Fermentation by gut microbiota cultured in a simulator of the human intestinal microbial ecosystem is improved by probiotic Enterococcus

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1 Functionl Foods in Helth nd Disese 2011; 10: Pge 389 of 402 Reserch Open Access Fermenttion y gut microiot cultured in simultor of the humn intestinl microil ecosystem is improved y proiotic Enterococcus fecium CRL 183 Kti Sivieri 1, Fernnd Binchi 1, Mri A. Tllrico 2, nd Elizeu A. Rossi 1 1 Deprtment of Food nd Nutrition, Lortory Study of Proiotics nd Preiotics, Fculty of Phrmceuticl Sciences of Arrqur, UNESP-Pulist Stte University, Arrqur, São Pulo,Brzil; 2 School of Engineering of São Crlos, University of São Pulo, So Pulo, Brzil Corresponding uthor: Sivieri, Kti, PhD, Professor, Deprtment of Food nd Nutrition, Arrqur/Ju, Km 1, Arrqur, São Pulo,Brzil Sumission dte: Septemer 05, 2011; Acceptnce dte: Octoer 07, 2011; Puliction dte: Octoer 08, 2011 Astrct Bckground: Enterococci re used in lrge numer of diry products, such s strter cultures in food supplements nd in foods considered functionl. In vitro gut fermenttion models present n unmtched opportunity of performing studies frequently llenged in humns nd nimls owing to ethicl concerns. A dynmic model of the humn intestinl microil ecosystem (SHIME) ws designed to etter simulte conditions intestinl microiot. Methods: The SHIME model ws used to study the effect of Enterococuus fecium CRL 183 on the fermenttion pttern of the colon microiot. Initilly, n inoculum prepred from humn feces ws introduced into the rector vessels nd stilized over 2 wk using culture medium. This stiliztion period ws followed y 2-wk control period during which the microiot were monitored. The microiot were then sujected to 4-wk tretment period y dding 10 8 CFU/mL of the Enterococcus fecium CRL 183 to vessel one (the stomch comprtment). Results: The ddition resulted into n overll increse of cteril mrker popultions (Enteroctericee, Lctocillus spp., Bifidocterium spp. nd Clostridium spp.), with significnt increse of the Lctocillus sp. nd Bifidocterium sp popultions. The shortchin ftty cid (SCFA) concentrtion incresed during the supplementtion period; this ws due minly to significnt increse in the levels of cetic, utyric nd propionic cids. Ammonium concentrtions incresed during the supplementtion period. Conclusions: Results showed tht the mjor effect of E. fecium CRL 183 ws found in the scendnt nd trnsverse colon.

