American Journal of Clinical Nutrition, Vol. 87, No. 1, 91-96,
January 2008
© 2008 American Society for Nutrition
ORIGINAL RESEARCH COMMUNICATION |
Molecular analysis of yogurt containing Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in human intestinal microbiota1,2,3
Raimundo García-Albiach,
María José,
Pozuelo de Felipe,
Santiago Angulo,
María-Isabel Morosini,
Daniel Bravo,
Fernando Baquero and
Rosa del Campo
1 From the Servicio de Microbiología, Hospital Universitario Ramón y Cajal and CIBER en Epidemiología y Salud Pública, Madrid, Spain (RG-A, M-IM, DB, FB, and RdC), and the Departamento de Microbiología (RG-A and MJPdF) and the Departamento de Métodos Cuantitativos (SA), Universidad San Pablo-CEU, Boadilla del Monte, Spain
2 Supported by a research grant from the "Grupo Leche Pascual, S. L." and by the CIBER en Epidemiología y Salud Pública (CIBERESP). RdC received a research contract from the Fondo de Investigaciones Sanitarias of the Spanish Health Ministry (05/0137).
3 Reprints not available. Address correspondence to R del Campo, Servicio de Microbiología, Hospital Universitario Ramón y Cajal. Ctra. Colmenar, Km 9,1. 28034 Madrid, Spain. E-mail: rosacampo{at}yahoo.com.
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ABSTRACT
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Background: Yogurt has traditionally been considered a probiotic-carrier food with health-promoting effects. Despite the universal assumption of this assertion, several researchers have evaluated the real capability of the yogurt bacteria Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to survive and proliferate in the human intestine and have found contradictory results.
Objective: This double-blind crossover study assessed the qualitative and quantitative effects of fresh and heat-treated yogurt on bacterial intestinal microbiota from healthy subjects.
Design: The subjects were divided into experimental (n = 63) and control (n = 16) groups. The experimental group consumed fresh and heat-treated yogurt for 15 d according to a crossover design with a washout period of 2 wk. Three different fecal samples per individual were recovered: at baseline, after fresh yogurt intake, and after heat-treated yogurt intake. Qualitative changes in microbiota were studied by denaturing gel gradient electrophoresis (DGGE) with universal and lactic acid bacteria (LAB) 16S-rRNA primers. Quantitative changes in LAB, Clostridium coccoides, Clostridium perfringens, and Bacteroides groups were analyzed by real-time polymerase chain reaction.
Results: A particular DGGE stable band pattern was observed in each sample. No significant qualitative differences were detected in any fecal sample. However, a significantly higher density of LAB and C. perfringens and a significant decrease in the density of Bacteroides was observed after consumption of both types of yogurt. Microbiota density was not significantly different between the fresh and heat-treated yogurt groups, except for LAB, which was significantly greater in the fresh yogurt group.
Conclusion: The main change in human microbiota observed after yogurt consumption was an increase in the density of LAB and C. perfringens to the detriment of Bacteroides. Bacterial changes were not different after the consumption of fresh and heat-treated yogurt.
Key Words: Yogurt • human microbiota • molecular analysis • real-time polymerase chain reaction • RT-PCR • denaturing gel gradient electrophoresis • DGGE • healthy individuals
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INTRODUCTION
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Mature human colonic microbiota have traditionally been estimated in at least 1013 microorganisms of different bacterial species, most of which are strictly anaerobic (1). Nevertheless, 99% of the total microbiota consist of only 30–40 different bacterial genera, the relative proportions of which vary (2, 3). Recent studies suggest that each individual host harbors a particular array of gut bacteria that remain relatively constant over time (4). Natural factors modifying normal intestinal microecology, such as diet changes, sex, or age, induce both quantitative and qualitative changes in the composition of intestinal microbiota (5).
Different molecular techniques using total microbiota DNA (microbiome analysis) have been developed to detect and identify microorganisms from complex microbial ecosystems without the necessity of being cultured. Genes encoding the 16S-rRNA are excellent phylogenetic markers for genus and species level determination and their sequences have rendered vast information about the composition of human intestinal microbiota, a particularly complex bacterial system. The denaturing gradient gel electrophoresis (DGGE) technique combines the polymerase chain reaction (PCR) amplification of the 16S-rRNA genes with subsequent electrophoresis of the PCR product in vertical gels in a denaturant gradient, which allows the visualization of even 1% of the dynamic changes in bacterial communities (6, 7). Complementary to qualitative changes explored by the DDGE method, the quantitative real-time PCR technique offers the possibility of analyzing the quantitative changes within a particular bacterial group (8).
