A total of 16 male, specific pathogen-free Sprague-Dawley rats (3 weeks old) were purchased from Hiroshima Laboratory Animal Centre (Hiroshima, Japan) and maintained according to the Guide for the Care and Use of Laboratory Animals of Hiroshima University (Hiroshima, Japan). The study was approved by the Research Ethics Committee of Hiroshima University (approval no.: C15-12). The rats were individually housed in an air-conditioned room at 23–24°C under a 12-h light/dark cycle (lights on from 08:00 a.m. to 8:00 p.m. Following acclimatization with a non-purified commercial rodent diet (MF diet for rat and mouse; Oriental Yeast Co., Ltd., Tokyo, Japan) for 3 days, the rats (mean body weight, 68.3±0.5 g) were randomly assigned to one of two groups (n=8 rats per group) based on experimental diet. The compositions of the experimental HF diets with and without FOS supplementation are summarised in Table I. In these diets, the 30% beef tallow and 10% FOS were in accordance with previous studies (14,15). The rats were randomly assigned to one of the two diets supplemented with or without 10% FOS (w/w; Wako Pure Chemical Industries, Ltd., Osaka, Japan). Equal quantities of the experimental diets were incorporated daily into food cups at 17:00 (9, 10, 12, 14 and 15 g on days 1, 2–4, 5–7, 8–12 and 13–14, respectively) to prevent differences in food intake. Food intake was determined from the food consumed each day until the next day's food was served. The weight of spilled food was recorded daily and appropriately incorporated into calculations of food intake. At the end of the feeding period, the rats were sacrificed by decapitation following brief exposure to 3–4% isoflurane gas (Wako Pure Chemical Industries, Ltd., Osaka, Japan) mixed with air in an anesthesia chamber. A total of 8 ml blood was collected from the neck, and serum was separated by centrifugation at 2,000 × g for 20 min at 4°C and stored at −80°C. The intestinal digesta was immediately isolated, weighed and stored at −80°C until the subsequent analyses of ethanol, organic acid and beneficial bacteria.

To determine ethanol levels by gas chromatography, 100 mg of the intestinal digesta and 100 µl serum were transferred into individual ice-cold 0.6 N perchloric acid solutions (PCA). The cecum PCA solution was homogenized on ice (Polytron RT-MR2100; Kinematica AG, Littau-Luzern, Switzerland), and the serum PCA solution was mixed for 10 sec on a vortex-mixer (17). The extracted samples were centrifuged at 10,000 × g at 4°C for 5 min, and the supernatant was stored as 1.0 ml samples collected in 20 ml glass vials at −80°C until measurement. 1-Propanol at a final concentration of 100 ppm was included as internal standard prior to analysis. A gas-chromatograph (GC-2014; Shimadzu Corporation, Kyoto, Japan) equipped with a flame ionization detector was used for analysis. A headspace autosampler (HT2000H; ALPHA M.O.S. Japan K.K., Tokyo, Japan) was used to load the analyses. DB-WAX capillary columns (1.0 µm thickness, 30 m length, 0.53 mm internal diameter) from Agilent Technologies Japan, Ltd. (Tokyo, Japan) were applied. Samples were incubated for 30 min at 65°C in the head-space autosampler. Samples (0.5 ml) were injected into the column at a rate of 1 ml/min with the injector maintained at 200°C and detector at 250°C. An initial temperature of 40°C was maintained for 1 min and then the column oven temperature was increased from 40 to 80°C at a rate of 8°C/min and then maintained at 80°C for 1 min. The column oven temperature was increased from 20 to 180°C at a rate of 20°C/min and then maintained at 180°C for 4 min. The flow rate of the carrier gas (N₂) was 1.0 ml/min (split ratio 10:1). Standard curves were linear across four different concentrations (0.00, 3.25, 6.50 and 13.00 mmol/l) of ethanol (R²>0.99), and used for the assay of ethanol levels in the samples.