2 Functionl Foods in Helth nd Disese 2011; 10: Pge 390 of 402 Key words: Gut microiot, Enterococcus, Gstrointestinl resource mngement, Simultor of Humn Intestinl Microil Ecosystem (SHIME) BACKGROUND: The gstrointestinl (GI) trct is colonized y vst community of symionts nd commensls tht hror complex nd diverse ecology of microorgnisms comprised of species with levels reching CFU/g of intestinl contents in the lrge intestine [1]. These microes hve fr-reching implictions on helth in tht they ffect immunity nd digestion of nutrients. Microil interctions contriute to the homeostsis of the gut cteril microiot nd destiliztion of this microorgnism ecosystem results in vrious GI disorders [2]. It hs een suggested tht proiotics help to mintin the GI equilirium of the indigenous microiot nd enefit the host s helth. They re thus defined s live microorgnisms, tht when dministered in n dequte mounts, confer helth enefit on the host [3]. Proiotic strins re considered s sfe nd non-pthogenic [4]. Enterococci re used in lrge numer of diry products, such s strter cultures in food supplements nd in foods considered functionl [5]. Currently, mny reserchers seek microorgnisms tht hve proiotic properties, s is the cse of, for exmple, Enterococcus fecium CRL 183. Our reserch group verified tht E. fecium CRL 183 hs the cpcity to survive in nd colonize the gstrointestinl trct of rts [6], one of the prerequisites for eing considered proiotic [7], since the viility of lctic cteri cn e lost on exposure to gstric cid nd to ile slts [8]. Our group lso oserved tht the consumption of 200 ml/dy of soymilk fermented with E. fecium CRL 183 nd Lctocillus helveticus susp. jugurti 416 y normocholesterolemic dult men, for period of 6 weeks, reduced the levels of totl cholesterol nd of the LDL frction nd led to n increse of out 10% in HDL-C levels [9]. Others eneficil helth effects, such s prtil inhiition of rest cncer [10], prevention of cncer colon [11] nd osteoporosis [12] hve lso een chieved with E. fecium CRL 183. However, the influence of E. fecium consumption on humn gut microil fermenttion hs een little investigted to dte. For tht reson, the im of our study ws to investigte the effects of E. fecium CRL 183 on the fermenttive cpcity of the simulted microiot of the colon. METHODS: Preprtion of the E. fecium CRL 183 cells: At weekly intervls, pure culture of E. fecium CRL 183 ws inoculted into MRS Agr culture medium (Acumedi, Bltimore). The cteri in the log phse ws centrifuged (4000 x g, 10min., 4ºC) nd wshed with sterile peptone wter. The E. fecium cells were kept t the concentrtion of 10 8 CFU/mL in sterile peptone wter until use [6]. Long-term SHIME run: The SHIME is simultor of the humn intestinl microil ecosystem [13,14] in which the environmentl conditions (ph, residence time, inoculum, nd temperture) re controlled to resemle those of in vivo experiments. A SHIME system consists of five doule-jcketed vessels, respectively simulting the stomch, the smll intestine, nd the scending, trnsverse nd descending colon, with totl retention time of 72 h (Figure 1). Rector setup

3 Functionl Foods in Helth nd Disese 2011; 10: Pge 391 of 402 nd the composition of the liquid feed (Tle 1), which entered the system 3 times per dy were previously descried y Possemiers et l. [15]. Figure 1. Schemtic representtion of the Simultor of the Humn Intestinl Microil Ecosystem (SHIME) The three colon vessels of the SHIME rector were inoculted with cteri from fecl smple of helthy 22-yer-old dult femle volunteer with no history of ntiiotic tretment 6 months prior to the study. Aliquots (10 g) of freshly fecl smples were diluted nd homogenized with 100 ml sterilized phosphte uffer (0.1 mol/l, ph 7), contining 1 g/l sodium thioglycolte s reducing gent. After removl of the prticulte mteril y centrifugtion, the superntnts were pooled nd 50 ml ws introduced into ech of the colon simultion vessels. Tle 1. Ingredients (g) for one liter of the sl feed utilized in Shime rector Ingredient Quntity necessry for 1L Arinoglctn 1.0 Pectin 2.0 Xiln 1.0 Potto strch 3.0 Glucose 0.4 Yest extrct 3.0 Peptone 1.0 Mucin 4.0 Cystein Sterile distilled wter The microil inoculum ws stilized over period of 2 weeks on crohydrte-sed medium nd llowed to dpt to the specific environmentl conditions of the scending, trnsverse nd descending colon in terms of ph rnge, retention time nd ville cron sources [15]. An initil stiliztion period of two weeks fter inocultion ws pplied to llow the intestinl cteri to dpt to the environmentl conditions present in the colon vessels nd to form stle microil community representtive of the one present in the gstrointestinl trct [20]. Upon stiliztion, the SHIME run included 2 weeks of sl period (to quntify ll stedy-stte cteril prmeters which were used s strting point to evlute the effect of specific tretment), 4 weeks of tretment period in which 3mL of 10 8

