nv opues eMdi,200Mdi,San uhr Hervert-Hern Authors Spain. Madrid, Go and 28040 Madrid, de Complutense Univ. pi.Drc nure oato Go Madrid, author 28040 to Madrid; inquiries de Direct Complutense Univ. Spain. Pharmacy, of Faculty I, Bromatology lordcsaotssadidcsadciei uoa thickness, mucosal in decline a induces and apoptosis reduces also eMdi,200Mdi,San uhrRte swt et fMcoilg II, Microbiology of Dept. Ram with Inst. is and Rotger Pharmacy Author of Spain. Faculty Madrid, 28040 Madrid, de n tes20;P 2000; (Martin-Carrion others and (Go and subjects LDL-cholesterol hypercholesterolemic plasma status in of pressure antioxidant reduction blood and cecal 2005) of Serrano and GADF enhancement with associated include properties intake in Physiological antioxidants material. and fiber single dietary a both of properties the combining others and (Seeram apoptosis and arrest 2005). cell-cycle an- also of include activities; mechanisms induction antiproliferative effects Suggested and beneficial anti-inflammatory, content. exert tioxidant, polyphenolic polyphenols tumors due their grape 2005)—are tract whereby to others asso- and part gastrointestinal diseases (Manach in of stress especially prevention oxidative and with cancer, ciated 2010) others of and (Velmurugan risk the ute erdcinwtotpriso sprohibited is permission without reproduction Further 10.1111/j.1750-3841.2011.02520.x doi: Boadilla Pablo-CEU, San Univ. Pharmacy, L Agis-Torres, Authors of Spain. Mart Faculty Madrid, Biology, Pozuelo 28668 Monte, Author of del 10/24/2011. Dept. Accepted with 4/24/2011, is Submitted 20110516 MS Introduction Go Isabel and Rotger, Rafael Mar Lactobacillus Stimulates Fiber Dietary Antioxidant Grape C rp nixdn itr br(AF santrlproduct natural a is (GADF) fiber dietary antioxidant Grape of consumption—reduction grape of effects beneficial The 02Isiueo odTechnologists Food of Institute 2012 AFwsas on ohv tmltv feton effect stimulative a have to found also was GADF ugs httecnupino itrc npatfoswt ihdeayfie n oyhnlcnetmyenhance may content polyphenol and fiber dietary high modulation. with microbiota foods through plant host in the rich of diet health showed a gastrointestinal results the of The consumption (RT-PCR). the reaction diets that chain with suggest polymerase wk evaluated 4 time was of for growth real proliferation fed quantitative bacterial were stimulates on rich using rats intake GADF is rats Wistar of GADF which adult of effect that (GADF), Male microbiota The fiber rats. fiber. cecal of dietary dietary the antioxidant cecum as on the GADF grape in or of profile cellulose capacity either bacterial the containing the study modify to to was polyphenols, microbial work gut in influence this may of and endogenous microbiota objective both colonic by for The modulated substrates ecology. be are residues can microbiota dietary gut Undigested The substrates. interact. exogenous products and metabolic of number large a and components, rcia Application: Practical Keywords: Abstract: aua nixdnsfo rps rp nixdn brcudb sda nigein o ucinlfosada a as and foods functional for ingredient compounds. bioactive an and as fiber used dietary of be intake could the fiber increase to antioxidant supplement Grape dietary grapes. from antioxidants natural ´ nzaewt hsooyDp.Scin aut fPamc,Ui.Complutense Univ. Pharmacy, of Faculty Section, Dept. Physiology with are ınez ´ aJos ıa iaewt urto n atonetnlHat nt et fNtiinand Nutrition of Dept. Unit, Health Gastrointestinal and Nutrition with are ni ˜ oul,AglAi-ors es Hervert-Hern Deisy Agis-Torres, Angel