Álvro Augusto Feitos Pereir for the review of the mnusript. Referenes Assoition of Offiil Anlytil Chemists (1995) Offiil Methods of Anlysis, 16th ed. Wshington, DC: AOAC. Bjerknes M & Cheng H (25) Gstrointestinl stem ells. II. Intestinl stem ells. Am J Physiol 289, G381 G387. 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