4 Functionl Foods in Helth nd Disese 2011; 10: Pge 392 of 402 CFU/mL of E. fecium CRL 183 were dded once per dy to the stomch comprtment. Finlly, 2-week wshout period without E. fecium ddition. Microiologicl nlysis At weekly intervls, throughout the entire experimentl period, (sl, tretment nd wshout), 5 ml- smples were collected from the rectors for microiologicl exmintions. Anlysis of the composition of the intestinl microiot ws sed on the enumertion of totl eroic nd neroic cteri, Enterococcus ssp., Lctocillus ssp., Bifidocterium ssp., Enterocteri nd, Clostridium ssp. One ml of smple tken from ech rector ws suspended into 99 ml peptone wter. Seril dilutions were prepred nd inoculted into selective culture medi: totl eroic nd neroic counts: - Stndrd Methods Agr (Acumedi, USA; 37ºC/48h); Enterococcus spp.: - KF Streptococcus Agr (Acumedi, USA; 37ºC/48h) [16]; Lctocillus spp.:- MRS Agr (Merck, Germny; 37ºC/48h, under neroiosis); Bifidocterium spp.:- Bifidocterium formulted medium BIM-25 (supplier, 37ºC/72h, under neroiosis) [17], Enterocteri:- McConkey Agr (Acumedi, USA; 37ºC/48h) nd Clostridium spp.: RCA Agr (Difco, Frnce; 37ºC/48h, under neroiosis) [18]. Anlysis of short-chin ftty cid nd mmonium Once week, throughout the entire experimentl period (sl, tretment nd wshout), smples were collected from the rectors for nlysis of short-chin ftty cids (SCFA) nd mmonium. The nlysis ws crried in triplicte. Every week, the levels of short-chin ftty cids (SCFA) were determined from smples collected from the rectors nd frozen to -20ºC. The SCFA were extrcted with diethyl ether nd determined using gs chromtogrph equipped with flme-ioniztion gs detector, cpillry split/splitless injector nd n HP-INNOWAX column with 30 m x 0.25 mm x 0.25 m inlet (Shimdzu GC2010), using hydrogen s crrier gs t flow rte of 1.56 ml/min. The tempertures of the column, injector nd detector were 170, 250 nd 280 ºC, respectively [19, 20]. The mmoni content ws determined using selective ion meter (710A model, Orion) coupled to n mmoni selective-ion electrode (Orion 95-12). The pprtus ws clirted using 0.1M stndrd mmonium chloride solutions, t the concentrtions of 10, 100 nd 1000 mg/l mmoni. Ech 25 ml of smple ws dded with 0.5 ml ISA solution ( ph-djusting Ionic Strength Adjuster (Orion) ph-djusting nd n ionic force solution). All mesurements were tken t 25 C [21]. Sttisticl nlysis Significnce of ll results ws investigted using the sttisticl softwre Sigm Stt 5.0. with one-wy ANOVA, nd individul mens were compred using the Tukey's test. RESULTS AND DISCUSSION: Microiologicl evlution It is estimted tht the gstrointestinl microiot hrors round cteri. This microiot undergoes oth qulittive nd quntittive chnges depending on the locle of coloniztion. The huge complexity of this microiot, which re often unculturle microorgnisms (30 to 70%) in culture medi, in ddition to eing locted in difficult-to-

5 Functionl Foods in Helth nd Disese 2011; 10: Pge 393 of 402 ccess res of the digestive trct, which would require invsive methods to collect them, re the limiting fctors for more precise nlysis [22]. An investigtive lterntive is the use of continuous or semi-continuous models simulting the lrge intestine. The continuous models ws vlidted sed on the intestinl contents of sudden deth victims [23]. Among the dvntges of this model re the ese-ofuse, the possiility to use rdioctive sustnces nd the low cost [24]. Tle 2 shows the microiologicl counts of the flsks tht simulted the scendnt, trnsverse nd descendnt colon of the SHIME rector. Using selective growth medi, the microiologicl nlyses reveled the influence of the dministrtion of E. fecium CRL 183 on the composition of the intestinl microil community. With regrd to the enterocteril popultion, no quntittive chnge ws oserved during the tretment period. However, Bedni et l., [25] oserved significnt increse in the numers of these microorgnisms in the feces of rts tht hd consumed pure cells of E. fecium CRL 183 during 30 dys. Tle 2. Averge plte count mesurements (±SEM), expressed in log CFU/ ml, for the different microil groups, SHIME comprtments nd periods Bcteril groups Colon scendns Colon trnsversum Colon descendns Bsl Enterococcus spp A ± A ± A ± 0.03 Enterocteri 7.30 A ± A ± A ±0.08 Lctocillus spp A ± A ± A ± 0.09 Bifidocterium spp A ± A ± A ± 0.03 Clostridium spp A ± A ± A ± 0.02 Totl eroes 7.63 A ± A ± A ± 0.01 Aneroes fculttive 6.68 A ± A ± A ± 0.0 Tretment Enterococcus spp B ± B ± A ± 0.54 Enterocteri 7.00 A ± A ± A ± 0.93 Lctocillus spp B ± B ± A ± 0.54 Bifidocterium spp B ± B ± A ± 0.66 Clostridium spp A ± A ± A ± 1.43 Totl eroes 8.45 A ± A ± A ± 0.07 Aneroes fculttive 8.82 B ± B ± B ± 0.56 Wshout Enterococcus spp AB ± A ± B ± 0.31 Enterocteri 5.37 B ± A ± B ± 0.25 Lctocillus spp B ± B ± A ± 0.01 Bifidocterium spp A ± CB ± B ± 0.09 Clostridium spp A ± A ± A ± 0.50 Totl eroes 7.86 A ± A ± A ± 0.08 Aneroes fculttive 8.08 AB ± AB ± AC ± 0.02 Different letters indicte significntly different results (P<0.05) in sme microil group nd sme comprtment (colon scendns, trnsversum or descendns).

