Molecular detection of Bifidobacterium animalis DN-173010 in human feces during fermented milk administration

Food Research International 39 (2006) 530–535 www.elsevier.com/locate/foodres

Molecular detection of Bifidobacterium animalis DN-173010 in human feces during fermented milk administration M.C. Collado a, Y. Moreno a, J.M. Cobo b, J.A. Mateos b, M. Herna´ndez a

a,*

Department of Biotechnology, Polytechnic University of Valencia, Camino de Vera 14, 46022 Valencia, Spain b Red INDE, Investigacio´n Nutricional Danone Espan˜a, C/Buenos Aires, 21, 08029 Barcelona, Spain Received 17 October 2005; accepted 21 October 2005

Abstract We tested different techniques to detect exogenous bifidobacteria (DN-173010) in feces; genus- and species-specific PCR technique; amplified ribosomal DNA restriction analysis (ARDRA) and fluorescent in situ hybridization (FISH) technique. A significant increase in the number of bifidobacteria in feces was observed during ingestion of fermented milk, and also, we detected a decrease in this number when the ingestion stopped. The number of bifidobacteria enumerated by culturing was 10–100-fold lower than by FISH technique. Bifidobacterium animalis DN-173010 can survive passage through the gastrointestinal tract and was detected viable in human feces. Combination of ARDRA and FISH was a powerful tool to detect exogenous bifidobacteria. The aim of this study is to demonstrate that Bifidobacterium DN-173010 can survive passage through the gastrointestinal tract and be recovered live in human feces. For this purpose, we have assessed different techniques to detect and quantify the number of bifidobacteria following fermented milk supplemented with B. animalis subsp. lactis DN-173010 administration and to confirm that this strain can survive passage through the gastrointestinal tract by recovering viable cells in human feces.  2005 Elsevier Ltd. All rights reserved. Keywords: Bifidobacterium; Fermented milk; ARDRA; FISH; Fecal samples

1. Introduction The use of live microbes as dietary adjuncts or ‘‘probiotics’’ is a subject of intense and growing interest. Probiotics have been defined as living organisms that, when included in the diet, have a favorable effect on the host (Fuller, 1991). Bifidobacteria are particularly important since they represent up to 91% of the total population of the intestinal tract in newborns and between 3% and 7% in adults (Biavati & Mattarelli, 2001). The contribution of these bacteriato good health has been recognized for quite some time and has led to widespread use of bifidobacteria as probiotics for maintaining or improving human and animal health (Stanton et al., 2001). This growing interest in the *

Corresponding author. Tel.: +34 96 387 74 23; fax: +34 96 387 74 29. E-mail address: [email protected], [email protected] (M. Herna´ndez). 0963-9969/$ - see front matter  2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.10.011

health benefits of bifidobacteria has prompted inclusion of these organisms in many dairy foods and led to increased consumption. This development requires control of the quantity of probiotics that the product contains, their capacity for survival in gastrointestinal conditions in order to arrive live at the end of the intestine in enough quantities (106–107 microorganisms per ml) (Bouhnik et al., 1992). For this reason, methods for specific identification of probiotic strains are necessary. Accurate identification techniques could serve in monitoring the progress of the population of specific probiotics through the gastrointestinal tract. Since there are inherent difficulties in obtaining definitive evidence for the suggested beneficial effects of bifidobacteria consumption, there is a great deal of speculation about the possible prophylactic and therapeutic properties of foods containing bifidobacteria. One difficulty is the presence of endogenous bifidobacteria in the gastrointestinal tract and human feces, which complicates the