Yogurt has traditionally been considered a probiotic-carrier food with health-promoting effects. Despite the universal assumption of this assertion, several authors have evaluated the real capability of the yogurt bacteria Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to survive and proliferate in the human intestine and have obtained contradictory results (9-11). However, the possible effect of living bacteria contained in yogurt on the composition of human intestinal microbiota has not been sufficiently explored. Moreover, the beneficial effects of fresh yogurt compared with those of heat-treated preparations were only observed in patients with lactose maldigestion (12).
The aim of this work was to analyze those changes occurring in the normal fecal microbiota of 63 healthy volunteers after sustained consumption of both fresh and heat-treated yogurt with the use of qualitative PCR-DGGE and real-time (RT)-PCR techniques.
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SUBJECTS AND METHODS
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Study design
A total of 79 healthy young subjects (n = 32 men and 47 women) with a mean age of 23.6 y were included and divided into 2 different groups: 63 in the experimental group and 16 in the control group. The healthy status of the individuals was confirmed by the Preventive Medicine Department on the basis of gastrointestinal health tests, large-spectrum blood tests, and immunologic tests [CD3, CD4, and CD8 lymphocytes; immunoglobulin (Ig) A, IgG, and IgM)]. Apart from the consumption of a daily measured amount of a specific type of yogurt, no special dietary constraints were imposed on the subjects, except that the subjects were instructed to abstain from consuming other types of yogurt or fermented milk preparations. After the subjects were found eligible and gave written consent to participate, a simple randomization scheme using random number tables was applied by an independent statistician. The subjects were then allocated to 2 categories as part of a crossover experimental design. In a first experimental period, 32 subjects ingested three 125-g units of commercial fresh yogurt containing a mean of 1.3 x 107 and 2 x 108 colony forming units (CFU)/g of L. bulgaricus and S. thermophilus, respectively, in a randomized sample of 10 yogurt units daily for 2 wk. In a 1-mo follow-up, none of the units contained <1 x 106 or 1 x 107 CFU/g, so the total bacterial intake was estimated at 
1 x 1011 cells. The same schedule, but with heat-treated yogurt (pasteurized), was applied to the other 31 subjects. Both types of yogurt used were chemically identical and came from the same manufacturer. The sterility of the heat-treated preparation was assessed in 10 yogurt samples that retained a lactic acid bacteria DNA concentration similar to that of the fresh preparation. This was a double-blind prospective study in which fresh and heat-treated yogurts were labeled with code names so that neither the subjects nor the microbiologists were aware of the type of preparation under study in each group. After a washout period of 2 wk without any yogurt intake, both groups were submitted to a crossover experimental period in which each group consumed the opposite type of yogurt used in the first experimental period of the study (heat-treated or fresh yogurt, respectively). The control group of 16 volunteers did not ingest any type of yogurt. Three different fecal samples per individual were recovered from the experimental group (a first baseline sample after 1 wk without yogurt in the diet, a second sample after completion of the first experimental period, and a third sample after the second experimental period of yogurt intake) and from the control group at the same time as recovered from the experimental group but without yogurt intake.
Fecal sample processing and detection of yogurt bacteria
Microbiome DNA extractions were performed with 0.5 g feces, washed twice with 5 mL phosphate-buffered saline (PBS)-EDTA (dilution 1/10), centrifuged at 13 400 rpm at 4 °C for 5 min, boiled for 15 min, and frozen at –20 °C for 1 h. The commercial system FastDNA Spin Kit Qbiogene, Irvine, CA, for soil was used for total DNA extraction. Vortex fecal homogenization was carried out for 6 min in continued cycles of 1 min. The Fast Prep Kit Qbiogene, Irvine, CA, was used for the mechanical break (Bio 101 Inc, La Jolla, CA). Finally, 100 ng DNA was used for each 16S-rRNA PCR reaction.
