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Real-time PCR was carried out using an AB 7300 system (Applied Biosystems, Foster City, CA) and sequence detection software (Version 1.3; Applied Biosystems, Foster City, CA). Each reaction was run in triplicate in a volume of 25 l in optical reaction plates (Applied Biosystems, Foster City, CA) sealed with optical adhesive film (Applied Biosystems, Foster City, CA). Amplification reactions were carried out with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) mixed with the selected primer set at a concentration of 0.5 M for each primer, and 2 l (~12 ng) of genomic DNA. To evaluate the efficiency (E) of the amplification of each primer set, DNA templates were pooled (50 ng/reaction) and serially diluted 8 fold. Amplification efficiency was calculated from the slope of the standard curve generated from plotting the threshold cycle (CT) versus logarithmic values of different DNA concentrations using the following equation (Denman and McSweeney, 2005):

E=10-1/slope (5)

Relative quantification was accomplished using following mathematical model (Pfaffl, 2001):

Ri = [(Etarget)ΔCTtarget (Controli - SARAi)]/[(Eref)ΔCTref (Controli - SARAi)] (6)

Where target is the 16S rDNA gene of interest, ref is Eubacteria, ΔCT is the CT deviation of the control vs treatment, i is the period, and Ri is the relative expression ratio of a target gene compared to a reference gene at a specific time point.

Statistical analysis

All data were expressed as mean+/- SE. Statistical significance was set at P<0.05 for all analyses. Log transformation was preformed when data were determined to be not normally distributed. Differences between treatments at baseline, midpoint and endpoint for lipids, cholesterol absorption and synthesis rates, fecal bile acid concentrations, body composition were compared by using the analysis of variance (ANOVA) model for determination of diet effects. When diet effects were found to be significant, Least Squares Means was used to identify differences between diet effects. Student's paired- t test was used to compare baseline and midpoint as well as baseline and endpoint within each diet. Differences of percent changes at endpoint relative to control between L. fermentum and L. reuteri treatments were also analyzed using Student's paired-t test.

The LSD multiple comparison test was conducted to detect significant differences among treatment groups in analyzing gut microbial composition parameters.

Data were analyzed with the use of SAS software (version 8.0; SAS Institute Inc, Cary, NC, USA).

Results

Two hundred and thirty seven subjects underwent blood screening sessions. Forty-eight subjects were initially recruited and thirty subjects (11 males and 19 females) completed the entire trial. Eighteen subjects dropped out due to difficulties with consuming study diets and/or with accommodating the setting of the study (n=2, dropped out on the first day), relocation to another city (n=1), problems with daily centre visiting (n=7), personal reasons (n=4), and difficulties with re-starting the study (n=4).

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