Fate and effects of Camembert cheese micro-organisms in the human colonic microbiota of healthy volunteers after regular Camembert consumption

International Journal of Food Microbiology 125 (2008) 176–181

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International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro

Fate and effects of Camembert cheese micro-organisms in the human colonicmicrobiota of healthy volunteers after regular Camembert consumption

Olivier Firmesse a, Elise Alvaro a, Agnès Mogenet b, Jean-Louis Bresson b, Riwanon Lemée c, Pascale Le Ruyet c,Cécile Bonhomme c, Denis Lambert c, Claude Andrieux a, Joël Doré a, Gérard Corthier a, Jean-Pierre Furet a,⁎,Lionel Rigottier-Gois a

a Unité d'Ecologie et Physiologie du Système Digestif, INRA, 78350 Jouy-en-Josas, Franceb Centre d'Investigation Clinique AP-HP/INSERM, Université René Descartes et Hôpital Necker-Enfants Malades, 75015 Paris, Francec Lactalis Recherche et Développement, 53089 Laval, France

⁎ Corresponding author. Tel.: +33 1 34 65 29 29; fax:E-mail address: [email protected] (J.-P. F

0168-1605/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2008.03.044

A B S T R A C T

Article history:

Keywords:Camembert cheeseLactic acid bacteriaHuman fecal microbiotaReal-time PCR quantification

was to determine i) if Camembert cheese micro-organisms could be detected infecal samples after regular consumption by human subjects and ii) the consequence of this consumption onglobal metabolic activities of the host colonic microbiota. An open human protocol was designed where 12healthy volunteers were included: a 2-week period of fermented products exclusion followed by a 4-weeksCamembert ingestion period where 2×40 g/day of Camembert cheese was consumed. Stools were collectedfrom the volunteers before consumption, twice during the ingestion period (2nd and 4th week) and onceafter a wash out period of 2 weeks. During the consumption of Camembert cheese, high levels of Lactococcuslactis and Leuconostoc mesenteroides were measured in fecal samples using real-time quantitative PCR,reaching median values of 8.2 and 7.5 Log10 genome equivalents/g of stool. For Ln. mesenteroides, persistencewas observed 15 days after the end of Camembert consumption. The survival of Geotrichum candidum wasalso assessed and the fecal concentration reached a median level of 7.1 Log10 CFU/g in stools. Except adecreasing trend of the nitrate reductase activity, no significant modification was shown in the metabolicactivities during this study.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

The bacterial inhabitants of the human digestive tract (DT), i.e. theintestinal microbiota, play an important role in the maintenance andthe improvement of health (Backhed et al., 2005). Some other bacteriatransited in DT via consumption of fermented milk such as cheeseswhich can containmore than 108 108 CFU/g of product (Gardiner et al.,1999). Among these bacteria ingested with food, those which exerteda beneficial effect on human health are called probiotics (Fuller, 1991;Guarner and Schaafsma, 1998). Most of them belong to lactic acidbacteria (LAB) such as Lactobacillus and Bifidobacterium genus(Sanders, 2000; Sullivan and Nord, 2002; Borriello et al., 2003; Saitoet al., 2004). Although the intestinal microbiota appear relativelystable over long period of time (Zoetendal et al., 1998), the ingestion ofprobiotics can involve a transitory increase of one or more bacterialspecies in the feces (Johansson et al., 1993; Garrido et al., 2005). Themost significant effects of probiotics consumption are control ofgastrointestinal balance, inhibition of pathogens growth, improve-ment of lactose intolerance, diarrhoea reduction, inflammatory

+33 1 34 65 24 62.uret).

l rights reserved.

abdominal affections and effects on the immune system (Marteauet al., 2003; Ouwehand et al., 2002; Mercenier et al., 2003).