M.C. Collado et al. / Food Research International 39 (2006) 530–535

task of differentiating these bifidobacteria from ingested bifidobacteria and unequivocally demonstrating the survival of ingested bifidobacteria through the gastrointestinal tract. Molecular based techniques can be used to improve the efficiency of detection and enumeration methods. Thus, PCR genus-specific, RAPD-PCR, amplified ribosomal DNA restriction analysis (ARDRA) and hybridization with specific DNA probes have been used to compare and identify bifidobacteria isolated from dairy products and human feces (Gueimonde et al., 2004; Satokari et al., 2003; Temmerman, Masco, Vanhoutte, Huys, & Swings, 2003; Ventura, Van Sinderen, Fitzgerald, & Zink, 2004; Ventura & Zink, 2002). For this study, we have selected Bifidobacterium animalis subsp. lactis strain DN-173010 contained in the most popular and consumed commercial dairy product in Spain. There are some studies about this strain (Duez et al., 2000; Lepercq, Relano, Cayuela, & Juste, 2004; Marteau et al., 2002; Tavan et al., 2002) but in this study we test different techniques to detect bifidobacteria (B. animalis subsp. lactis DN-173010) in human feces and describe the results of an in vivo study in which healthy human subjects consumed this product with viable bifidobacteria. This paper describes the results of an in vivo study in which healthy human subjects consumed a probiotic product with a viable bifidobacteria DN-173010 strain. The aim of this study was to demonstrate that B. animalis subsp. lactis DN-173010 can survive through the gastrointestinal tract and to obtain accurate data on the fate of ingested bifidobacteria in humans as a first step towards assessing the physiological importance of ingested bifidobacteria. We have monitored total bifidobacteria in fecal samples of healthy subjects throughout the course of a controlled DN-173010 administration using different techniques including selective culture techniques, molecular typing based on amplified 16S rRNA gene and digestion with different restriction enzymes of Bifidobacterium isolates. 2. Materials and methods 2.1. Subjects Healthy volunteers (n = 12) aged 25–40 years of age (mean 32.5 years), who did not include dairy products in their regular diet, participated in the study. Throughout the study period, the subjects were asked to avoid other fermented milks, oligosaccharides and any food or antibiotic that may influence the fecal microbiota other than used in the study. Otherwise there were no specific dietary restrictions. Also, the subjects did not have any history of milk allergy or intolerance nor other symptoms during the study. A total of 10 subjects ingested commercial fermented milk and two out of these 10 subjects were monitored as controls; negative control (individual without product ingestion) and positive control (subject with continuous product ingestion for three months prior the study).

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The volunteers provided written informed consent prior to the start, and the study was conducted based on the principles of the World Medical Association Declaration of Helsinki (1964) (Ethical Principles for Medical Research Involving Human Subjects). 2.2. Fermented milk administration Commercial fermented milk with B. animalis subsp. lactis DN-173010 produced by Danone Espan˜a S.A. was administered to healthy subjects. Also, the product contains Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains. Fermented milk was administered at weekly intervals up to 2 weeks before their expiry dates. The number of viable bifidobacteria in 250 ml of the fermented product was 1.4 · 109 CFU/ml. These results were obtained by traditional plate counting using specific bifidobacteria media (BFM agar; Nebra & Blanch, 1999) following incubation at 37 C for 48 h under anaerobic conditions. The study lasted 12 weeks, with the following schedule: control period (4 weeks with no ingestion of any fermented milk with bifidobacteria), 250 ml product administration period (4 week period with administration of fermented milk) and post-administration period (4 week period with no administration of bifidobacteria). During the bifidobacteria administration period, subjects consumed 250 ml of the DN-173010 fermented milk. Throughout the entire experimental period the volunteers did not consume any liquid milk products or fermented dairy products prepared using lactic acid bacteria (LAB). 2.3. Examination of fecal samples Fecal analysis was performed at the end of control period (1st sample) and every week during the administration (2nd, 3rd, 4th and 5th sample) and post-administration (6th, 7th, 8th and 9th sample) periods. Samples were collected fresh in sterile plastic recipients, refrigerated and processed without further delay. After thorough mixing, the fecal sample (2 g wet weight) was weighted and serial 10-fold dilutions from 1010 to 108 were prepared in PBS buffer (130 mM sodium chloride, 10 mM sodium phosphate, [pH 7.2]) and plated on the appropriate agar media. Bifidobacteria were enumerated on BFMAgar (Nebra & Blanch, 1999) and the plates were incubated at 37 C for 72 h in an anaerobic jar filled with an atmosphere of oxygen free CO2 using anaeroGen sachets (Oxoid, Hampshire, England). Total enterobacteria were enumerated on McConckey (Merck, Darmstadt, Germany) agar and plates were incubated aerobically at 37 C for 24 h. All colonies grown on McConckey were assumed to be enterobacteria. Duplicate plate values were averaged and bacterial densities were expressed as the log of the number of CFU/g wet weight of feces. After incubation at 37 C for 3–4 days in anaerobic conditions, 10–12 colonies from BFM media agar were selected and re-plated on the same media. Colonies were examined and those showing

