Am J Clin Nutr-1990-Marteau-685-8

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//-->Effect of chronic ingestion of a fermenteddairy productcontainingLactobacillusacidophilusandBifidobacteriumbifidumon metabolicactivities of the colonic florain humans3PhilippeMarteau,PhilippePochart,BernardFlouri#{233}, PierreJehan-Fran#{231}oisDesjeux,and Jean-ClaudeRambaudPellier,LeilitaSantos,ABSTRACTduring,andafteringLactobaci//usmesophilicwk. HydrogendasetionswhichandNinehealthyvolunteerswerestudiedbefore,contain-decreaseduronidase,theconcentrationsandof threenitroreductase)fecalenzymesthathave(/3-gluc-theca-ingestingacidophi/us,a fermenteddairyproductBijidobacteriumbijidum,lactisandproductionswereazoreductase,cultures(Streptococcusand methane/3-glucosidaseS cremoris)andmeasuredfecalasandfor 3-galactosi-indicatorsactivitiespacityto convertprocarcinogensto carcinogensin the colon.Theseresultssuggestedbeneficialeffects from the consumptionofLacidophilusand promptedinterestin studyingthe effectsof commercialfermentedproductscontaininglactic acid bac-teria.Downloaded fromajcn.nutrition.orgby guest on January 21, 2015of fermentationcapacityof thecolonicflora.Fecalconcentra-The aim of the presentwork was to studyin healthyvolun-alsoteers the effectsof a 3-wk periodof ingestionof a commercialassessed.Hydrogenandmethaneproductions,fecalf3-galacto- dairyproductcontainingLacidophilusandBifidobacteriumsidase,/3-glucuronidase,andazoreductaseactivitiesdidnotbijidumfrom humanoriginon fecal enzymaticactivitiesimpli-cated in the transformationofprocarcinogensin the colon,andchangeoverthree3-wkperiodswhereasfecal /3-glucosidaseac-tivityincreased(42 ± 6, 9 1 ± 12, and 40 ± 6 IU/gP<N,0.01)on fermentationcapacitiesoftheflora.of nitroreductase,maybe implicatedazoreductase,in colonicand/3-glucuronidase,carcinogenesis,wereandnitroreductaseIU/gdecreased(0.87N,P<±0.13,0.54±0.11, and0.57± 0.08685-8.0.05).AmJ C/inNutr1990;52:SubjectsSubjectsandmethodsKEYWORDSColonicdairymetabolism,products,Lactobaci//uscolonicflora,acidophilus,fecalenzymes,fermentedNinewhichhealthyadultsgavewas approvedby theinformedconsentEthicsCommitteefor thestudy,of Saint-LazareBifidobacteriumHospital.y (rangeThe30-42subjectsy) andwereweresix menidentifiedandasthreelactosewomenaged34onabsorbersIntroductionTheis alsothe basis ofa negativebreath-hydrogenload of 50 g lactosein water (7). Nonefloralikelytestoftheafter ingestionsubjectshadof atakencolonicveryexhibitsmanyphysiologicaltobe involvedin pathologicalpseudomembranousFermentationcolitisandcolonicis oneof the mostimportantfunctionsstatessuchcarcinogenesis(1,2).of theseinterestand producers,asin roomantibioticsforair(8).theexhalingprevious1 ppm3 mo.aboveSevensubjectsthe methaneweremethaneconcentrationfunctionsDairyTheYoplait,productproductParis)wascontainingacommercialfermentedmilk(Ofilus,andits alteration(3). Thus,the colonicThemightthereflorabe responsibleis considerablecane modulated,bfor antibiotic-associatedin understand-diarrheaing howetaryflora,videsLacidophi/usstrainA 1,Bbifidumparticularlypropertiesor gas production,dietaryinfluenceby di-of theIFromthe INSERMU290FonctionsIntestinales,M#{233}tabolisme etH#{244}pitalSaint-Lazare,Paris; the