6 Functionl Foods in Helth nd Disese 2011; 10: Pge 394 of 402 In previous studies conducted y our reserch group [26], no quntittive chnge ws oserved in the microiot of Enterococcus spp contined in the feces of rts tht hd een dily fed with E. fecium CRL 183 during 30 dys. However, in the SHIME rector, during the tretment period, sttisticlly significnt increse in the Enterococcus ssp counts occurred in the scendnt (1 log cycle) nd trnsverse (2 log cycles) colons. Proly, E. fecium CRL 183 hs greter cpcity to colonize nd dhere to the scending nd trnsversl regions of the colon. According to Jin et l [27], E. fecium occupies inding sites in the mucosl cells of the scendnt colon, which llow their dherence. The concentrtion of the Lctocillus ssp popultion incresed significntly y two logrithmic cycles in the scendnt colon nd y three log cycles in the trnsverse colon during the tretment phse. However, in the descendnt colon, there ws no sttisticlly significnt ltertion during the sme phse. According to Mrteu [28], the colon is the primry microil coloniztion site, region consisting of different niches nd ecosystems. The ph in the scending colon is out 5.6 to 5.9, vlue tht fvors the growth of Lctocillus ssp. According to Coudeyrs nd Forestier [29], the scendnt nd trnsverse regions of the colon hror microiot tht is very similr to tht of the stomch, with predominnce of fculttive eroes nd neroes. The dominnt genus in this region is Streptococcus ssp, ut Lctocillus ssp. nd Enterococcus ssp lso eing found here, oth of which re species tht re generlly present in the intestinl lumen contents. The results show tht the dministrtion of E. fecium stimulted the growth of Lctocillus ssp in the scendnt nd trnsverse colons. In the descendnt colon, the popultion of ifidocteri ccounts for out 3 to 5% of the totl microiot in this region [29]. Over the sl period, higher count ws oserved (10 8 CFU/mL) in the descending colon, s compred to the scendnt nd trnsverse regions (10 5 UFC/mL). However, the tretment with E. fecium stimulted the growth of the popultion of ifidocteri only in the scending nd trnsverse colon regions. Bedni et l., [25] oserved tht the nimls tht received suspension of pure E. fecium CRL 183 culture presented n increse in the fecl ifidocteri popultion in the feces. As for the popultion of Clostridium spp., there ws no sttisticl significnt difference during the tretment period in the three regions of the colon evluted. Bcteri elonging to this genus my e hrmful due to their metolic ctivity nd the pthogenic chrcter of some species [30]. The species elonging to the Clostridium genus my e involved in inflmmtory processes of intestinl diseses [31]. Bedni et l, [25] lso oserved tht there ws no ltertion in the fecl popultion of Clostridium ssp in rts tht consumed cultures of E. fecium CRL 183. With regrd to totl eroes, there ws no sttisticlly significnt chnge in the popultion of these microorgnisms during the tretment period. As for the fculttive eroes, significnt increse ws noted in the scendnt, trnsverse nd descendnt colons. According to Coudeyrs nd Forestier [29], one of the min differences etween the microiot found in the feces nd tht in the colon is relted to the fculttive neroes, which re undnt in the colon nd prcticlly sent from the feces. A comprison etween the sl, tretment nd wshout periods llows to stte tht, in generl mnner, ll the microil groups evluted hd their concentrtions reduced in the post-tretment period. According to Doré nd Corthier [32], the dominnt intestinl microiot is resistnt to modifictions. The dministrtions of proiotics or preiotics my temporrily chnge intestinl homeostsis. Furthermore, it my e stted tht E. fecium CRL