Denaturing gel gradient electrophoresis
Positive 16S-rRNA amplicons were separated in vertical electrophoresis polyacrylamide gels (8%) at 60 °C; the urea-formamide denaturating gel gradient (33–43%) was submitted to 130 V for 330 min. Gels were visualized with ethidium bromide. For the Lactobacillus-DGGE experiments, the control included a mixture of DNA from Lactobacillus acidophilus ATCC 4356, Lactobacillus reuteri ATCC 23272, Lactobacillus brevis ATCC 14869, Lactobacillus casei ATCC 393, and L. bulgaricus. When the whole microbiota DGGE gels were performed, DNA from Bifidobacterium bifidum ATCC 29521 and Bifidobacterium infantis ATCC 15697 were added to those controls used for the Lactobacillus-DGGE experiments. The primers and conditions used are listed in Table 1
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TABLE 1. Primers and conditions used in the denaturing gel gradient electrophoresis (DGGE) and real-time polymerase chain reaction (RT-PCR) experiments1
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To assess the reproducibility of the DGGE experiments, different assays using the same DNA sample and DNA from the same sample, extracted at 4 different times, were evaluated. Similarities among the electrophoretic band patterns were analyzed by using Phoretix 5.0 software (Nonlinear Dynamics Ltd, All Saints Newcastle, United Kingdom), and dendrograms were constructed based on the Dice coefficient. Profiles with coefficients from 0.9 to 1 were considered to be indistinguishable, from 0.8 to 0.9 were considered to be closely related, from 0.7 to 0.8 were considered to be related, and <0.7 were considered to be unrelated.
Real-time polymerase chain reaction
Oligonucleotides and optimized PCR conditions used in this study are summarized in Table 1
. We developed different group-specific assays for the lactic acid bacteria (LAB), Bacteroides-Prevotella-Porphyromonas group, Clostridium coccoides group, Clostridium perfringens group, and species-specific for Bacteroides vulgatus. Quantitative PCR experiments were performed with a LightCycler 1.5 apparatus with the use of the LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Indianapolis, IN). All PCR tests were carried out in duplicate with a final volume of 20 µL containing 50 ng of each fecal DNA preparation. The thermal cycling conditions used were as follows: an initial DNA denaturation step at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 15 s, primer annealing at optimal temperature (see Table 1) for 20 s, extension at 72 °C for 30 s, and an additional incubation step at 80–85 °C for 30 s to measure SYBR Green I fluorescence. Finally, melt curve analysis was performed by slowly cooling the PCRs from 95 to 60 °C (0.3 °C per cycle) with simultaneous measurement of the SYBR Green I signal intensity.
Statistical analysis
All real-time PCR reactions were performed in duplicate, and the results are expressed as the mean of both experiments. SPSS version 11.5 software was used for statistical data analysis. ANOVA was applied to analyze the number of bands obtained with the DGGE experiments, whereas nonparametric tests (Kruskal-Wallis, Friedman, and Wilcoxon's Mann-Whitney U test) were used to compare the RT-PCR results.
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RESULTS
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Qualitative analysis
The high reproducibility of the DGGE experiments was obtained when the same DNA was amplified in separate experiments and also when DNA from the same sample was extracted at different times. Although differences in the number of bands could be observed, a Dice coefficient of 0.94 was obtained, which indicated high reproducibility.
As expected, the mean number of bands, considering all electrophoretic profiles, was higher for DGGE assays with universal primers (7.68 at baseline, 7.78 after fresh yogurt intake, and 7.81 after heat-treated yogurt intake) than with LAB primers (4.36 at baseline, 4.65 after fresh yogurt intake, and 5 after heat-treated yogurt intake) (Figure 1). Statistically significant differences were not detected with any type of primer in comparisons of baseline samples with samples collected after fresh or heat-treated yogurt.
The analysis of the DGGE gels obtained with universal (whole microbioma) primers indicated a high heterogeneity in electrophoretic patterns among the different individuals. On the contrary, almost the same pattern was observed in the same individual with the 3 different fecal samples successively obtained on days 1, 15, and 45 (Figure 2). No specific differences in band pattern attributable to the subjects’ sex were detected. Of the 79 patients studied, 15 had almost identical patterns, 9 of which were shared by both men and women, which indicated the absence of any sex association.
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FIGURE 2.. Dendrograms of electrophoretic band patterns obtained in the denaturing gel gradient electrophoresis experiments with universal and specific lactic acid bacteria (LAB) primers in 3 different fecal samples collected from 7 subjects (as representation of all subjects). 0, baseline sample; P, sample after fresh yogurt consumption; A, sample after heat-treated yogurt consumption. The number 38 corresponds to a control patient with no yogurt intake.