D#{233}partement dc Microbio-pro-Nutrition,Facult#{233} Pharmacie,deUniversit#{233}ParisI,Ch#{226}te-Xand, logic-Immunologic,nay-Malabry,France;and the Hospitalda universidadede Coimbra,moreover,is moredirectlyrelevantto the flora’seffectson hu-Coimbra,Portugal.mansthanare the classicalbacteriologicalcounts(4). Among2Supportedin part by Minist#{232}ree la Recherchedet de Ia Technolo-the foodproductsthathavebeenstudiedas potentialmodula-giegrant 88G064 1.torsof the colonicmicroflora,fermenteddairyproductscon-suchas bacterialan alternativeenzymemethodactivitiesfor assessing3factors.determinationofmetabolicAddressreprintrequeststo P Marteau,FaubourgINSERMU290,H#{244}pitalParis,tamingtion.sivelyviableThus,studiedmicroorganismsGoldintheet al (5)effectsandhaveGoldinLactobacil/usofreceivedandacidophilustheGorbachmost(6)inratsSaint-Lazare,exten- France.alien-107bisrueduSaint-Denis,75010andmenandNuirshowedl990;52:685-8.thatoralPrintedadministrationin USA.© 1990of someAmericanstrainsSocietyReceivedAcceptedOctober12, I 989.for publicationDecemberNutrition6, 1989.A,nJC/infor Clinical685686TABLE1CompositionMARTEAUElALmol substrate/L.The reaction,whichproceededat 37 ‘C, wasofthefermenteddairy productstoppedafter5,10, and15 mm by adding0.5mL of a cold1mol Na2CO3/Lsolution.Aftercentrifugationfor 10 mmatCarbohydrate(lactose (mg/g)544000xgand 4 ‘C, the absorbancyofthesupematantwas readLipids (mg/g)97at 405 nm in a Zeiss modelPM 2K spectrophotometer(CarlProtein(mg/g)47Zeiss,Inc, lhornwood,NY). After the linearityofthereactionEnergy(J/g)5293was checked,the increasein absorbancewas comparedwith aLaciobacmllusacidophilusstrain A I (bacteria/g)10’BijidobaciermumbifidumstrainBl (bacteria/g)10’standardcurveobtainedwith p-nitrophenol.The enzymeac-Mesophiliclactic culturesMO’ (bacteria/g)10’tivitieswere calculatedaccordingto the InternationalUnits(IU)system;1 IU is the amountof enzymethat hydrolyzes1‘Streptococcus/actisand Scremoris.molsubstrate/mmat 37 ‘C. Fecalnitrogendeterminedwasaccordingto the Kjeldahlmethodand resultsofenzymeactiv-ities are expressedas IU/g N. Hydrogenconcentrationin end-strainBl,andmesophiliclacticcultureMO(Streptococcusexpiratory-breathsampleswas measuredby use of an electro-lactisandS cremoris).The compositionis describedin Ta- chemicalcell (ExhaledHydrogenMonitor,GMIMedicalLtd,ble1.Renfrew,Scotland),and methaneconcentrationwas measuredby use ofgas-liquidchromatography(IGL,Intersmat,Courtry,StudydesignFrance).The studycomprisedthreeconsecutiveperiods,each 3 wklong. Duringperiods1 and 3 the subjectsate their normaldietCakulationsand statisticalanalysisbut werenot allowedto consumefermenteddairyproducts.hydrogenand methaneexcretionsafter lactu-Duringperiod2 at the end of their meals(threetimesper day)Totalexcesswere determinedby integratingthe area underthethe subjectsingested100 g of the fermenteddairyproduct. lose loadingof intervaldelta breathhydrogenand methaneconcen-Duringthe morningbeforethe last day ofeachperiod,the sub- curvestrationsand expressedas ppmhfor the total testperiod..Re-jects evacuatedtheir feces into containersin the laboratoryandas I ± SEM.PairedI tests wereusedforthese were immediatelymaintainedin