7 Functionl Foods in Helth nd Disese 2011; 10: Pge 395 of stimulte the growth of some microil groups, such s; Lctocillus ssp., nd Bifidocterium ssp in the scendnt nd trnsverse regions of the colon, exerting little influence in the descending region. Ammonium concentrtion The intestinl microiot is complex ecosystem composed of interdependent cteri. Certin cteri stilize themselves solely s function of metolites produced y other cteri, nd together they trnsform complex polymers into simple molecules [29]. The finl metolites from the digestion of glycides y the intestinl cteri re short-chin ftty cids, H 2 nd CO 2. On the other hnd, the degrdtion products of proteins re short-chin ftty cids, rnched ftty cids, phenolic derivtives, indolic derivtives, polymines nd mmoni. The deleterious metolites re mines nd mmoni. These metolites my e either sored y the ody or excreted through urine [28]. Studies hve demonstrted tht high concentrtions of mmoni ct s tumor-promoting gents in the colon, since they re toxic to the epithelil cells of the intestine [22]. Figure 2 shows the concentrtion of mmonium ion in the sl, tretment nd wshout phses. Figure 2. Averge mmonium ion production (ppm) in shime run, during sl, tretment nd wshout period. Sttisticlly significnt differences mong the smples were investigted with one-wy ANOVA (smples with the sme letter on the top of the r re not sttisticlly different, P<0.05). During the tretment period with E. fecium, the concentrtion vlues of concentrtion of mmonium ion incresed significntly in ll the regions investigted (Figure 2). Similr results were oserved y Bedni et l [21], who noted significnt increse in the concentrtion of mmoni in tht hd een fed dily doses of E. fecium CRL 183 cells. Pomessier et l., [15] nd Pyne et l., [33] lso oserved n increse in the mmoni content in n experiment utilizing dynmic simultor of the humn microil ecosystem. Urese is expressed y mny cteri nd medited y the hydrolysis of ure into mmoni, which serves source of cteril nitrogen. According to Lukoová nd Kuncová

8 Functionl Foods in Helth nd Disese 2011; 10: Pge 396 of 402 [34], some strins of E. fecium hve urese ctivity, which would explin the increse in the concentrtion of mmoni during the dministrtion of E. fecium cells. Fermenttion cpcity One of the most importnt fctors for the persistence of the intestinl microiot is the diet, which does not only provides the host with nutrients, ut lso the intestinl microorgnisms [18]. In this sense, the crohydrtes tht re not digested in the colon re metolized y microorgnisms into SCFA, prticulrly cette, utyrte nd propionte. The formtion of short-chin ftty cids is of gret importnce, since they re sources of energy nd serve s microil sustrte, in ddition to eing relted to severl orgnic, locl nd systemic effects [35]. The production of SCFA depends on the sustrtes ville nd the microorgnisms present in the gstrointestinl trct [36]. Figure 3 depicts the production of cette during the periods of sl, tretment nd wshout in the Shime vessels. During tretment with E. fecium, significnt increse occurred in the production of cette in ll the rectors nlyzed, however, the gretest concentrtion of this cid occurred in vessel one, which simultes the scending region of the colon. In the wshout period, the levels of cette diminish, however, only in the trnsverse colon these levels differ sttisticlly from the sl period. A significnt increse in the concentrtion of utyrte ws oserved in the vessels simulting the trnsverse nd descendnt colon (Figure 4). Butyrte is considered the preferred fuel of the epithelil cells of the colon, which derive 70% of their energy from the oxidtion of this sustrte. Butyrte lso reduces the expression of proinflmmtory cytokines of tumor necrosis fctor-α (TNF-α), TNF-β, interleukine-6 (IL-6) nd IL-1β through ctivtion of the nucler growth inhiiting fctor kb (NF-kB) (23). In ddition, it hs een proposed tht utyrte reduces the risk of colon cncer due to is ility to inhiit the genotoxic cpcity of nitrosmines nd of hydrogen peroxide, s well s to induce different levels of poptosis, differentition nd cesstion of the cellulr cycle of colon cncer in niml models [36]. Figur 3. Averge SFCA production (cette) during the SHIME run, in the sl, tretment nd wshout period, respectively. Sttisticlly significnt differences mong the smples were