The soft cheese Camembert contained large numbers of livingmicro-organisms such as Streptococcus thermophilus, Lactobacillus sp.,Lactococcus lactis, Leuconostoc, Hafnia alvei and Geotrichum mainly onthe surface and contributing to the flavor, texture and appearance ofthe cheese. Lay et al. (2004) suggested that Camembert consumptioncould have a probiotic effect: in human microbiota-associated rats,following cheese consumption, changes were observed on themetabolism of intestinal microbiota, particularly a decrease inazoreductase activity and an increase in mucolytic activities. Themajor micro-organisms from Camembert cheese were enumerated atsub-dominant level between 105 and 108 108 CFU/g of fecal sample(Lay et al., 2004). Therefore, it was relevant to determine if similareffects could be observed in humans. Until now, to our knowledge, notrials with human subjects have yet evaluated whether the livingmicro-organisms of Camembert cheese that is widely consumed inFrance and throughout the world could have beneficial effects onhuman health. To optimise the use of probiotics, it is essential todetermine whether they can survive in the gastrointestinal tract (GIT)after consumption by human volunteers. To monitor the survival andthe eventual persistence of bacterial strain or species during thepassage through the GIT, accurate methods for their specific detection

Table 116S rRNA gene-targeted primers used in this study

Species Primer Sequence(5′–3′)

Position Genbank16S rRNAsequencereference

Reference

L. lactis Llac05

AGC AGT AGGGAA TCT TCGGCA

359–379

AF515226 Furet (personalcommunication)

Llac02

GGG TAG TTACCG TCA CTTGAT GAG

501–478

S. thermophilusSthm03

TTA TTT GAAAGG GGC AATTGCT

195–216

X68418 Furet et al., 2004

Sthm08

GTG AAC TTTCCA CTC TCACAC

474–454

Ln. mesenteroidesLmes01

ATG CAA GTCGAA CGC ACAGC

56–75 M23035 This study

Lmes02

AAA TGT TATCCC CAG CCTTGA G

157–137

Lb. paracasei Lpara01

GTG CTT GCACCG AGA TTCAAC ATG

60–83 D79212 Furet et al., 2004

Lcas08

TGC GGT TCTTGG ATC TATGCG

185–165

Lb. fermentum Lferm05

GTG GCG GACGGG TGA GTAA

121–139

AF522394 This study

Lferm06

CAT CCA GAAGTG ATA GCGAGG AG

260–238

Lb. plantarum Lpla03

GCT AAT ACCGCA TAA CAACTT GGA

176–199

AB104855 This study

Lpla04

TGC CAT GGTGAG CCG TTA C

295–277

177O. Firmesse et al. / International Journal of Food Microbiology 125 (2008) 176–181

are required. The methods used for tracing a particular strain orspecies introduced into a complex microbial ecosystem should enablethe distinction of the introduced bacteria from the endogenous fecalmicrobiota. Here, to enumerate Camembert cheese LAB, we adapted areal-time quantitative PCR (Q-PCR) derived from the recent work ofMatsuki on human microbiota (Matsuki et al., 2004) who used Q-PCRto quantify sub-dominant bacteria in human feces.

The aims of the present study were to determine i) if Camembertcheese micro-organisms could be detected in human feces afterconsumption and ii) if the metabolic activities of the human intestinalmicrobiota could be influenced by Camembert cheese consumption.

2. Materials and methods

2.1. Subjects

Twelve healthy volunteers (6 men, 6 women; median age: 28.5 years, range: 19–40 years) were recruited into the study at NeckerHospital, Paris, France. They had no antibiotic treatment for 3 monthsprior to the study and no acute or chronic disease or gastrointestinalproblem. During the investigation period, the restriction with regardto diet was the exclusion of fermented products. All participants gavewritten informed consent to participate in the trial, according to theprocess approved by the Institutional Ethic Committee (CCPPRB —

Necker).

2.2. Study design and samples collection

This study was an open human protocol. The total experiment timewas 8 weeks. A 2-week adjustment period of fermented productsexclusion was carried out, followed by 4-week consumption period ofCamembert cheese and a 2-week wash out period. Throughout thestudy treatment, the subjects have consumed daily 2×40 g of cheese,either cheese spread containing dead bacteria during the exclusionand wash out period, or Camembert cheese during the ingestionperiod. All volunteers have consumed the same batch of Camembertcheese. Five fecal samples were collected: one during initial period (I)before the exclusion period, another (Ex) at the end of the exclusionperiod, one after 2 weeks (C1) and one after 4 weeks (C2) in theingestion period (2nd and 4th weeks) and one (W) at the end of thewash out period. Ten grams of feces were collected for enumeration ofG. candidum of dairy product origin. Fecal aliquots of 0.2 g werecollected and stored at −80 °C until analyses.