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morphological characteristics of bifidobacteria were subcultured on Tryptone–Peptone–Yeast Extract (TPY) agar (Scharlau Chemie, Barcelona, Spain). Stock cultures were prepared in TPY supplemented with 10% glycerol and stored at 20 and 80 C. 2.4. FISH analysis of fecal samples The enumeration of Bifidobacterium cells present in feces was carried out by fluorescent in situ hybridization (FISH) using a 16S rRNA oligonucleotide probe specific to the Bifidobacterium genus (Bif662) as described by Langendijk et al. (1995). Probe was synthesized and labelled with FITC by MOLBIOL (Berlin, Germany). An aliquot (1 ml of fecal homogenate in PBS) was centrifuged (1000 rpm, at 4 C for 10 min) and fixed with 4% paraformaldehyde at 4 C overnight. The preparations were stored at 20 C until they were analyzed. Fixed samples were centrifuged again, washed with PBS buffer and finally resuspended in 1:1 PBS/ethanol (v/v). An aliquot of 5 ll fixed bacteria was placed on a gelatine-coated slide, dehydrated (50%, 80%, 100% ethanol) and hybridized as described by Amann, Ludwig, and Schleifer (1995). Slides were visualized by Olympus BX50 epifluorescence microscope mounted with U-MWIB and U-MWIG filters. Results are presented as the average of the 20 fields counted per sample and per staining.

2.5.2. Amplified rDNA restriction analysis (ARDRA-PCR) A specific 1350 bp 16S rRNA gene fragment from Bifidobacterium strains was amplified by PCR using the primers Lm26 and Lm3 (Kaufmann et al., 1997). Amplified DNA was concentrated by adding ethanol and resuspended in 15 ll of TE buffer. DNA was digested with 10 U of different restriction endonucleases MboI and BamHI (New England BioLabs, UK) in a final reaction (one for each enzyme) volume of 20 ll at 37 C for 3 h following the procedure described by Ventura et al. (2001). Restriction reaction was stopped by adding 3 ll of stopmix solution (50 mM EDTA, 0.3% Ficoll, 0.3% bromophenol blue). The digestion products were visualized by 3% agarose gel electrophoresis in TAE buffer (40 mM Tris– acetate, 2 mM EDTA, pH 8.3) stained with ethidium bromide (0.5 ll/ml) and visualized under UV light. 2.6. Statistical analysis The numbers of bacteria were converted to log colonyforming units (CFU)/g wet weight and expressed as means ± SD (standard deviation). Results obtained during study were analyzed using the SPSS 11.0 software (SPSS Inc, Chicago, IL, USA) and data were subjected to oneway ANOVA. Results were considered statistically significant if P < 0.05 using ANOVA test. 3. Results and discussion

2.5. Molecular identification of Bifidobacterium spp. isolates from fecal samples 2.5.1. Genus- and species-specific PCR The confirmation of the identity of presumptive isolates on BFM agar with positive characteristics of bifidobacteria was carried out by PCR using genus-specific primers LM26 and LM3 with sequences 5 0 -GAT TCT GGC TCA GGA TGA ACG-3 0 and 5 0 -CGG GTG CTI ICC CAC TTT CAT G-3 0 , according to the procedure of Kaufmann, Pfefferkorn, Teuber, and Meile (1997), which amplified a specific 1350 bp fragment of the 16S rRNA gene. The application of the oligonucleotide pair Bflact2-Bflact5 (Bflact2 5 0 -GTG GAG ACA CGG TTT CCC-3 0 and Bflact5 5 0 -CAC ACC ACA CAA TCC AAT AC-3) according to Ventura, Reniero, and Zink (2001) resulted in a 680bp amplification fragment only with DNA derived fromB. animalis subsp. lactis, whereas absolutely no PCR product could be detected with those primers for any other Bifidobacterium such as Bifidobacterium infantis (ATCC 15697), Bifidobacterium longum (ATCC 15707), Bifidobacterium bifidum (ATCC 2952) and Lactobacillus strains such as L. delbrueckii subsp. bulgaricus NCFB 1006, L. delbrueckii subsp. delbrueckii ATCC 9649, Lactobacillus casei ATCC 393 and Lactobacillus acidophilus ATCC 9224. Amplified fragments were separated by 1.5% (w/v) agarose gel electrophoresis in TAE buffer and visualized by staining with ethidium bromide (0.5 lg/ml) and photographed under UV light (wavelength, 260 nm).