9 Functionl Foods in Helth nd Disese 2011; 10: Pge 397 of 402 investigted with one-wy ANOVA (smples with the sme letter on the top of the r re not sttisticlly different, P<0.05) Figur 4. Averge SFCA production (utyrte) during the SHIME run, in the sl, tretment nd wshout period, respectively. Sttisticlly significnt differences mong the smples were investigted with one-wy ANOVA (smples with the sme letter on the top of the r re not sttisticlly different, P<0.05) Figure 5. Averge SFCA production (propionte) during the SHIME run, in the sl, tretment nd wshout period, respectively. Sttisticlly significnt differences mong the smples were investigted with one-wy ANOVA (smples with the sme letter on the top of the r re not sttisticlly different, P<0.05)

10 Functionl Foods in Helth nd Disese 2011; 10: Pge 398 of 402 Butyrte hs een studied in clinicl pplictions, prticulrly inflmmtory owel diseses of the colon. While ville dt re not entirely conclusive, this sustnce ppers to hve useful therpeutic role complementry to tht of stndrd drugs. A ody of experimentl dt lso suggests tht this short-chin ftty cid my exert preventive ction ginst colorectl cncer, ut for the moment this is still hypothesis tht remins to e verified. More generlly, utyrte hs een shown to e useful in certin types of dirrhe, prticulrly chronic forms, y promoting sorption of wter nd electrolytes [37]. Previous studies conducted using n niml model llowed to oserve tht the consumption of pure cells of E. fecium CRL 183 inhiited the development of colon cncer [11]. Within this context, one my ssume tht this inhiition my e connected with the production of utyrte oserved in in vitro experiments. With regrd to propionte, significnt increse ws oserved in the concentrtions in the vessels 2 nd 3, tht simulte the trnsverse nd descendnt colon during the tretment with E. fecium. In the wshout period, reduction in the concentrtion of this cid ws found to hve occurred, however only in the descendnt colon this vrition ws sttisticlly significnt (Figure 5). c Figure 6. Averge SFCA production during the SHIME run in the tretment period. Sttisticlly significnt differences mong the smples were investigted with one-wy ANOVA (smples with the sme letter on the top of the r re not sttisticlly different, P<0.05) Figure 6 refers to the production of SCFA (cette, propionte nd utyrte) during the tretment phse in the rectors tht simulte the scendnt, trnsverse nd descendnt colon. The cid with the gretest production ws cette. Compring the three comprtments of the colon, it is oserved tht there ws significnt difference etween the scendnt colon nd the trnsverse nd descending colons for this cid. As for the other cids investigted (utyrte nd propionte) there were no significnt differences in the concentrtions etween the regions of the colon. The sme pttern ws oserved in the wshout period, tht is, upon