2.3. Geotrichum candidum enumeration

Survival of G. candidum was determined for each fecal sampleobtained at I, Ex, C1, C2, and W. Fecal samples were homogenized andserially diluted 10-fold in 9 mL of Tryptone Salt (Merck, Darmstadt,Germany) and 0.1 mL of each dilution was evenly spread on plates ofCzapeck-Dox medium (Warcup, 1950). The plates were incubated inaerobic conditions at 22 °C for 3 days, after which G. candidum CFUwere counted. Results were expressed as the logarithm of number ofCFU per gram of stool.

2.4. Extraction of DNA from fecal samples, cheese and bacterial cultures

(i) Fecal samples. Total cellular DNA from 0.2 g fecal samples wasextracted using the GNOME® kit (BIO 101, La Jolla, CA)according to the manufacturer's instructions with modifica-tions to improve the rate of extraction and to limit the presenceof PCR inhibitors from feces (Firmesse et al., in press). Fecalsamples were thoroughly homogenized in Cell SuspensionSolution and after addition of Cell Lyses/Denaturing Solutionthey were incubated at 55 °C for 120 min. To improve cellularlyses, 750 µL of 0.1-mm-diameter silica beads (Sigma-Aldrich,

St. Louis, MO) were added, and the tube was shaken atmaximum speed for 10 mins in a Beadbeater (Biospec,Bartlesville, OK). Polyvinylpolypyrrolidone (15 mg) was addedto the tube, vortexed and centrifuged at 20,000 ×g for 3 min.The first supernatant was recovered. The pellet was washedwith 400 µL of TENP (50 mM Tris [pH 8], 20 mM EDTA [pH8],100 mM NaCl, 1% polyvinylpolypyrrolidone) and centrifuged at20,000 ×g for 3 min. The second supernatant was added to thefirst supernatant. The washing step was repeated twice torecover two other supernatants. Nucleic acids were precipi-tated by addition of 1 volume of isopropanol, stored at −20 °Cfor 20 min and centrifuged at 20,000 ×g for 10 min. The pelletwas homogenized in 400 µL of distilled water and 100 µL ofSalt-Out Mixture was added as was indicated thereafter in theGNOME® protocol.

(ii) Cheese and bacterial cultures. The bacterial strains of L. lactisCNRZ1342, S. thermophilus CNRZ1358, Leuconostoc mesenter-oides CNRZ749 and lactobacilli (Lb. plantarum CNRZ1008, Lb.fermentum CNRZ64 and Lb. paracasei CNRZ62) obtained fromthe CNRZ Collection of LAB (INRA, UBLO, Jouy-en-Josas, France)were used in Q-PCR as positive and negative controls and tomake standard curves. The Lactobacilli and Ln. mesenteroideswere grown in MRS (De Man et al., 1960) at 37 °C, and L. lactisand S. thermophilus were grown in M17 (Terzaghi and Sandine,1975) at 37 °C and 42 °C, respectively. Total DNA from bacterialcultures and cheese was extracted using the Wizard genomicDNA purification kit (Promega, Madison, WI), in accordancewith the manufacturer's instructions.

Table 2Specificity of the 16S rRNA gene-targeted primers

Species Strain Otherdesignation

PCR results

Llac Sthm Lmes Lpara Lferm Lpla

L. lactis CNRZ1342T

IL 1403T + − − − − −

S.thermophilus

CNRZ1358T

NCDO 573T − + − − − −

Ln.mesenteroides

CNRZ 749T NCDO 523T − − + − − −

Lb. paracasei CNRZ 62T NCDO 151T − − − + − −Lb.fermentum

CNRZ 64 NCDO 335 − − − − + −

Lb. plantarum CNRZ1008T

ATCC 14917T − − − − − +

T: type strain; +: positive result; −: negative result.

Table 3Comparison of bacterial populations in Camembert cheese and spread cheese by real-time quantitative PCR

Species Log10 genome equivalents/gram ofcheese determined by Q-PCR

ΔLog10

Camembert Cheese spread

L. lactis 8.9±0.2 6.8±0.1 2.1Ln. mesenteroides 7.6±0.1 5.6±0.2 2.0S. thermophilus 7.4±0.3 7.9±0.2 −0.5Lb. fermentum 3.2±0.2 4.5±0.0 −1.3Lb. paracasei 3.9±0.2 5.0±0.1 −1.1Lb. plantarum 4.2±0.2 4.3±0.3 −0.1

Q-PCR: real-time quantitative PCR.