3.1. Analysis of fecal samples The results of B. animalis subsp. lactis DN-173010 ingestion on human bifidobacteria and Enterobacteriaceae are shown in Table 1. The plate counts for the control subjects during the entire study were: for the positive control; 7.70 ± 0.42 log CFU bifidobacteria/g and 7.34 ± 0.47 log CFU enterobacteria/g, and for the negative control; 6.98 ± 0.24 log CFU bifidobacteria/g and 7.12 ± 0.34 log CFU enterobacteria/g. During the consumption period, we detected a significant increase (P < 0.05) in the number of bifidobacteria on BFM agar plates and a decrease (P < 0.05) in bifidobacteria counts during the post-administration period. The number of total enterobacteria varied throughout the experiment, depending on the individual but the number of enterobacteria increased (P < 0.05) when the ingestion of B. animalis subsp. lactis DN-173010 stopped (Table 1). 3.2. FISH analysis of fecal samples The mean total numbers of bifidobacteria in the control period obtained using FISH technique were 8.82 ± 0.59 log cells/g (mean ± standard deviation of log cells/g wet feces), this number depending on the individual. The results obtained during administration and post-administration periods are shown in Table 1. The fermented milk contains 109 cells/g B. animalis subsp. lactis DN-173010

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Table 1 Total fecal bifidobacteria and enterobacteria concentrations in 10 healthy subjects before, during and after ingestion of B. animalis DN-173010 contained in fermented milk Time (week)

Control period 1 week administration period 2 weeks administration period 3 weeks administration period 4 weeks administration period 3 weeks post-administration period 4 weeks post-administration period

Plate counts (log CFU/g wet feces) (n = 10)

FISH counts (log cells/g wet feces) (n = 10)

Bifidobacterium spp. (BFM agar)

Enterobacteraceae (McKonkey agar)

Bifidobacterium spp. (Bif 662 probe)

6.22 ± 0.34 8.45 ± 0.71a 8.09 ± 0.70a 8.70 ± 0.78a 8.86 ± 0.44a 8.35 ± 0.37a

6.86 ± 0.64 6.36 ± 1.11 6.06 ± 0.86b 6.25 ± 0.89b 6.10 ± 1.25b 7.37 ± 1.36a

8.82 ± 0.59 9.73 ± 0.47 9.82 ± 0.79a 10.37 ± 0.66a 10.41 ± 0.81a 10.12 ± 0.90a

6.95 ± 3.71

7.39 ± 0.54a

8.50 ± 0.42

Mean ± SD (standard deviation). a Significantly higher than the control group (P < 0.05). b Significantly lower than the control group (P < 0.05).

14

14

12

12

10

10

8

8

6

6

4

4

2

2

0

Plate counts (Log CFU/ml)

FISH counts (Log cells/ml)

and all the cells give positive signals with the Bif662 probe. The detection limit of FISH for DN-173010 strain was 104 cells/g. All throughout the study, the feces of the positive and negative controls were monitored. During the consumption period, we detected a significant increase (P < 0.05) in the number of bifidobacteria in feces, these significant changes are in line with the results obtained using plate counts. After 2 weeks of post-administration period, the probe Bif662 counts revealed a progressive decrease in the number of Bifidobacterium spp. even on the original levels of bifidobacteria found in the control period. The initial bifidobacteria counts in feces using plate counts were between one and two orders of magnitude less than those obtained by FISH (Fig. 1).