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The individual pattern observed with the universal DGGE experiments was even more heterogeneous when specific primers for the LAB group were applied (Figure 2). Specific bands for the yogurt bacteria L. bulgaricus or S. thermophilus were undetectable in the LAB-DGGE experiments. Selected shared and individual bands from all profiles were sequenced, and the bacterial identifications are shown in Table 2.
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TABLE 2. Bacterial identification after sequencing the shared and the particular bands obtained with the universal and lactic acid bacteria (LAB) primers in the denaturing gel gradient electrophoresis (DGGE) experiments
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Quantitative analysis
Results obtained in the RT-PCR experiments with the different primers are shown in Table 3. Statistical differences in several bacterial densities (baseline samples and samples after yogurt intake) were observed between the 63 experimental subjects. A significantly higher density of LAB (P 
0.001) and of C. perfringens (P = 0.006) was observed in the samples after fresh yogurt intake than in the baseline sample.
The total number of 16S rRNA copies in the Bacteroides-Porphyromonas-Prevotella group was significantly lower in the fresh (P = 0.02) and heat-treated (P = 0.001) yogurt samples than in the baseline sample. A significant decrease was also observed in the B. vulgatus load with quantitative experiments for both types of yogurts (P = 0.001 and P = 0.007, respectively). The study design annulled the possible carryover effect of the first type of yogurt consumed on the second, because half of the volunteers started the experiment consuming one type of yogurt and the other half the other type of yogurt. Statistical analysis corroborated the absence of a carryover effect. Similarly, the temporal effect was also eliminated by the statistical analysis of the control subjects’ results. Taking account quantitative data after intake of fresh and heat-treated yogurts, significant differences (P = 0.05) were only detected for the LAB group, which were more numerous after fresh yogurt consumption (Table 3).
The relation between concomitant changes (increase or decrease) among different groups of bacterial organisms was also evaluated. Only a global decrease in the total amount of both the B. vulgatus and the Bacteroides-Porphyromonas-Prevotella groups was observed. The apparent density of organisms among bacterial groups in all baseline samples was calculated on the basis of total RT-PCR media results. Density was lowest for the C. perfringens group, followed by the LAB group (22.7 times that for C. perfringens), B. vulgatus (416 times), and the C. coccoides group (2136 times). The highest density was found in the Bacteroides-Prevotella-Porphyromonas group, which was 2520 times that of C. perfringens.
Baseline data were pooled and converted into a normalized distribution for all individuals studied. Considering this distribution, the population was divided into low, middle, and high bacterial density for each bacterial group (15th, 15–85th, and 
85th percentiles). For each group, the genome limits that defined the middle bacterial density group were as follows: 1–75 for LAB, 0.25–10 for C. perfringens, 330–7000 for C. coccoides, 209–7500 for Bacteroides, and 25–1300 for B. vulgatus.
For this study, each subject was used as his or her own control. RT-PCR results showed an overall increase in the proportion of the LAB and the C. perfringens groups after intake of either fresh or heat-treated yogurt. This increase was higher in the group of subjects that initially presented a low density (15th percentile) of these groups of organisms (Figure 3). However, the initial high-density group (85th percentile) also experienced a relevant increase in LAB and C. perfringens groups after yogurt consumption.
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FIGURE 3.. Variation in normalized distribution of total quantitative values of bacterial densities in the 63 healthy subjects after yogurt consumption. Left column: A, subjects with an initial low density (15th percentile) of each of the 5 bacterial groups analyzed; B, lactic acid bacteria (LAB) organisms increased despite their low initial density (most of the group was now in the >85th percentile), whereas the Bacteroides group remained in the <15th percentile. Middle column: A, subjects with middle densities (15th–85th percentile) of each of the 5 bacterial groups; B: yogurt intake scarcely altered the normal distribution. Right column: subjects with the highest initial densities of the different bacterial groups in which LAB and Clostridium. perfringens were high in the baseline sample and remained high throughout the study period, whereas Bacteroides vulgatus density was reduced to the 15th–85th percentile. The nonparametric Friedman, Mann-Whitney U, and Wilcoxon's tests showed that the increase in the LAB and C. perfringens groups and the decrease in the Bacteroides group were statistically significant.
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Global RT-PCR data demonstrated that Bacteroides density decreases after yogurt consumption. This decrease was mostly marked in the initial high density group (85th percentile) (Figure 3). In the C. coccoides group, global density data did not change after yogurt intake, which is also reflected in the distribution.