Pozuelo, ´ e rp nixdn itr fiber, dietary antioxidant grape h iet sahgl ciebooia ytmweeeihla el,mcoit,nnietbedietary nondigestible microbiota, cells, epithelial where system biological active highly a is digesta The erez-Jim ´ nzadohr 08) AFintake GADF 2008b). others and enez ´ rp nixdn brcmie urtoa n hsooia rpriso itr brand fiber dietary of properties physiological and nutritional combines fiber antioxidant Grape nyCjld Investigaci de Cajal y on i(-al [email protected]). (E-mail: ni ´ ˜ ni ˜ R rwhi a Cecum Rat in Growth pzOia n Mu and opez-Oliva, ´ nSntra(IRYCIS), Sanitaria on ´ Lactobacillus Lactobacillus atbclu reuteri Lactobacillus polyphenols , andez ´ noz- ˜ ne,Mar andez, ´ ni ˜ n lgtyafcstecmoiinof composition the affects slightly and aebe on osiuaebeneficial pomace stimulate grape to from and growth found proliferation extracted been the polyphenols have enhance connection, to this and In line of cell gut adhesion of human adhesion a and growth bacteria to the inhibit gut factors to shown beneficial promoting been have of as survival act also or also (Hervert-Hern proliferation, but are growth, bacteria polyphenols for pathogenic proliferation the some Dietary inhibit stimulus may of and 2008). cytokine microbiota colonic through for others substrates system and a immune and of digestion, (Gotteland the lactose inhibition of of mainly improvement stimulation pathogens, properties, of health range wide attributed with isms the on defined and be (Saura-Calixto compounds could bioactive Go and pattern fiber healthy dietary of a basis recently that has digestion it proposed the regard, this and been In dietary enzymes 2004). Rowland digestive undigested and (Heavey to of process resistant form are by the that ecology residues in microbial gut available the substrates influence of providing activity can metabolic diet and and the profile cells) of the mucosa composition The hormones, substrates. gut exogenous enzymes, 2006, mucines, others (intestinal and (Lopez-Oliva the colon between distal hypoplasia 2010). rats, the epithelial Wistar and of of mucosa proximal degrees colonic in varying density inducing crypt and depth, crypt h rboi fetcudb nacdwe usata amount substantial a when enhanced be could effect prebiotic the Lactobacillus endogenous both by modulated be can microbiota gut The i2009). ni ˜ ´ aEvr L Elvira ıa nvitro in and atbclu rhamnosus Lactobacillus o.7,N.2 2012 2, Nr. 77, Vol. and ne n Go and andez atbclu cdpiu nvitro. in acidophilus Lactobacillus ´ (Hervert-Hern pzOia mlaMu Emilia opez-Oliva, ´ Bifidobacterium i21) eea ri polyphenols fruit Several 2011). ni ˜ ne n tes20) Indeed, 2009). others and andez ´ r eeca u microorgan- gut beneficial are r ora fFo Science Food of Journal Pra n tes2008). others and (Parkar Bifidobacterium noz-Mart ˜ atbclu acidophilus Lactobacillus hs findings These ´ ınez, nvitro in species. Salmonella and H59

H: Health, Nutrition & Food . , B. 260 C; 30 ◦ and Lactobacil- spp. (Fu- Bacteroides B. vulgatus B. longum Instrument Lactobacillus , C; and a final R 1 and diluted -GGCCAGT for ◦ Bifidobacterium   = -ATGAAAGCT  for . MRS Broth and B. vulgatus  550 and 5 , were incubated in ). GADF (2.4, 5, or  (ATCC 25922), were B. vulgatus Lactobacillus were grown in Brain -GGTTTATGCAGCAA  for B. vulgatus  B. longum C, and 45 s at 72 (Bej and others 1991). These ◦ C. PCR products were separated and 5 L. acidophilus ◦ B. vulgatus -ACGATGAATACTCGCTGTTT L of these dilutions were inoculated   Escherichia coli μ C in an orbital shaker (100 rpm), either E. coli and ◦ -GATTCTGGCTCAGGATGAACGC- was extracted by the method described by  cultures, supplemented with 0.05% (w/v) SPIN Kit for Soil (MP Biomedicals, Santa for -cysteine for L of PBS. Genomic DNA was extracted l R  C, 1 min at 60 strains were grown to OD μ ◦ E. coli 2hat37 C) and washed twice with 1 mL of PBS-EDTA, ◦ ± -CATGTTCCTCCGCTTGTGC-3  B. longum L. acidophilus PCR Beads (GE Healthcare, U.K.) in a Gene Amp B. longum (ATCC 8482) and -CTGATAGGACGCGACCCCAT-3  and 5 TM , and in some cases of  ,and 5 min at 4 spp. and The primers corresponding to 16S rRNA sequences used in Toevaluate the effect of GADF on bacterial growth, Cecal content from 10 rats belonging to each group, either -GAGGCAGCAGTAGGGAATCTTC-3 and 5 ×   -cysteine for the latter species. Cultures of E. coli Mahenthiralingam and others (1996) usingThe a amount FastPrep of Instrument. genomic DNA extracted was estimated by OD Heart Infusion (bioMerieux,Agar Madrid, (bioMerieux) was Spain); also used Columbia for Blood jita and others 2002); 5 the following program: a denaturing step of 2 min at 95 PCR System 2400 (Perkin Elmer, Boston, Mass., U.S.A.) with control or GADF, wasglass pooled rod and inusing homogenized the with 300 FastDNA a sterile PCRandRT-PCRwere:5 spp. and (MP Biomedicals) for 10 spurposes, at a DNA speed of setting the of 5.5. reference For strains calibration of 1:200 in fresh medium;in 50 Falcon 50 mLsupplemented tubes with with 520 mg/mL), mL EPP of (1.2 the mg/mL),(control), same NEPP were medium (2.4 mg/mL), added (MRS, orincubated to 24 nothing the corresponding tubes, which were Primers for DNA amplification uate the effectsvulgatus of GADF. Theseused strains, as as referencetion well strains (RT-PCR). as for real time polymerase chain reac- Rogosa Agar (Pronadisa, Madrid,lus Spain) were used for l longum anaerobiosis. Effects of GADF fiber on bacterial growth Extraction of bacterial genomic DNA primers were checkedTo by - G normal o PCR with PuReTaq Ready- and the homogenization step was done in a FastPrep Ana, Calif., U.S.A.) followingsome the modifications: samples recommended were protocolg previously with centrifuged (12000 in an aerobic orplate anaerobic count atmosphere. in Growth triplicate was and plotted measuredper as by mL colony forming of units culture (CFU) on a decimal logarithmic scale. 5 by agarose gelby electrophoresis UV and transillumination DNA (300ethidium bands bromide nm) solution were after for visualized 20 staining min. with 5 mg/mL GGCTACAGGAAGGCC-3 3 cycles of 15 s at 95 GC-3 extension step of 10 min at 72 TACTACCTCTATCCTTCTTC-3 CGAGACGTCA-3 spp. (Gueimonde and others 2004); and 5 growth . . . 2g 3.10 3.01 0.46 0.11 0.03 0.89 0.49 0.19 0.19 ± ± ± ± ± ± ± ± ± ± 10) and (ATCC ´ enez and = 77.60 62.07 Dry matter C until mi- n 375.50 ◦ ´ erez-Jim Lactobacillus L. acidophilus Vol. 77, Nr. 2, 2012 C, 60% humidity and with 12-h ´ enez and others 2008a). ◦ r (ATCC 23272) were used to eval- 3). 1 ≥ n ± ´ erez-Jim (ATCC 15707), a SD ( ± mol μ ( c d Lactobacillus reuteri b Proximate composition and antioxidant capacity of grape Journal of Food Science Bifidobacterium longum Male Wistar rats with an average body weights of 215 GADF is a natural product obtained from red grapes (Cenci- trolox equivalents/g dry matter) Determined as indigestible fraction (Saura-Calixto and othersDetermined 2000). by ABTS method (P Some polyphenols and resistant protein are included in this value. Data are expressed as mean Component (g/100 g) c d a b Protein 11.08 Soluble dietary fiberExtractable polyphenols 15.53 4.23 Fat 