178 O. Firmesse et al. / International Journal of Food Microbiology 125 (2008) 176–181

2.5. Design of PCR primers

The primers used in this study are listed in Table 1. Their designwas performed with sequences of the gene of the 16S rRNA retrievedfrom the EMBL/GenBank database. Multiple alignments with thesequences were constructed with the program ClustalW provided byEMBL/EBI (http://www.ebi.ac.uk/clustalw) and potential primer targetsites for Q-PCR were assessed using the PrimerExpress software v1.0(Applied Biosystems, Foster City, CA). The oligonucleotides wereprovided by MWG-Biotech (Ebersberg, Germany).

2.6. Real-time quantitative PCR

PCR amplification and detection were performed with an ABIPRISM® 7000 SDS (Applied Biosystems, Foster City, CA). Each reactionmixture (25 µL) was composed of 12.5 µL SYBR®-Green PCR MasterMix kit (Applied Biosystems, Foster City, CA), 2.5 µL of specific primersat the final concentration of 0.2 µM and 10 µl of DNA extracted from0.2 g of each fecal sample. The amplification program consisted of onecycle at 50 °C for 2 min, one cycle at 95 °C for 10 min and 40 cycles ofamplification (95 °C for 15 s, 60 °C for 1 min). For each set of primers,the threshold cycle (Ct) of each sample was then compared to astandard curve made from serial DNA dilutions (ten to ten) of asolution of known concentrations of amplicons of conserved regionson the 16S rRNA gene. The result was expressed as a numerical valueof genome equivalents per gram of feces. The presence of inhibitors ineach sample was estimated using Taqman® Exogenous InternalPositive Control and the Taqman® Universal PCR Master Mix kit(Applied Biosystems, Foster City, CA) as described by Furet et al.(2004). To determine the detection limit of 16S rDNA amplification byQ-PCR assays in fecal sample, DNA extracted from a known amount ofL. lactis CNRZ1342 was added in serial dilutions from 9.0 to 4.0 Log10bacterial cells/g of stool.

2.7. Biochemical analyses

Enzyme activities were measured as previously described(Andrieux et al., 2002) using a thermo-regulated anaerobic chamber(H2, CO2, N2) (10:10:80). Samples were diluted 1/20 using pre-reducedphosphate-buffered saline (PBS) (pH 6.7). α- and β-galactosidase, α-and β-glucosidase, β-glucuronidase, β-N-acetyl-galactosaminidaseand α-L-fucosidase activities were measured by determining the rateof p-nitrophenol release from p-nitrophenyl-glycosides. Azoreductaseactivity was determined using amaranth (5 mM) as substrate.Neuraminidase activity was measured using 4-methylumbelliferyl-N-acetylneuraminic acid as substrate. Nitrate reductase was deter-mined by the generation of nitrite. Enzyme activities were expressedas µmol of metabolised substrate per min and per g of protein.

Protein concentration was determined in triplicate by the methodof Lowry et al. (1951) using 1/500 fecal dilution in Na2CO3 (2%) andNaOH (0.1 N). Bovine serum albumin was used as the standard.

Short chain fatty acid concentration of fecal samples were analyzedin duplicate after water extraction of acidified samples using gaschromatography (Perkin-Elmer 1020 GC, Applied Biosystems)(Andrieux et al., 2002). Ammonia was determined using the Berthelotmethod adapted by Drospy and Boy (1965).

2.8. Statistical analyses

Results of quantification by Q-PCR were plotted as boxes andwhiskers and analysis of variance were performed using the one-wayANOVA or the paired t-test (equivalent to the one-way ANOVA forcomparison of 2 samples) programs from StatView® software (AbacusConcepts, Berkeley, CA). The box and whisker plot shows the median(horizontal line), interquartile range (box contains 50% of all values),the whiskers (represent the 10th and 90th percentiles), and the

extreme data points are indicated as circles. When ANOVA indicatedsignificant treatment effects, values were compared using the Neu-man–Keuls test with a Bonferroni adjustment. Statistical significancewas accepted at Pb0.05.