0 1

2

3

4

5

6

7

8

9

Sampling FISH counts Bif662 Plate counts BFM agar

Fig. 1. Total bifidobacteria counts in the feces of 10 healthy volunteers after consumption of B. animalis DN-173010 containing fermented milk using plate count method and FISH technique. Sampling 1 = control period; sampling 2–5 = administration period and sampling 6–9 = postadministration period (bars: counts using genus-specific probe Bif 662; line: counts using plate count method).

3.3. Molecular identification of Bifidobacterium spp isolates from fecal samples 3.3.1. Genus- and species-specific PCR All presumptiveBifidobacterium strains isolated from the commercial fermented milk and feces were identified by genus-specific PCR (Kaufmann et al., 1997) and fluorescent in situ hybridization technique using a Bifidobacterium genus-specific probe as described by Amann et al. (1995). In every case, a 1350-bp 16S rDNA fragment was detected confirming the correct assignation of the strains to the genus Bifidobacterium (data not shown). Moreover, all the strains isolated on BFM agar gave a positive signal when hybridized with the genus-specific probe Bif662 for Bifidobacterium spp. With both molecular techniques we could identified all bifidobacteria isolated from commercial fermented milk and from human feces at genus level. Nevertheless, the FISH technique is faster than PCR since results are obtained in few hours. To evaluate the presence of B. animalis subsp. lactis in commercial fermented milk and in human feces we used B. animalis subsp. lactis specific PCR primers according to Ventura et al. (2001). A 680-bp fragment was amplified for all bifidobacteria isolated from commercial fermented milk confirming the correct assignation of the bifidobacteria strains in commercial fermented milk as belonging to the species B. animalis subsp. lactis which agrees with the labelling on bacterial strains stated by the producers. Throughout the study, we monitored the positive and negative controls and the rest of individuals using genusand species-specific PCR and FISH. All bifidobacteria isolated from the positive control amplified specific fragments corresponding to Bifidobacterium genus and B. animalis subsp. lactis species by PCR. Besides, these bifidobacteria gave also positive signal when hybridized with the Bif662 probe. For the negative control, there were positive signals when the Bif662 probe was used but we failed to detect any species-specific B. animalis subsp. lactis fragment amplification by PCR.

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Fig. 2. 16S rRNA restriction fragment length polymorphism of B. animalis DN-173010 from a representative subject after 4 weeks of administration period. (M) 100 bp ladder marker; (E) bifidobacteria spp.; (P) B. animalis DN-173010 profile present in commercial fermented milk.

Specific 680 bp fragment of B. animalis subsp. lactis was detected in the feces of all the subjects after the second week of fermented milk administration in 30–32% of bifidobacteria isolated from human feces. The proportion of selected colonies with amplified 680 bp fragment rose quickly during feeding until a maximum of 80–90% and became the predominant strain of bifidobacteria after 4 weeks of consumption (90% isolates gave positive amplification of species-specific PCR). This result suggested that these fragments corresponding to B. animalis subsp. lactis could come from the strain DN-173010 present in the fermented milk administrated. After five weeks of non-consumption of the product, we were unable to detect any specific B. animalis subsp. lactis amplification from the strains isolated in fecal samples. Our results suggested that this strain can survive passage through the gastrointestinal tract. Nevertheless, when DN-173010 consumption was stopped, the proportion of the amplified B. animalis specific fragment in the strains isolated from feces diminished gradually and was not detectable after 5 weeks. 3.3.2. Amplified rDNA restriction analysis (ARDRA-PCR) The restriction enzymes MboI and BamHI were selected for the digestion of the 16S rRNA fragment, since this allows a clear differentiation between the B. animalis subsp. lactis, strain and the rest of the Bifidobacterium species according to their restriction profiles. We used this technique to compare the restriction profile of the DN-173010 strain contained in commercial fermented milks with the profiles obtained for the bifidobacteria isolated from human feces following product ingestion. DN173010 strain contained in the commercial fermented milk shows only one reproducible restriction profile which is different from the other profiles of bifidobacteria isolated from fecal samples. Thus, four bands were observed with the use of MboI (molecular weights from 550 to 150 bp) and three bands with molecular weights ranging from 1400 to 50 bp were exhibited when BamHI enzyme was used.