anaerobicconditionsbysults are expressedstatisticalcomparisonsamongthe threeconsecutivesperiods.meansofananaerobicsystem(Anaerocult,Merck,Darmstadt,FRG) and assayed within2 h ofevacuation.At 0800that samemorning,after an overnightfast, the subjectsingested10 g lac-tulosein200mL tap water.End-expiratory-gassampleswereResultsobtainedfor hydrogenand methanemeasurementsby use of amodifiedHaldane-Priestleytube(Duphalac,Duphar,Paris) Azoreductaseand /3-glucuronidaseactivitiesdid not changeand 60-mLplasticsyringesequippedwith three-waystopcocks throughoutthe three periods.In contrast,nitroreductaseactiv-(9). Threebasal sampleswere takenat 1 5-mmintervalsbefore ity droppedsignificantlyin period2 and remainedat alow leveland at 30-mmintervalsduringthe 8-h periodafter the ingestion duringperiod3 (Table2).oflactulose.The purposeofthistest was to get an index for theBreathhydrogenand methaneexcretionafter ingestionof 10fermentationpropertiesofthcflora in the right colon.At 0800 g lactuloseand after a 10-g lactuloseenemawere not affectedon the last day ofeachperiod,after anotherovernightfast, theby consumptionofthedairy product(Table3). /3-galactosidasesubjectsreceiveda 100-mLenemacontaining10 g lactulose, activitydid not changebut /3-glucosidaseactivitysignificantlywhichwas subsequentlyfollowedby measurementsof the cx-increasedin period2 and returnedto baselinelevels in periodcretionof hydrogenandmethanein breathevery30 mmfor36(Table3).h.The purposeofthistest was to set an index for the fermenta-In vitro the dairy productshoweda high /3-glucosidaseactiv-tioncapacitiesof the florain the left colon,whichmaydifferity, whichwas relatedto the presenceBofbifidum./3-glucosi-from those oftheflora in the right colon(9). To avoid any car-dase activityof the differentstrainsisolatedfromthe productry-overeffects from the lactuloseingested,we used a small dose is indicatedin Table4.of lactulosewiththe subsequentlactuloseenema,whichisknownto be very rapidlyand locallyfermentedin the rightcolon.TABLE 2Azoreductase,nitroreductase,and 3-glucuronidaseactivitiesfecesinAnalytica/proceduresbefore (period 1), during(period2), and after(period3) a 3-wk perioddairyNitroreductaseand azoreductaseactivitieswere measuredinduring which all subjects ingested100 gofthe fermentedproduct three timesper day’anaerobicconditionsas previouslydescribed(5). Thereleaseofp-nitrophenolfromwasthishydrolysisusedofp-nitrophenylas theindicatorsample/3-r-galacto-Downloaded fromajcn.nutrition.orgby guest on January 21, 2015Periodp-ni-/3-galac-1PeriodIU/gN2Period3pyrannoside,tro-13-D-glucuronidep-nitrophenyl-fl-D-glucopyrannoside,/3-glucosidase,andpurposeandof fecaltosidase,tively(10)./3-glucuronidase1 g of fecalactivity,wasrespec-suspendedNaC1/L,Cell,Forin 30 mL buffer7.4) and sonicatedics & MaterialInc)(0. 1 molNa2HPO4/L,by use ofa probein anicebath.One0. 1 5 molsonicator(VibramilliliteroftheAzoreductaseNitroreductasepHfl-glucuronidaseSon-0.96± 0. 