For the analysis of intestinal microflora, bacterial genomic DNA was isolated from the cecum digesta using an UltraClean™ Fecal DNA extraction kit (MO BIO Laboratories; Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's instructions. The purified DNA was eluted in 100 µl elution buffer (5 mM Tris-HCl, pH 8.5), and the quality and quantity of DNA were determined by measuring the absorbance at 260 and 280 nm using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Samples were stored at −20°C. Real-time quantitative polymerase chain reaction (qPCR) was performed to investigate the variation in the total number of bacteria, including of Bifidobacterium spp. and Lactobacillus spp. The 16S rRNA primers for Bifidobacterium spp. and Lactobacillus spp. used for qPCR have been described previously (14). The total volume of reagent mixture for each PCR was 20 µl [4.4 µl distilled water, 10 µl Master mix (Takara Bio, Inc., Otsu, Japan), 2 µl Plus solution (Takara Bio, Inc., Otsu, Japan), 0.8 µl of each forward and reverse primer and 2 µl DNA]. The reaction conditions were as follows: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, 55°C for 15 sec and 72°C for 30 sec. The fluorescent products were detected at the last step of each cycle. Melting curve analysis was performed following amplification to distinguish the targeted PCR product from the non-targeted PCR products. Data were analysed using the second derivative maximum method of the StepOneTM Real-time PCR software version 2.3 (Applied Biosystems; Thermo Fisher Scientific, Inc.). The copy numbers of Bifidobacterium spp. and Lactobacillus spp./µl were determined for the standard plasmid solution of these bacteria [(cut standard plasmid mixture ng/µl) × (molecules bp/1.0 × 10⁹ ng) × (1/660 DNA length bp/plasmid)=plasmid copies/µl] (14). Real-time qPCR products were run as five 10-fold serial dilutions of each standard mixture to compare the threshold cycle number with the copy number of the target sequence and to generate standard curves for the quantification of unknown samples. Typically, standard curves were linear across five orders of magnitude (R²>0.98).

Cecum SCFAs were measured according to an internal standard method using high-performance liquid chromatography (HPLC; L-2130; Hitachi, Ltd., Tokyo, Japan) equipped with an Aminex HPX-87H ion exclusion column (7.8 mm internal diameter × 30 cm; Bio-Rad, Laboratories, Hercules, CA, USA) (18). Briefly, 500 mg cecum digesta was homogenised in 5 ml 50 mmol/l H₂SO₄ containing 10 mmol/l 2,2-dimethyl butyric acid as an internal standard and subsequently centrifuged at 17,000 × g at 2°C for 20 min. The supernatant was ultrafiltered, and the filtrate was applied to the HPLC column.

Data are expressed as the mean ± standard error of the mean. Data were analyzed by Student's t-test after statistical outliers were removed using the Smirnov-Grubbs rejection test. In addition, association of cecum ethanol levels with cecum Bifidobacterium and SCFA levels was assessed using Spearman rank correlation analysis. The data analysis was performed using Excel Statistics 2010 for Windows (Microsoft Corporation, Redmond, WA, USA). For all tests, P<0.05 was considered to indicate statistical significance.

Dietary manipulation had no significant effect on total food intake over 14 days or on final body weight (P>0.05; Table II).

The weights of the ileum digesta were unaffected by FOS intake (control vs. FOS: 0.30±0.02 vs. 0.26±0.05 g; P>0.05). Ethanol levels per gram of ileum digesta were significantly increased 3.5-fold by FOS intake (P<0.01; Fig. 1A). In the total ileum digesta, FOS intake significantly increased ethanol levels by 3.6-fold (P<0.01; Fig. 1B). The weights of the cecum digesta were markedly increased 4.2-fold by FOS intake (control vs. FOS: 1.33±0.06 vs. 5.54±0.48 g; P<0.01). The ethanol levels per gram of cecum digesta were significantly increased 1.9-fold by FOS intake (P<0.01; Fig. 1C). In the total cecum digesta, ethanol levels were markedly increased by FOS intake by 7.9-fold (P<0.01; Fig. 1D). Serum ethanol levels were marginally but significantly increased by FOS intake (control vs. FOS: 2.63±0.05 vs. 2.84±0.07 mM; P<0.05).

Compared with the control group, the copy numbers of Bifidobacterium per gram of cecum digesta were markedly increased 4.2-fold in the FOS group (P<0.05; Table II). However, the numbers of Lactobacillus per gram of cecum digesta were unaffected (P>0.05). The numbers of Bifidobacterium were positively correlated with ethanol level per gram of cecum digesta (r=0.626, P=0.017; Fig. 2).

Succinate and acetate levels per gram of cecum digesta were decreased by FOS intake (2.5- and 2.7-fold decrease, respectively; P<0.05; Table II). By contrast, FOS intake significantly increased lactate, propionate and n-butyrate levels per gram of cecum digesta (2.4-, 1.7- and 1.4-fold, respectively; P<0.05; Table II). In the total cecum digesta, the levels of lactate, propionate, n-butyrate and total SCFAs were markedly increased by the FOS diet (10.3-, 7.3-, 6.0- and 4.0-fold, respectively, P<0.01). The ethanol levels per gram of cecum digesta were negatively correlated with acetate levels (r=−0.558, P=0.025; Fig. 3A) and positively correlated with lactate levels (r=0.691, P=0.006; Fig. 3B). However, ethanol levels were not significantly correlated with the level of propionate (r=0.449, P=0.081; Fig. 3C) or butyrate (r=0.493, P=0.052; Fig. 3D).