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DISCUSSION
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The healthy properties of yogurt have traditionally been attributed to its biological effects, promoted by the intake of living bacteria responsible for milk fermentation (18). This classic view implies that the viability of yogurt bacteria is maintained in the gut after yogurt intake. In support of this hypothesis, it can be expected that heat-treated yogurt, without viable organisms, might lack the beneficial effect of fresh preparations on lactose maldigestion (12). This view is also corroborated by the Codex Alimentarius (Codex Standard for Fermented Milks, 243–2003) (19), which recommends the name "yogurt" only for fermented milk with living bacteria. Our group recently challenged the hypothesis of survival of the classic yogurt bacteria L. bulgaricus and S. thermophilus in the human gut (9). Nevertheless, such negative results might not necessarily be applicable to other strains of the LAB group. In this study, we explored changes in gut microbiota after yogurt consumption, knowing in advance that bacteria contained in yogurt do not survive once ingested. We also explored whether any effect on microbiota composition could be expected after heat-treated yogurt consumption in comparison with that after fresh yogurt intake. Results obtained with both fresh and heat-treated yogurts were not different, except for the density of the LAB group, which strongly suggests that changes in microbiota are mainly due to the properties of the yogurt itself, which do not require bacterial viability. This finding suggests that yogurt should also be considered a prebiotic-like food, although this theory must be demonstrated following the criteria of Gibson and Roberfroid (20). Nevertheless, probiotic effects cannot be discarded for specific yogurt strains able to survive and reproduce in the human intestinal tract.
The DGGE analysis showed that the individual composition of intestinal microbiota remained stable during the study period, and no significant differences were observed after the exposure to either type of yogurt. The DGGE analysis corroborated previous results indicating that classic bacteria used for yogurt fermentation were unable either to survive after ingestion or to colonize the intestinal tract (9). DGGE experiments using universal primers allowed us to identify particular band patterns among the different individuals, although we consider that the low number of bands detected with these primers hinders the precise analysis of qualitative changes.
On the contrary, we observed significant changes when we performed the quantitative analysis. After yogurt intake, a clear reduction in the Bacteroides population and a concomitant increase in the LAB population were observed. Note that the study was designed to evaluate changes in the proportion of different bacterial groups after yogurt intake, and the results do not necessarily reflect the actual number of organisms in each of the groups. This number is certainly difficult to calculate because of the heterogeneity in the number of rRNA copies inside each bacterial group (16). In a comparison of fecal samples collected after the intake of fresh and heat-treated yogurts, we detected a significant difference only in the LAB group, the density of which was more numerous after the intake of fresh yogurt (P = 0.05). For the other bacterial groups studied, the quantitative analysis showed no significant differences after intake of both types of yogurts.
Considering that all subjects were healthy, we normalized the data obtained at baseline to classify the population into low, middle, and high bacterial group densities that corresponded with the 15th, 15–85th, and 85th percentiles, respectively. Using these baseline considerations, we analyzed the relevance of each bacterial density group to the qualitative changes after yogurt consumption. In the case of LAB and C. perfringens, the global increase observed was at the expense of the low-density group, whereas the decrease in Bacteroides was most marked in the initial high-density group. These results indicate that yogurt consumption contributes to the normalization of bacterial density groups.
Healthy persons constitute most of the yogurt-consuming population and represent one of the targets for which presumptive intestinal health benefits are claimed by the dairy industry. In conclusion, the results obtained in young subjects in the present study may be applied to the global healthy population. It would be of interest to apply a similar experimental approach to investigate the possible microbiotic changes in patients with different gut diseases.
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ACKNOWLEDGMENTS
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We are grateful to Víctor Abraira (Unidad de Bioestadística, Hospital Universitario Ramón y Cajal), to Clotilde Vazquez (Servicio de Dietetica y Nutricion, and to Francisco Javier Yuste (Servicio de Salud Laboral, Hospital Universitario Ramón y Cajal) for their contribution to the project design and to Santos del Campo for his help with the RT-PCR experiments.
The authors’ responsibilities were as follows—RG-A, MJPdF, and DB: conducted the hands-on work; M-IM, FB, and RdC: drafted the manuscript; and SA: performed the statistical analysis. The authors had no conflict of interests.
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Received for publication February 19, 2007.
Accepted for publication August 17, 2007.
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ABSTRACT
INTRODUCTION
, controls (no yogurt consumption); 
, yogurt consumers. Significant differences were not detected between either type of yogurt and the baseline sample (ANOVA).