Fermented Milk Containing Lactobacillus casei Strain Shirota Preserves the Diversity of the Gut Microbiota and Relieves Abdominal Dysfunction in Healthy Medical Students Exposed to Academic Stress

Abstract

Stress-induced abdominal dysfunction is an attractive target for probiotics. To investigate the effects of the probiotic Lactobacillus casei strain Shirota on abdominal dysfunction, a double-blind, placebo-controlled trial was conducted with healthy medical students undertaking an authorized nationwide examination for academic advancement. For 8 weeks, until the day before the examination, 23 and 24 subjects consumed an L. casei strain Shirota-fermented milk and a placebo milk daily, respectively. In addition to assessments of abdominal symptoms, psychophysical state, and salivary stress markers, gene expression changes in peripheral blood leukocytes and composition of the gut microbiota were analyzed using DNA microarray analysis and 16S rRNA gene amplicon sequence analysis, respectively, before and after the intervention. Stress-induced increases in a visual analog scale measuring feelings of stress, the total score of abdominal dysfunction, and the number of genes with changes in expression of more than 2-fold in leukocytes were significantly suppressed in the L. casei strain Shirota group compared with those in the placebo group. A significant increase in salivary cortisol levels before the examination was observed only in the placebo group. The administration of L. casei strain Shirota, but not placebo, significantly reduced gastrointestinal symptoms. Moreover, 16S rRNA gene amplicon sequencing demonstrated that the L. casei strain Shirota group had significantly higher numbers of species, a marker of the alpha-diversity index, in their gut microbiota and a significantly lower percentage of Bacteroidaceae than the placebo group. Our findings indicate that the daily consumption of probiotics, such as L. casei strain Shirota, preserves the diversity of the gut microbiota and may relieve stress-associated responses of abdominal dysfunction in healthy subjects exposed to stressful situations.

Importance: A novel clinical trial was conducted with healthy medical students under examination stress conditions. It was demonstrated that the daily consumption of lactic acid bacteria provided health benefits to prevent the onset of stress-associated abdominal symptoms and a good change of gut microbiota in healthy medical students.

FIG 1

Experimental design.

FIG 2

Effect on the time-dependent changes of psychological and physiological stress markers during the experimental period. STAI-state score (A), VAS measure feelings of stress (B), salivary cortisol (C), and salivary alpha-amylase activity (D). One subject in the L. casei strain Shirota (LcS) group was excluded from the analysis because no questionnaires were collected. Values indicate the means ± SE. Data were analyzed by Dunnett's multiple-comparison test within a group and two-way ANOVA between the groups. #, P < 0.05 between the two groups; *, P < 0.05; and **, P < 0.01 versus baseline.

FIG 3

Effect on the time-dependent changes of abdominal dysfunction during the experimental period. Total scores of abdominal dysfunction (A), total GSRS scores (B), changed score of “abdominal discomfort and pain” from the preintervention period in subjects without symptoms during the preintervention period (the placebo group, n = 9; the L. casei strain Shirota [LcS] group, n = 14) (C), and changed score of “abdominal discomfort and pain” from the preintervention period in subjects with symptoms during the preintervention period (the placebo group, n = 15; the L. casei strain Shirota group, n = 8) (D). One subject in the L. casei strain Shirota group was excluded from the analysis because no questionnaires were collected. Values indicate the means ± SE. Data were analyzed by Dunnett's multiple-comparison test within a group and two-way ANOVA between the groups. #, P < 0.05; ##, P < 0.01 between both groups; *, P < 0.05 versus preintervention period.

FIG 4

Effects on the changes of gene expression in peripheral blood leukocytes. Normalized intensity of gene expression changes (A) and the number of genes with a change in expression of more than 2-fold compared with that at baseline (P < 0.05, unpaired Student's t test) (B). Gene expression change in peripheral blood leukocytes was determined by microarray analysis, and 19,194 probes, having fluorescence intensities higher than the cutoff value of 50 among all samples, were analyzed. Data were analyzed by Fisher's exact test. #, P < 0.05; ###, P < 0.001 between the two groups. LcS, L. casei strain Shirota.

FIG 5

Effects on the composition of the gut microbiota. Composition of phyla (A), number of observed species (B), percentage of Bacteroidetes in the phyla (C), and percentage of Bacteroidaceae in the families (D). (B to D) Values indicate the means ± SE. Data were analyzed by Wilcoxon's signed-rank test within a group and Mann-Whitney U test between the groups. #, P < 0.05 between the two groups. LcS, L. casei strain Shirota.