3. Results

3.1. Validation of primers specificity

The specificity of the primer pairs was tested in silico and wasvalidated experimentally using DNA extracts from type strains oftarget and non target species (Table 2). PCR assays were performed on1.0 ng DNA solution of each bacterial strain using Q-PCR. A PCRproduct was obtained for the corresponding target species. Con-versely, no significant cross-reaction of specific primers was observedwith the non target species. The amplification specificity of the variousprimer pairs was confirmed on human fecal samples using theanalysis by the dissociation curves showing that only targeted specieswere amplified (data not shown).

3.2. Quantification of bacterial population in Camembert cheese andcheese spread

Quantification of bacterial strains was performed on the twocheeses consumed during this study (Table 3). The Camembertcheese was composed of L. lactis, S. thermophilus and Ln. mesenter-oides, reaching 8.9±0.2, 7.4±0.3 and 7.6±0.1 Log10 genome equiva-lents/g of Camembert cheese, respectively. Low levels weremeasuredfor Lb. fermentum, Lb. plantarum and Lb. paracasei, reaching 3.2±0.2,4.2±0.2 and 3.9±0.2 Log10 genome equivalents/g of Camembertcheese, respectively. Camembert compared to cheese spread, havehigher contents of L. lactis and Ln. mesenteroides, (reaching +2.1 and+2.0 Log10 units). The differences were not significant with S. ther-mophilus and Lb. plantarum between the two cheeses. Camembertcompared to cheese spread, have lower contents of Lb. fermentum

Fig. 1. Fecal concentration of G. candidum candidum obtained in 12 volunteers duringthe periods of exclusion (Ex), ingestion (C1 and C2) and wash out (W) of Camembertcheese. G. candidum bacteria were grown at 22 °C for 3 days in Czapeck-Dox medium,after which G. candidum CFU were counted. ⁎Pb0.05, median values significantlydifferent from Ex and W.

179O. Firmesse et al. / International Journal of Food Microbiology 125 (2008) 176–181

and Lb. paracaseiwhich were not thus estimated by Q-PCR during thehuman trial.

3.3. Survival of G. candidum in fecal samples

After the exclusion period (Ex) preceding the Camembert cheeseconsumption (Fig. 1), no G. candidum was detected on plates ofCzapeck-Dox medium suggesting that G. candidum was absent orlower than the detection limit (2 Log10 CFU/g of stool). During theingestion period of Camembert cheese (C1 and C2), a plateau of7.1 Log10 Log10 CFU/g of stool was observed. After the Camembertcheese consumption, the survival of G. candidum in stools decreasedrapidly, at the initial level (inferior at 2 Log10 Log10 CFU/g of stool) afterthe wash out period (W). G. candidum must be considered as a goodmarker for Camembert cheese consumption.

3.4. Bacterial quantification in fecal samples using Q-PCR

Detection and quantification of bacterial species from the Camem-bert cheese were established in the fecal microbiota. The dilution for

Fig. 2. Real-time Q-PCR quantifications of Camembert species in fecal samples of 12 subjects cdifference at Pb0.05.

which no PCR inhibitor was detected in the fecal samples was 1/100;therefore the detection limitwas 5.0 Log10 Log10 genome equivalents/gof stool.

(i) Exclusion period. Significant decreases of bacterial populations(Pb0.05) were observed between the two fecal samples (I and Ex) forL. lactis, Ln. mesenteroides and Lb. plantarum (Fig. 2). No decrease(P=0.595) was observed for S. thermophilus during the exclusionperiod.

(ii) Camembert cheese consumption period. The statistical analysis(one-way ANOVA test) carried out on the amount of L. lactis andLn. mesenteroides during the 4 sampling times (Ex, C1, C2 and W)showed significant increases in the bacterial population (Pb0.0001,Fig. 3). The quantities of genome equivalents found in the samplescorresponding at the ingestion period of Camembert cheese (C1 andC2) were significantly higher (Pb0.05) than those obtained during theexclusion (Ex) and wash out (W) periods, reaching median values of8.2 and 7.6 Log10 Log10 genome equivalents/gram of stool, respectively.After the wash out period, persistence was observed forLn. mesenteroides. The number of genome equivalents measured inW sample is higher than in Ex sample. This was not observed withL. lactis.

For Lb. plantarum, we observed a significant increase in thegenome equivalents quantification during the second week of in-gestion period (C1), reaching a median value of 7.2 Log10 Log10 genomeequivalents/gram of stool. No significant differences were observedbetween the quantification results obtained for the C2 sample duringthe fourth week of Camembert cheese consumption and thoseobtained in the exclusion (Ex) and wash out (W) periods.