7.69 Insoluble dietary fiber Nonextractable polyphenols 17.51 Ash 5.25 Antioxidant capacity Dietary fiber (Harlan Iberica, Spain) were housedandkeptinaroomat22 in individual metabolic cages antioxidant dietary fiber H60 Bacterial strains and cultures Animals and diets Materials and Methods Samples Grape antioxidant dietary fiber stimulates of polyphenols is associated withobjective dietary of fiber. In the this present context,intake the of work an was antioxidant to dietaryon fiber study the rich the bacterial in profile influence polyphenols in (GADF) of the the cecum of animals. crobiology analysis. Rats were killed by exsanguination. 4356), and following the procedure describedothers elsewhere (P 2008a, Table 1). in Table 1. GADFanimal was diet. used Dietary astion fiber (Saura-Calixto a and was source others determinedextractable of 2000). polyphenols dietary Extraction as (EPP) and as fiber indigestible well measuretractable in as of frac- polyphenols determination the (NEPP) of from nonex- to GADF Saura-Calixto were and made others (2007). according Theof antioxidant the capacity extracts (AC) was estimated in terms of radical scavenging activity bel variety, vintage year 2005,composition La Mancha, of Spain). The GADF proximate was previously evaluated and presented Louis, Mo., U.S.A.) and immediately frozen at –80 Directive 86/609 EEC (European Community). Atstudy, the the end of rats the werexylazine anesthetized (10 mg/kg). with The ketamine peritonealline cavity (90 mg/kg) was incision opened and by and aaseptically mid- the collected cecum and mixed(phosphate-buffered was with saline removed. 10% Cecal 10 (w/v)tetraacetic content of mM acid was PBS–EDTA 1 pH mM) 7.4, (Bioreagent ethylene degree; diamine Sigma-Aldrich, St. light/dark cycles. Two treatment groupswere were fed used ( eitherFood the and water control were freely diet available.isocaloric The experimental or and diets were were the manufactured byU.S.A.) GADF having Dyets identical diet Inc. (Bethlehem, fiber for Pa., contentin 4 the (50 type g/kg wk. of diet) fibermentation but was (cellulose approved by varying or the University GADF; committee Table and 1). followed Animal experi- Table 1– H: Health, Nutrition & Food tmltdtegot fboth also used, NEPP of concentration and growth EPP lowest the Fractions the stimulated dynamics. to saturation a respect suggesting with mg/mL) significant units, 20 log and not 2 (5 with (about GADF tested mg/mL of 2.4 were concentrations at Higher GADF 1A). of Figure presence the in spp. mlfiainb lwcoigfo 5t 60 to after 95 determined from cooling to slow was by analysis the amplification of curve Specificity melt extraction. reactions, DNA RT-PCR before count plate by merated acidophilus L. n MLdtbsswr erhdwt h LS software BLAST the with searched were (www.ncbi.nlm.nih.gov/BLAST). databases Gen-Bank in EMBL Biosys- stored sequences and (Applied rRNA 16S Sequencer with Similarities 377DNA an tems). in PRISM Kit ABI Reaction automated Ready PRISM ABI sequencing the Cycle using Terminator primers, bigDye same the with amplification PCR with RT-PCR by i ApidBoytm)i nlvlm f20 of volume (10 final dilution a DNA in Biosystems) (Applied Mix ec rpeec fGD r hw nFgr .Teewas There 1. Figure in ( shown increase are significant GADF of a presence or sence U.S.A.) Ill., Chicago, (SPSS, Windows if for significant considered were differences P cases both and In organism Differences 2). of (Figure 1B). type and 1A same the Figure the in anaerobic; within or fiber (aerobic condition dietary with ANOVA of One-way