190.87±0.1333± 51.44 ± 0.330.54±0.1 lt41 ± 60.72 ± 0.120.57±O.08t31 ± 5appropri-‘i±SEM;n=9.tSignificantlydifferentfromperiodP1,<0.05.atelydilutedsamplewasthencombinedwith0.5mLof 0.13COLONICMETABOLISMANDFERMENTEDMILK687DiscussionThisdairycreasestudyproductinfecalconfirmscontainingthatregularconsumptionviableLacidophi/usactivity.Inofainducesaddition,in thefermenteda de-it suggestscoloniccompoundsTABLE43-glucosidaseactivitiesbacterialstrainsofthetesteddairyproductandofitsdifferentnitroreductaseActivityDairy product(mIU/g)Bbijidum(strainB 1) (mIU/10’ cells)Lacidophi/us(strainAl) (mIU/1O’cells)MesophilicculturesMO’ (mIU/lO’cells)‘thatingestionofviableactivity,of cellulose.canharmfulincolonB bifidumwhichconvertaminescanceris responsibleis implicatednitrated,(112)1for an increasefer-in /3-glucosidasementationNitroreductaseintothispotentiallyenzymearomaticbuttheis stillexactI312.90.80.3roleofOurinhumansunknown.S/actisandS cremorms.resultsareand Aycboenzymaticin keepingwith thoseof Goldinet al(13), who observeda decreaseactivityinthefecesafteringestionand Gorbachin nitroreductaseofLactobacilli.(6)sistedof an increasein fecal /3-galactosidaseactivity(14,15).However,in contrastwith the studyof Goldinand Gorbach Thus,the currentstudy was performedon lactoseabsorberstoavoidthe modificationsoftheintestinalflora that couldresult(6), the decreasein nitroreductaseactivitystill persisted3 wkafterthe cessationof the dairyproductingestion,suggesting fromthe arrivalof lactoseinto the colon.The significantin-moreprolongedmodificationsof the colonicflora. Theseau-creasein fecal/3-glucosidaseactivityin our subjectscontrastswith the decreasein this enzymeobservedby Ayeboet al (13)thorsalsonoticeda decreasein /3-glucuronidaseand azoreduc-ingestinga dairyproductcontainingL acidophi/ustase activitiesin the feces (6); we couldnot confirmthesemodi-in subjectsalone.Thisdiscrepancycouldbe due to differencesin theficationsalthoughweusedthesamedoseof Lactobacilli.Differencesin the strainsofLactobacilliarc likelyto be respon-strainsofLactobacillusused or to the presenceofbijidum,Bsible for these discrepencies.Indeed,these authorsmentionedwhichexhibiteda high /3-glucosidaseactivityin vitro.The interpretationof our resultsin termsof potentialim-thatonlysomestrainsofLacidophiluswere able to inducesuchmodifications(5).provementof humanhealthmustbe approachedcautiously.Fermentationis one ofthemost importantfunctionsof theIndeed,enzymescan play dual roles. For example,nitroreduc-colonicfloraandresultsin theproductionof volatilefattytase can inactivatesome carcinogensproducedin the liver (16,acids,lacticis a goodstudyweacid,andgas(hydrogen,carbondioxide,andIn thesome- 17),andenhanced/3-glucosidaseactivity,whichis alsotumorsrespon-observedDownloaded fromajcn.nutrition.orgby guest on January 21, 2015timestion,entmethane).didGasindicatornotproduction,ofcolonicobserveanyparticularlyfermentationsignificanthydrogen(3).changescxcre- siblepres-catedfor thein thecycadincreasednuttoxicityincidencein rats(18),seemsto be impli-ofcolorectaltreatedwith chemicalcarcinogensand fed bran-con-gen or methaneproductionin responseto the lactuloseloadtamingdiet (19).We concludethatchronicingestionof a fermenteddairyby the florain the rightor left colonafterthe dairyproductadministration.Enzymesimplicatedin colonicfermentationsproductcontainingL acidophi/usandB bijidumofhumanori-are mainlyoxidasesthat can be stimulatedby exogenouslyde-gin leads to