No differences were observed for S. thermophilus (P=0.1311). Thegenome equivalents number was stable in the four periods, reaching amedian level between 7.9 and 8.6 Log10 Log10 genome equivalents/g ofstool.

3.5. Bacterial enzyme activities and short chain fatty acid (SCFA) fecalconcentration

Enzyme activities and SCFAwere determined in fecal samples alongthe study (Table 4). Large inter-individual variations were observedwith the 15 metabolic markers followed. No significant change of

orresponding to initial period (I) and directly after the exclusion period (Ex). ⁎Significant

Fig. 3. Real-time Q-PCR quantifications of bacterial species of Camembert cheese found in fecal samples at the different periods of study (Ex: exclusion (), C1 and C2: ingestion ofCamembert cheese () and W: wash out. ⁎Significant difference at Pb0.05 between median values of C1 or C2 and Ex and W periods. ⁎⁎Significant difference at Pb0.05 betweenmedian values between W and Ex periods.

180 O. Firmesse et al. / International Journal of Food Microbiology 125 (2008) 176–181

bacterial enzyme activities occurred as a result of Camembert cheeseconsumption. A decrease in nitrate reductase after 4-weeks ofingestion was observed and was more pronounced for half of theindividuals, where the initial level was high.

4. Discussion

The consumption of Camembert cheese did not significantlymodify the microbiota enzyme activities and more particularly themucolytic activities or the fermented metabolic profile as previouslydescribed in themodel of humanmicrobiota-associated rats (Lay et al.,2004). However, we observed a decrease of the nitrate reductaseactivity, especially when this activity was initially high. High nitratereductase activity might be potentially harmful to health due toconversion of nitrate in toxic nitrite by the intestine (Rowland, 1992).No changes were detected in the metabolism of human fecalmicrobiota strengthening the idea that data observed in animalmodels should be validated in human trials. However, effects in theupper parts of the digestive tract (ileum, colon) could not be excluded.

The second major result of this human study concerns the transit ofCamembert cheese micro-organisms. In this study, we chose to include

Table 4Enzyme activities (µmol/min/g of protein) and SCFA in human feces before (Ex), during(C1 and C2) and after (W) the consumption of Camembert cheese. (Mean values withtheir standard errors for the twelve subjects)

Fecal sample Ex C1 C2 W

Enzyme activities Mean SEM Mean SEM Mean SEM Mean SEM

β-galactosidase 145.9 22.8 133.4 20.4 145.8 20.7 143.9 21.7α-galactosidase 39.1 5.3 39.0 5.1 43.7 5.3 40.7 4.5β-glucosidase 12.2 1.4 11.5 1.1 13.6 1.8 11.6 1.6β-glucuronidase 6.3 1.0 5.5 1.0 6.8 1.4 6.1 1.0Neuraminidase 0.9 0.2 0.9 0.2 0.9 0.1 0.9 0.3β-N-acetylgalactoaminidase 5.0 0.8 4.9 0.6 5.6 0.8 4.4 0.6α-l-fucosidase 3.9 0.4 3.8 0.4 4.2 0.6 3.6 0.5Nitrate reductase 3.4 0.8 3.5 1.2 1.5 0.6 2.6 0.9Azoreductase 1.6 0.1 1.5 0.3 1.2 0.2 1.5 0.2Total SCFA (µmol/g) 67.9 9.3 68.3 9.1 75.4 7.8 54.9 7.2Acetate (%) 58.0 1.3 58.5 1.1 57.7 1.3 57.1 1.2Propionate (%) 15.6 0.6 16.4 0.5 16.5 0.4 16.3 0.7Butyrate (%) 15.2 0.9 13.6 0.9 15.7 1.3 14.1 1.3Valerate+Caproate (%) 6.8 0.9 7.4 0.8 5.5 0.7 7.6 0.8Iso-acids (%) 5.2 0.5 5.8 0.5 5.5 0.6 6.8 0.8

cheese spread consumption in the control group to avoid a completeelimination of cheese products which may influence the effects onmicrobiota metabolisms. G. candidum was absent in the Exclusion andWash out periods and detected at high level during the Ingestionperiod. This shows its capacity to resist during the gastrointestinaltransit and demonstrated that G. candidum must be considered as agood marker for Camembert cheese consumption. Our results are ingood agreement with those observed in rat model associated withhuman microbiota (Lay et al., 2004).