It was proved that the increase of the bifidobacteria counts agrees with an increase in the proportion of appearance of the B. animalis subsp. lactis DN-173010 profile in the molecular tests (Fig. 2). After 2 weeks of fermented milk administration we can observe that the same DN173010 profile appears in 40% of bifidobacteria isolated from human feces. This number was increased to 60% after 3 weeks of administration and 90% after 4 weeks of fermented milk ingestion. Our results demonstrate that colonies of bifidobacteria of subjects consuming DN-173010 strain have a 16S rDNA ARDRA that is identical to that of DN-173010, suggesting that this strain can survive passage through the gastrointestinal tract. B. animalis subsp. lactis DN-173010 was detected in the feces of all the subjects after the second week. The proportion of selected colonies with digestion of 16S rRNA gene fragment matching that of DN173010 rose quickly during feeding first and became the predominant strain of bifidobacteria after 4 weeks of consumption (Fig. 2). Nevertheless, when DN-173010 consumption was stopped, the proportion of the ingested organism in the feces diminished and was not detectable after 5 weeks. 4. Conclusions In this study, 10 healthy subjects were administered B. animalis subsp. lactis DN-173010 fermented milk. We monitored the increase of total bifidobacteria in feces during DN-173010 fermented milk consumption using a conventional culture method prior to molecular analysis. We employed a molecular method to distinguish between endogenous bifidobacteria and an unmodified, ingested food-grade bifidobacteria. Total bifidobacteria increased significantly during consumption in all the subjects. The observations presented demonstrate that colonies of bifidobacteria selected from the feces of subjects consuming DN-173010 strain have a 16S rDNA ARDRA profile that is identical to that of DN-173010, suggesting that this strain can survive passage

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through the gastrointestinal tract. B. animalis subsp. lactis DN-173010 was detected in the feces of all the subjects after the second week of ingestion using specific PCR and ARDRA techniques. The proportion of selected colonies with digestion of 16S rRNA gene fragment matching that of DN-173010 rose quickly during feeding. Nevertheless, when DN-173010 consumption was stopped, the proportion of the ingested organism in the feces diminished and was not detectable after 4–5 weeks non-consumption. Using conventional plate counts we can observe an increase in the total fecal bifidobacteria population following the consumption of DN-173010 strain. We can also observe the same results using molecular techniques such as ARDRA (amplified rDNA restriction analysis). This suggests that this increment in the total bifidobacteria population was brought about by an increase in the DN173010 strain in feces, whereas the lower reduction in total fecal numbers after stopping consumption of the DN173010 strain showed that this strain may act as a prebiotic for endogenous bifidobacteria. This increase in the total bifidobacteria population, which was presumably induced by DN-173010 strain consumption, also suggests that the DN-173010 strain can survive passage through the gastrointestinal tract. Therefore, continued product ingestion is required to maintain the appropriate number of B. animalis subsp. lactis DN-173010 to cause beneficial effects for the host, since they remain in the intestinal tract for approximately 4–5 weeks. In this study, we have demonstrated that ingested DN173010 bifidobacteria can survive passage through the gastrointestinal tract into the feces. Results show that it was possible to detect and recover ingested DN-173010 bifidobacteria strain in the feces of all subjects using molecular techniques suggesting that these bacteria can survive through the gastrointestinal tract. Procedures used in this study are effective and rapid to detect and to quantify bifidobacteria in human feces. Also, the application of the specific-PCR and FISH techniques in the analysis of human feces can be a very useful tool for rapid monitoring of the B. animalis subsp. lactis species (product claims, bacterial counts, strains and species identification). Both techniques allowed us a good, fast, precise detection and identification of bifidobacteria in feces and in dairy products, and could be routinely used to detect and identify ingested bifidobacteria. Acknowledgement An FPU/MEC (Spain) scholarship to M.C. Collado is fully acknowledged. References Amann, R. I., Ludwig, W., & Schleifer, K. H. (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews, 59, 143–169.

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