type 2. and the 1 Figure of the legends the in tailed 95 fec rmr n 10 and primer, each of Real-Time Each Fast 5 U.S.A.). contained Calif., 7900HT reaction Carlsbad, a Biosystems, (Applied using System PCR Plates Reaction 96-Well tical iue1Efc fgaeatoiatdeayfie GD)o atra rwh esrdb lt on n xrse ndcmllg fCUprm of mL indicated per the CFU at of cultures logs the decimal to 24 in added incubated expressed were were and (control) and count nothing mg/mL) plate or (in polyphenols), by concentrations (nonextractable measured NEPP growth, polyphenols), bacterial (extractable on EPP (GADF) GADF, fiber culture. dietary antioxidant grape of 1–Effect Figure Discussion and Results analysis Statistical (RT-PCR) reaction chain polymerase time real Quantitative stimulates fiber dietary antioxidant Grape l fec apewscmae oasadr uv aeby made curve standard a to compared was (10 sample diluting each of old ro asrpeetteS ftetilct atra onso sas(for assays 2 of counts bacterial triplicate the of SD the represent bars Error control; < atra onsfo eoi n neoi utrsi h ab- the in cultures anaerobic and aerobic from counts Bacterial de- as deviations, standard and mean as expressed were Results MicroAmp on out carried were reactions RT-PCR ◦ othoc post ntesm 90Teupet h rget detected fragments The equipment. 7900HT same the in C .5 h ttsia nlsswr odce sn PS15.1 SPSS using conducted were analyses statistical The 0.05. nvivo in + : P < − ofroits a sdt n ifrne u to due differences find to used was test Bonferroni 3 , 0.001, sa eeasse ya nardStudent’s unpaired an by assessed were assay o10 to .longum B. .acidophilus L. − Bifidobacterium ++ 1 − μ 7 o10 to eoi N bandfrom obtained DNA genomic ) fdltdDA 1 DNA, diluted of L : P μ P ,and < LofPowerSYBR ≤ − .0)o h rwhof growth the of 0.001) .2wt eadt AF24mg/mL; 2.4 GADF to regard with 0.02 3 a ulctd h yl thresh- cycle The duplicated. was ) .coli E. u h nraeo rwhwas growth of increase the but , Lactobacillus p.pieswr eune by sequenced were primers spp. hs atrawr enu- were bacteria These . μ R Lofa5 ± pce ( species ◦ re C Master PCR Green n eetn to reheating and C 2hat37 Lactobacillus μ μ ,adeach and L, P Lactobacillus R .vulgatus B. solution M ◦ < atOp- Fast na ria hkr(0 p)i eoi PnlA raarbc(ae )conditions. B) (Panel anaerobic or A) (Panel aerobic in rpm) (100 shaker orbital an in C 0.001), t -test § rwh... . . growth : P , < 0.001, rmtecclsmls igebnso h xetdsz were size expected the of bands Single samples. cecal the from reliable a be to microbiota proven cecal quantifying has the for which and RT-PCR, method 1) by either Table containing analyzed g/kg, diets were (50 with GADF fed or were cellulose rats of groups Two 1B). plored. (Figure bacteria these of either of of signif- growth addition no the produced the in increase mg/mL) (20 Again, icant concentration conditions. higher a aerobic at in GADF obtained those to of case the in GADF, with induced the acidophilus to L. similar less or more was N rmclue fterfrnesrisof strains with reference amplification the PCR of normal longum cultures a in from checked DNA first was were assay quantify PCR This to 2008). primers vulgatus others B. specific and using (Delroisse by samples complemented fecal rat in con- anaerobic the of effect with possible which a in out ditions rule To 1B). (Figure of increase growth ( EPP the than whereas ever, efficient more slightly being NEPP ahpromdwt eia iuin fteetatdDA ntecase the reactions, in PCR ∗ DNA; extracted different the 2 of of Data dilutions in decimal content. obtained 3 cecal with results of performed