During the exclusion period, the levels of the targeted bacteriadecreased suggesting a regular undetermined LAB food consumptionbefore the exclusion period. The diet excluding fermented foodproductswas necessary to better assess the fate andpotential influenceof the Camembert cheese micro-organisms. After 2 weeks of cheesespread ingestion, we have not observed high levels of the targeted LABspecies (especially L. plantarum present in similar quantity as inCamembert cheese). This suggests that the dead or non revivablebacteria from the cheese spread are not able to support the passagethrough the gastrointestinal tract and thus enable to obtain a LAB basallevel in human feces. During Camembert cheese consumption, thelevels of L. lactis, Ln. mesenteroides and Lb plantarum (at least during ashort time for this bacterium) increased significantly. Although, wecannot estimate a rate of bacterial recovery, the values of L. lactis andLn. mesenteroides in the feces were in accordance with the highquantities contained in the Camembert cheesemicrobiota. In contrast,the level of Lb. plantarum in feces and in the Camembert cheese wasnot as well correlated: 7.2 Log10 Log10 in human feces versus 4.3 Log10Log10 in Camembert cheese. The large quantity in feces could beexplained either by a multiplication of Lb. plantarum from theCamembert in the digestive tract or by an influence of the cheeseconsumption on the endogenous Lb. plantarum species of the colonicmicrobiota. In the Lactobacillus genus, several strains are considered asprobiotics with beneficial effects on human health identified (Isolauriet al., 2002; Mercenier et al., 2003; Ouwehand et al., 2002; Saavedra,2001; Mater et al., 2005). Ln. mesenteroides is currently isolated fromfermented dairy products and hitherto not described as inhabitants ofintestinal microbiota. Studies have shown that Ln. mesenteroidesexpressed enzymes of medical and biotechnological interest asalteransucrase synthesizing oligosaccharides, for which prebioticproperties have been shown (Sanz et al., 2005). The Camembert LABcan potentially combine different beneficial effects as it was already

181O. Firmesse et al. / International Journal of Food Microbiology 125 (2008) 176–181

demonstrated with VSL#3 probiotic (Kuehbacher et al., 2006). Itcannot be excluded that LAB Camembert cheese could have probioticeffects different from the one investigated here on microbiotametabolism.

In this study, we have developed a Q-PCR approach, which enabledto reach detection limits of about 6 Log10 Log10 genome-equivalent/gof stool. This approach is increasingly used to assess the compositionof the dominant and sub-dominant fecal microbiota (Malinen et al.,2005; Rinttila et al., 2004; Matsuki et al., 2004; Ott et al., 2004) and toallow the follow-up of probiotic bacteria through the digestive tract(Rochet et al., 2006; Bartosch et al., 2005). This technique was appliedto the complex microbiota of Camembert cheese by performing themonitoring of LAB species in healthy human volunteers. To developthe Q-PCR method, three issues were considered: i) the in vitrospecificity to detect bacterial species of the Camembert cheese, ii) thein vivo selectivity for the introduced Camembert bacteria and not forthe endogenous bacteria of the colonic microbiota, and iii) theremoval of the PCR inhibitors coming from the colonic samples. Thein vitro specificity was ensured by developing in silico 16S rRNAprimers validated with a set of target and non target strains. The invivo selectivity and the removal of PCR inhibitors were detected withnatural samples from Camembert cheese and human feces. However,we cannot exclude that the Q-PCR basedmethod could detect not onlyviable bacteria but also non viable bacteria or free DNA of deadbacteria. To overcome this shortcoming, we worked with RNA insteadof DNA and we combined reverse transcription with quantitative PCRto detect viable bacteria as described recently by Oozeer et al. (2005).

This present study demonstrated that the complex microbiota ofCamembert cheese could be detected after digestive transit in fecalsamples of human subjects. The role of such consumption on humanhealth and especially the humanmicrobiota equilibrium remains to bedetermined.

Acknowledgements

The authors thank the manufacturers Lactalis “Beurre et Fromage”for providing the Camembert (Camembert Président). We also thankPhilippe Langella and Sylvie Rabot for the critical reading of themanuscript.

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