metabolicmodificationsof the colonicflora. Fur-rivedsubstrates.We showedin previousstudiesthat chronic ther studiesare neededto determinethe mechanismsof theseingestionoflactuloseinduceda colonicadaptationthatcon-modificationsandthefateofthesebacteriain thegastrointesti-in hydro-in rodentsnaltract.TABLE3BBreath-hydrogenand -methaneexcretionafter receiptof 10 g lactuloseReferencesper os(right colon microflora)or by enema(left colonmicroflora)I . SavageDC. Gastrointestinalmicroflorain mammaliannutrition.and fecal fl-galactosidaseand $-glucosidaseactivitiesbefore (period1),AnnuRev Nutr1986;6:l55-78.during(period2), and after(period3) a 3-wk period duringwhich all2. SimonGL,GorbachSL. Intestinalflora in health and disease.Gas-subjectsingested100 g ofthefermenteddairy productthree times pertroenterology 1984;86: I74-93.day’3. Rao SSC, EdwardsCA, AustenCJ, Bruce C, Read NN. Impairedcolonicfermentationof carbohydrateafter ampicillin.Gastroen-Period1Period2Period3terologyl988;94:928-32.Area underdelta breath-4. MooreWEC,HoldemanLV. Discussionofcurrentbacteriologicalhydrogen profileinvestigationsoftherelationshipsbetweenintestinalflora, diet and(ppm.h)colon cancer.CancerRes 1975; 35:3418-20.Right-colonmicroflora247±42167±8025±1665. GoldinBR,SwensonL, Dwyer J, Sexton M, GorbachSL. Effect ofLeft-colonmicroflora40±248±259 ±4diet andLaciobaci//usacidophi/ussupplementson humanfecalArea underdelta breath-bacterialenzymes.JNCIl98O;64:255-60.methaneprofile6. GoldinBR, GorbachSL. The effect of milk and Lactobacillifeed-(ppm.h)ing on humanintestinalbacterialenzymeactivity.Am J Clin NutrRight-colonmicrofloral984;39:756-6 I.Left-colonmicroflora69±2154 35±63±337. NewcomerAD,McGillDB,ThomasPJ, HofmannAF. Prospec-fJ-galactosidase IU/g N)(152±181 1832±103 19±tive comparisonof indirectmethodsfor detectinglactasedefi-fJ-glucosidase(IU/g N)42±69 1 ± 1 2t40±6ciency.N Engi J Med 1975;293:1232-6.8. Bond JH, EngelRR, LevittMD.Factorsinfluencingpulmonary‘i±SEM;n=9.methaneexcretionin man. J Exp Med 197 1; 133:572-88.tSignificantlydifferentfrom period1 and periodP<0.01.3,9. Florent C, Flouri#{233} Pellier P, Maurel M, LeblondA,RambaudB,688MARTEAUElALand methane productionresponseto a lactu-in15. FlorentC, Flouri#{233} LeblondB,A, RautureauM, BernierJJ, Ram-man.Gastmentemlogyl987;92:A1392 (abstr).baud JC. Influenceof chroniclactuloseingestionon the colonic10. Yuen CT, Price RG, RichardsonAC, Praili PFG. Colorimetricsmetabolismof lactulosein Man (an in vivo study).J Clin Investassays for N-acetyl-fl-D-glucosaminidaseand fl-D-galactosidasein1985;75:608- 13.humanurine using newly developedw-nitrostyrylsubstrates.Clin 16. WilliamsiR,GranthamPH, Marsh HH, WeisbergeriH,Weisber-ChimActs 1982; 124:195-204.ger EK. Participationofliver fractions and ofintestinalbacteria in1 1. Drasar BS, RenwickAG, WilliamsRT. 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New York: AcademicPress, 1983:129-46.zoleTherformsN-(2-hydroxyethyl)-oxamicl979;2O8:406-10.acid.J PharmacolExp17.WheelerLA, Soderbergdroxy-4-acetyl-aminobiphenylFB,Goldmanby theP. TheintestinalreductionmicrofloraofN-by-of theonDownloaded fromajcn.nutrition.orgby guest on January 21, 2015 [ Pobierz całość w formacie PDF ]