11 Functionl Foods in Helth nd Disese 2011; 10: Pge 399 of 402 cesstion of the tretment with E. fecium, the production of SCFA diminished, ut the tendency etween the regions of the colon remined unchnged (Figure 7). Similr results were oserved y Vn de Wiele et l., [19] in experiments using the Shime rector. c Figur 7. Averge SFCA production during the SHIME run in the wshout period. Sttisticlly significnt differences mong the smples were investigted with one-wy ANOVA (smples with the sme letter on the top of the r re not sttisticlly different, P<0.05) The production of SCFA in the tretment period my e explined y the increse in the concentrtion of lctocilli nd ifidocteri oserved over the sme period of time (Tle 2), which use the rekdown of the strch present in the sl medium with the consequent production of SCFA. According to Vn de Wiele et l., [20] the increse in the synthesis of ftty cids cretes more cid intestinl environment, which is importnt for the coloniztion resistnce ginst potentilly pthogenic microorgnisms. On the other hnd, the SCFA re importnt sources for the colonocytes, in ddition to stimulting the sorption of wter nd sodium nd modulting intestinl motility [38]. This study indictes tht the consumption of E. fecium my influence the gut microiot in eneficil wy. The concentrtion of SCFA incresed, wheres concentrtion of the hrmful putrefctive metolite (NH 3 ) ws lso ugmented. Apprently, E. fecium hd greter influence on the gut microiot of the scendnt nd trnsversl colon. Limittions ssocited with in vitro systems [39] lso should trigger further reserch to study the eneficil effects of E. fecium consumption in vivo. Arevitions: short-chin ftty cids (SCFA), Simultor of the humn intestinl microil ecosystem (SHIME), gstrointestinl (GI);

12 Functionl Foods in Helth nd Disese 2011; 10: Pge 400 of 402 Competing interests: No competing interests. Acknowledgements: This work ws supported y Fundção de Ampro à Pesquis do Estdo de São Pulo (FAPESP) (2009/ ). Authors Contriutions Sivieri, Kti, PhD is the principle investigtor for this study providing oversight nd contriuted fundmentl conceptuliztion for the reserch, writing grnt proposl nd mnuscript. Binchi, Fernnd, performed the microiologicl nlysis for the study, Rossi, Antonio Elizeu, PhD is the reserch coordintor for the study. Adorno, Mri Angel Tllrico, performed the SCFA nlysis. REFERENCES 1. Berg RD. The indigenous gstrointestinl microflor. Trends Microiol 1996; 4: Riud P. Bcteril intertions in the gut. In: Fuller R., editor. Proiotics: the scientific sis. London: Chpmn & Hll; p Food nd Agriculture Orgniztion of United Ntions; World Helth Orgniztion. FAO/WHO (2001). Aville from. Evlution of helth nd nutritionl properties of proiotics in food including powder milk with live lctic cid cteri. Report of joint FAO/WHO expert consulttion, Cordo, Argentin. ftp://ftp.fo.org/es/esn/food/proioreport_en.pdf 4. Slminen S, Von Wright A, Morelli L. Demonstrtion of sfety of proiotics review. Int J Food Microiol 1998; 44: Klein G. Txonomy, ecology nd ntiiotic resistnce of enterococci from food nd the gstro-intestinl trct. Int J Food Microiol 2003; 88: Sivieri K, Cno VS, Vlentini SR, Rossi EA. Demonstrtion of the cellulr viility nd sfety of Enterococcus fecium CRL 183 in long-term experiments. Le Lit 2007; 87: Lund B, Admsson I, Edlund C. Gstrointestinl trnsit survivl of n Enterococcus fecium proiotic strin dministered with or without vncomycin. Int J Food Microiol 2002; 77: Bezkoroviny A. Proiotics: determinnts of survivl nd growth in the gut. Am J Clin Nutr 2001;73: Rossi EA, Vendrmine RC, Crlos IZ, Oliveir MG, Vldez GF. Efeito de um novo produto fermentdo de soj sore lípides séricos de homens Adultos normocolesterolêmicos. Arch Ltinom Nutr 2003; 53: Kinouchi F.L. (2006). Iogurte de soj como codjuvnte no trtmento de câncer de mm. Arrqur, 85p. (Tese, UNESP). 11. Sivieri K, Spinrdi-Brisn ALT, Brisn LF, Bedni R, Puly ND, Crlos IZ, Benztti RC, Vendrmini RC, Rossi EA. Proiotic Enterococcus fecium CRL 183 inhiit chemiclly induced colon câncer in mle Wistr rts. Eur Food Res Technol 2008; 228: Bedni R, Rossi EA, Leper JS, Wng CC, Vldez CF. Efeito de um novo produto fermentdo de soj, enriquecido com isoflvons e cálcio, sore o tecido ósseo de rts. Arch Ltinom Nutr 2006; 56:

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