Male C3H/HeN (Japan SLC Inc.) and C57BL/6J (Charles River Laboratories Japan) mice with a body weight of 20 - 23 g at the time of experiment were used in the in vivo and ex vivo studies. Mice were housed in a cage with free access to chow and water at 23˚C ± 2˚C under a 12 h light-dark cycle (Lights on: 08:00 - 20:00). The animals were allowed at least 1 week to acclimatize to their housing conditions before experiments. The Animal Care and Use Committee of Frontier Laboratories of Value Creation, Sapporo Breweries Ltd. approved all animal care and handling procedures. 

L. brevis SBC8803 were cultured for 24 h at 30˚C in broth containing 2% maltose, 1.4% yeast extract, 0.5% sodium acetate, and 0.005% MnSO₄·5H₂O. Bacteria were collected by centrifugation at 8000 × g for 10 min at 10˚C (Himac CR21G; Hitachi Koki Co. Ltd.) and washed 3 times with deionized water. After washing, bacteria were heat-killed at 105˚C for 10 min and lyophilized.

The rat endocrine RIN-14B cell line (ATCC, CRL-2059) was suspended in RPMI1640 medium supplemented with 10% fetal calf serum (Gibco, Life Technologies) at 1 × 10⁵ cells/0.5 mL, seeded in 24-well plates, and cultured for 3 d at 37˚C in a 5% CO₂ incubator (MCO-20 AIC; SANYO Co.). The medium was removed, and the cells were washed with Hank’s balanced salt solution (HBSS) containing Ca and Mg, (Wako Pure Chemical Industries, Ltd.). After washing, HBSS was removed and replaced with 0.5 mL HBSS containing samples (0 - 0.3 mg/mL), and the cells were further incubated for either 30 min or 60 min at 37˚C. The assay solution was collected and centrifuged for 5 min to remove any detached cells. Serotonin concentrations in supernatants were measured using a serotonin-ELISA kit (Enzo Life Science). Results are represented as mean ± SD (n = 4 - 6). Statistical evaluation of the results was performed using one-way analysis of variance (ANOVA).

The sample was suspended in sterile water and then orally administered using a stomach sonde (feeding needle) to male C57BL/6J mice (2 mg of sample/mouse). At 0, 15, 30, 60, and 90 min after administration, blood samples were collected from mice and treated with heaprin, followed by centrifugation with 5000 rpm for 10 min at 4˚C. Plasma 5-HT concentrations were measured using 5-HT ELISA kits. Results are represented as the mean ± SD (n = 5). Statistical evaluation of the results was performed using one-way ANOVA.

Male C3H/HeN and C57BL/6J mice were killed. An intestinal loop (approximately 5 cm), composed of duodenum and part of the jejunum, was then removed from the small intestine. Each end of the intestinal loop was ligated with silk sutures and the loops filled with 0.1 ml sterilized water containing 2 mg/mL sample. To measure releasing effects, the loops were incubated for 60 min at 37˚C in a 5% CO₂ incubator. The loops were placed into the center section of the organ culture dish (3.5 cm in diameter) in 3 mL of RPMI 1640. RPMI 1640 media in the culture dish were collected at 60 min to determine 5-HT release from the lumen to the medium bathing the loops. The 5-HT level in the medium was measured using the 5-HT-ELISA kit. Results are represented as mean ± SD (n = 5). Statistical evaluation of the results was performed using one-way ANOVA.

The in vitro experiments were performed to evaluate 5-HT release from the rat endocrine RIN-14B cells, which were used as a tentative model for the investigation of enterochromaffin (EC) cell function [10]. As shown in Figure 1 (a), 5-HT release from RIN-14B cells increased depending on the concentration of SBC8803 (0 - 0.3 mg/mL) after incubation for 30 min (0 ppm vs. 30 ppm; P < 0.05, 0 ppm vs. 100 ppm and 300 ppm; P < 0.01). Moreover, maximum 5-HT release from RIN-14B cells was observed after incubation for 60 min (Figure 1 (b)). Subsequently, we performed an in vitro study using cellulose powder (100 ppm) as a physical stimulus in the 60 min reaction. The 5-HT concentration at 60 min after cellulose powder addition was equal to that obtained after addition of sterile water (Figure 2). Thus, cellulose powder had no effect on 5-HT release from these cells. On the other hand, 5-HT release was observed after addition of heat-killed L. brevis SBC8803 cells (SBC8803 vs. sterile water and cellulose powder; P < 0.01).

We determined 5-HT concentrations in blood after administration of heat-killed L. brevis SBC8803. Heatkilled L. brevis SBC8803 was suspended in sterile water and administered using a stomach sonde (feeding needle) to C57BL/6J mice (2 mg of SBC8803/mouse). This dose was equivalent to the daily intake of food containing 0.05% L. brevis SBC8803. At 0, 15, 30, and 60 min after administration, plasma 5-HT concentrations were determined using 5-HT ELISA kits. As shown in Figure 3,

plasma 5-HT concentrations gradually increased after L. brevis SBC8803 administration (0 min vs. 60 min; P < 0.05). Subsequently, we performed an in vivo administration study using cellulose powder (2 mg/mouse) as a physical stimulus. The 5-HT concentration at 90 min after cellulose powder administration was equal to that observed after administration of sterile water (Figure 4), which was similar to that observed in the in vitro study using RIN-14B cells. 

On the other hand, administration of heat-killed L. brevis SBC8803 led to higher plasma 5-HT concentrations than did sterile water or cellulose powder (SBC 8803 vs. sterile water; P = 0.08). These in vivo studies results (Figures 3 and 4) showed that oral administration of heat-killed L. brevis SBC8803 in mice increased 5-HT concentrations in the blood.

Finally, we carried out ex vivo experiments using the intestinal loop, which is composed of the duodenum and part of the jejunum from male C3H/HeN and C57BL/6J mice. We injected 0.1 mL of sterile water containing heat-killed L. brevis SBC8803 (2 mg/mL) into the intestinal loop. We then evaluated the 5-HT-releasing ability of L. brevis SBC8803 by using the procedure described in the Material and Methods section. In C57BL/6J mice, injection of L. brevis SBC8803 led to 5-HT release from the intestinal cells into the RPMI1640 medium, whereas the amount of 5-HT released did not differ statistically from the amount release after injection of sterile water into intestinal loops, as shown in Figure 5. However, injection of heat-killed L. brevis SBC8803 into the intestinal loop of C3H/HeN mice led to release of significantly

higher concentrations of 5-HT from the intestinal cells compared to that released after sterile water injection (SBC8803 vs. sterile water; P < 0.01).