the g each of per CFU mean group. of the control logs the represent decimal rats in in and of expressed (GADF) group are fiber the Results dietary in antioxidant RT-PCR grape by with bacteria fed cecal of 2–Quantification Figure Lactobacillus P < et h feto AFi hsooia odtoswsex- was conditions physiological in GADF of effect the Next, of growth affect significantly not did GADF Bifidobacterium 0.001. .acidophilus L. §§ , : .acidophilus L. P ≤ Bjadohr 91.Tepiesdsge o RT- for designed primers The 1991). others and (Bej .2wt eadt EPP. to regard with 0.02 p. r3asy (for assays 3 or spp.) h tmlto a lal lower. clearly was stimulation the o.7,N.2 2012 2, Nr. 77, Vol. p. needn N xrcin eeanalyzed. were extractions DNA independent 2 spp., .longum B. rw naarboi,wt oprberesults comparable with anaerobiosis, in grown ,and Lactobacillus .coli E. a utvtd h sa a repeated was assay the cultivated, was .longum B. n lowt N extracted DNA with also and , r ora fFo Science Food of Journal p.and spp. .reuteri L. ). ∗ : P < Bifidobacterium .0 ihrgr to regard with 0.001 P yteefractions these by < .vulgatus B. .0) How- 0.001). .coli E. .longum B. H61 , final spp. and B.

H: Health, Nutrition & Food by ˜ ni I, Escherichia ,and Escherichia coli ˜ ni I, Saura-Calixto F. 2008a. ´ andez acknowledges ˜ noz-Martinez E. 2006. Induction Bacteroides vulgatus mice. Neoplasia 12:95–102. spp. in rat fecal samples by real-time isolates recovered from patients with colonization with cranberry juice and + ˜ ni I, Mu spp., ˜ ni I. 2009. Stimulatory role of grape pomace Lactobacillus . This study supports the hypothe- ˜ Mycobacteria ni I. 2000. Effects of dietary fibre- and polyphenol- growth. Int J Food Microbiol 136:119–22. ˜ ni I, Bravo L. 2000. In vitro determination of the in- Helicobacter pylori ˜ ni I, Munoz-Martinez E. 2010. Grape antioxidant dietary spp. and Pseudomonas aeruginosa in vitro ˜ ni I. 2007. Intake and bioaccessibility of total polyphenols in a ˜ ni I. 2011. Dietary polyphenols and human gut microbiota: a review. La1 in children. Nutrition 24:421–6. Bifidobacterium ˜ ni I. 2009. Definition of the Mediterranean Diet based on bioactive com- Lactobacillus acidophilus observed ´ ´ andez D, Go andez D, Pintado C, Rotger R, Go ´ ´ enez J, Serrano J, Tabernero M, Arranz S, Diaz-Rubio ME, Garcia-Diz L, Go enez J, Arranz S, Tabernero M, Rubio ME, Serrano J, Go D, AGL-2008-01633. D. Hervert-Hern in tissue samples from patients with inflammatory bowel diseases. J Gastroenterol 37: + The present research was performed under the financial support ˜ ni I, Serrano J. 2005. The intake of dietary fiber from grape seeds modifies the antioxidant Saura-Calixto F. 2008b. Effects offactors. grape antioxidant Nutrition dietary 24:646–53. fiber in cardiovascular disease risk 149:S115–20. pounds. Crit Rev Food Sci Nutr 49:145–52. Updated methodology to determineextraction, antioxidant measurement capacity and expression in of plant results. foods, Food oils Res Int and 41:274–85. beverages: antiproliferative, apoptotic and antioxidantpomegranate activities tannin of extract are punicalagin, enhancedpomegranate ellagic in juice. combination acid J with Nutr and other Biochem polyphenols a 16:360–7. as total found in JP. 2008. Flavanol99:782–92. monomer-induced changes to the human faecalextract microflora. prevents intestinal Br tumorigenesis J in Nutr APCmin/ digestible fraction in48:3342–7. foods: an alternative to dietarywhole diet. fiber Food Chem analysis. 101:492–501. J Agric Food Chem rich grape products1183–8. on lipidaemia and nutritional parameters in rats.colonic microflora J and Sci human gut Food health. Agric Int 80: J Food Microbiol 124:295–8. multiplex polymerase chain reaction: comparisonfor with water defined quality substrate monitoring. and Appl plating Environ methods Microbiol 57:2429–32. Quantification of status in rat cecum. J Sci Food Agric 85:1877–81. H, Salazar G. 2008.Lactobacillus Modulation johnsonii of procedure for quantification of bifidobacteria in70:4165–9. human fecal samples. Appl Environ Microbiol 18:323–1. Food Rev Int 27:154–69. polyphenols on of epithelial hypoplasia in rat cecalNutr and distal Res colonic 26:651–8. mucosa by grape antioxidant dietary fiber. fibre reduced apoptosis and inducedrat a proximal pro-reducing colonic shift mucosa. in Br the J glutathione Nutr redox 103:1110–7. state ofpolymorphic the DNA typingcystic of fibrosis. J Clin Microbiol 34:1129–35. efficacy of polyphenols in81:230S–42S. humans. I. Review of 97 bioavailability studies. Am J Clin Nutr PCR. Microbiol Res 163:663–70. S, Orpianesi C, Crescimicroflora, A. oxidative damage 2005. Red andMutat wine gene Res polyphenols 591:237–46. expression influence profiles carcinogenesis, of intestinal colonic mucosative in analysis F344 rats. ofcoli bacterial DNA509–16. from ´ ´ erez-Jim erez-Jim Salminen S, Isolauri E. 2006.Saura-Calixto Intestinal F, Go colonization, microbiota, and probiotics.Saura-Calixto J F, Pediatr Garcia-Alonso A, Go sis that the consumptiondietary of fiber a and dietgastrointestinal polyphenol rich health content in of may plant thecrobiota help foods host profile. with to by high promoting enhance a the beneficial mi- P of the Spanish Ministry ofof Science I and Innovation, Natl. Program predoctoral fellowships from the Mexican institutions CONACyT and SEP. Tzounis X, Vulevic J, Kuhnle GG, George T, Leonczak J, Gibson GR, Kwik-Uribe C, Spencer Velmurugan B, Singh RP, Kaul N, Agarwal R, Agarwal C. 2010. Dietary feeding of grape seed Saura-Calixto F, Serrano J, Go Seeram NP, Adams LS, Henning SM, Niu Y, Zhang Y, Nair MG, Heber D. 2005. In vitro Parkar SG, Stevenson DE, Skinner MA. 2008. TheP potential influence of fruit polyphenols on firming the stimulativeLactobacillus effect on proliferation of 2 species of Acknowledgments References Bej AK, McCarty SC, Atlas RM. 1991. Detection of coliformDelroisse bacteria JM, and Boulvin AL, Parmentier I, Dauphin RD, Vandenbol M, Portetelle D. 2008. Gotteland M, Andrews M, Toledo M, Munoz L, Caceres P, Anziani A, Wittig E, Speisky Gueimonde M, Tolkko S, Korpimaki T, Salminen S. 2004. New real-timeHeavey quantitative P, PCR Rowland I. 2004. GastrointestinalHervert-Hern cancer. Best Practice Res ClinHervert-Hern Gastroenterol Lopez-Oliva ME, Agis-Torres A, Garcia-Palencia P, Go Lopez-Oliva ME, Agis-Torres A, Go Mahenthiralingam E, Campbell ME, Foster J, Lam JS, SpeertManach DP. C, 1996. Williamson Random G, amplified Morand C, Scalbert A, Remesy C.Martin-Carrion 2005. Bioavailability N, and Saura-Calixto bio- F, Go Dolara P, Luceri C, De Filippo C, Femia AP, Giovannelli L, CaderniFujita G, Cecchini H, C, Eishi Silvi Y, Ishige I, Saitoh K, Takizawa T,Go Arima T, Koike M. 2002. Quantita- growth . . . and Lac- Bifi- com- E. coli 2 depend- 10 E. coli (but not of × ,and Bifidobacterium Bifidobacterium E. coli C). , were less clear. . Varying effects ◦ Lactobacillus and and species present in the species and may cause in vivo 0.05) increase of growth Lactobacillus pseudolongum (accession > Bifidobacterium in the cecum of rats, con- . This is important in that ). Changes in , P 4 strain (82.9 Bifidobacterium Lactobacillus 10 Lactobacillus , but in the case of Vol. 77, Nr. 2, 2012 × r C) was not coincident with that of ◦ Bifidobacterium Bifidobacterium E. coli )-catechin. Changes in these popula- B. longum Lactobacillus + of the GADF activity over several species C), which was coincident with the peak Bifidobacterium ◦ Bifidobacterium ). On the other hand, populations of ,and spp. primers. This sequence gave the high- 1 10 in vivo L. acidophilus in vitro × and . ) over Clostridium in the treated group was signifi- and clearly correlates with the observed effects of GADF but not of decreased by less than 1 log in the GADF-fed group compared with 1.4 counts were not significant. Lactobacillus , 3 spp. with respect to the control group (2.42 in vitro and a decrease in the population Bifidobacterium 10 in vivo Journal of Food Science × L. reuteri Our major finding was that the increase in the lactobacilli popu- GADF intake produces a significant increase of beneficial This finding supports the hypothesis that the diet can have To clarify this discrepancy, the DNA sample from the con- The results for GADF effects on B. vulgatus detected for the reference ing on the polyphenol concentration,Bifidobacterium and a very slight decline of were more commonin in the feces control.Bifidobacterium of The polyphenol-treated predominancecant. of rats Other results reported than by Tzounisbatch and others culture (2008) with in colonic a microbiota fecal fermentingshowed tea an polyphenols increment of a gut microbialnant profile over detrimental inof bacteria which the host, may beneficial promote a be bacteriaIsolauri beneficial beneficial 2006), immune predomi- function and to (Salminen help and theresults to are prevent health gastrointestinal supported disorders. by Our apolyphenols study on to the administration ratsers of fed red 2005), wine with which a reported high-fat that diet (Dolara and oth- lation on Conclusions H62 Grape antioxidant dietary fiber stimulates detected in each casequantification (data in not both shown). groups ResultsRats of of rats fed are the with summarized RT-PCR GADFtobacillus in showed Figure a 2. roughly one-log increase of the GADF group (82.7 pared with 3.52 Lactobacillus dobacterium (4.4 PCR. Although we cannot ruletion out of a the different microbiotagiven starting in that composi- each each group group, was this composedviduals. possibility In of any is 10 case, randomly not this chosen finding likely analysis indi- shows that a much more thorough ofthisgenusisrequired. est identity (95%)nr with AB107570.1). Identification withaccurate such a enough, short and sequencedetected is so different not species we in can each control only and GADF conclude ratspecific that group. effects RT-PCR on different changes in the predominance of these species, detectable by RT- trol group wasrRNA amplified and sequenced with the same 16S the peak of thefrom dissociation the control curves group of (85.1 the amplification products of GADF oncolonic the microbiota different that may beruled detectable out, by and RT-PCR cannot so be thismal possibility dissociation was investigated. curves Therefore, ther- werethe performed uniformity post-RT-PCR to of verify theand amplification control products groups. in TheseBacteroides both dissociation the curves GADF were uniform for tions were more complex duringshowing fermentation opposite of behavior of (–)-epicatechin, both during fermentation of ( There was a slight but not significantin ( vitro

H: Health, Nutrition & Food