Baseline measures and compliance
There were no significant differences between groups for age, trait measures of anxiety, stress reactivity, neuroticism or cognitive status as assessed by digit span (see Table 1). This suggests that groups were well matched between the two prebiotic and placebo conditions.
Out of 48 participants, three participants did not complete the full course of prebiotic/placebo intake and were excluded from all analyses, leading to the final sample of 45 participants. Participants completed a checklist each day to monitor prebiotic/placebo intake, and none reported missing more than two intakes (0 missed = 41, 1 missed = 3, 2 missed = 1).
Hormonal contraceptive use and menstrual cycle
Of the 23 female participants, 13 used hormonal methods of contraception. Menstrual cycle phase was reported by 16 females, and an additional two female participants reported no or very infrequent menses due to hormonal contraceptive use—these were coded as a separate group. Cycle phase was analysed according to the following phases (based on averages from Wolfram et al. 2011; Fehring et al. 2006): menses (days 1–6), follicular phase (days 7–12), ovulation phase (days 13–19), luteal phase (day 20–end of cycle). In order to test the between-subjects effects of contraceptive use on the waking cortisol response, repeated-measures ANOVAs were performed on the cortisol level upon waking (first sample) and the cortisol area under the curve with respect to ground (AUC-g) including the factor supplement group to test for potential interactions.
There was no significant difference in the number of females who took hormonal contraceptives between the groups (χ
2 = 3.45, p > 0.1). The effect of hormonal contraceptive on the first sample of salivary cortisol after waking showed a trend for lower levels in females taking contraceptives compared to those who were not (mean (SD) = 8.20 (3.42) vs. mean (SD) = 10.65 (4.37), F(1,17) = 3.74, p = 0.07); however, this difference was stable across days of testing and treatment groups (all interactions’ p values >0.7). The effect of hormonal contraceptives on cortisol AUC-g was not significant (F(1,17) = 3.01, p > 0.1), and there were no significant interactions with testing day or treatment group (all p > 0.1).
The number of participants who were in a particular cycle phase at the time of testing did not differ between the groups (χ
2 = 8.75, p > 0.1). Due to insufficient power, the effects of menstrual phase on cortisol or attention were not tested with ANOVAs.
Salivary cortisol did not differ significantly between groups at baseline but was significantly lower following B-GOS compared with placebo (Fig. 1). This was shown by an ANOVA interaction effect of pre- versus post-treatment, group (placebo, FOS or B-GOS) and sampling time point (in minutes post-waking) on salivary cortisol levels, followed up with separate group × time point ANOVAs for each day of sampling (day 0: main effect of group F(2,41) = 1.08, n.s.; day 21: main effect of group F(2,41) = 4.20, p < 0.05, followed up with Sidak-corrected contrasts: placebo vs. GOS, p = 0.02, all others p > 0.1). Main effects analyses of the day × group × time ANOVA confirmed that cortisol levels were increased post-waking 15, 30, 45 and 60 min after waking (significant main effect of time F(2.41,98.63) = 58.61, p < 0.001, planned follow-up contrasts all significant at p < 0.001). The main effects of day of sampling and treatment group were not significant (p > 0.1). Gender was not entered as a factor of interest due to insufficient power.
The lowered CAR in the B-GOS compared to the placebo group on day 21 of supplement administration was also confirmed when analysing area under the curve with respect to ground (Fig. 2; day × group ANOVA on square-root-transformed salivary cortisol values: day × group interaction [F(2,41) = 3.52, p = 0.039], followed up with separate group ANOVAs for pre/day 0 [F(2,41) = 1.24, n.s.] and post/day 21 [F(2,41) = 4.12, p = 0.023, follow-up contrasts: placebo vs. B-GOS p = 0.019, placebo vs. FOS p > 0.1, FOS vs. B-GOS p > 0.1]).
Emotional Test Battery
Attentional dot-probe task
There was a significant group × emotion × masking condition interaction in the visual dot-probe task (group × emotion × masking condition [F(2,41) = 3.14, p = 0.05]). As can be seen in Fig. 3, this effect was driven by decreased attentional vigilance to negative versus positive information in the unmasked condition (Fig. 3b), with no significant main effects or interactions in the masked condition (Fig. 3a; valence × group interaction in unmasked: F(2,41) = 4.29, p = 0.02; masked: F(2,41) = 0.85, p > 0.1). Follow-up analyses with separate ANOVAs for prebiotic group compared with placebo in the unmasked condition confirmed this effect as driven by increased positive versus negative vigilance after B-GOS compared to placebo, while the FOS group did not perform differently to placebo (B-GOS vs. placebo: valence × group F(1,27) = 6.94, p = 0.014, FOS vs. placebo: valence × group F(1,27) = 3.20, n.s.).
There were no significant effects of prebiotic treatment on measures of accuracy (main effect of group: F(2,42) = 1.71, n.s., emotion × group interaction F(7.83,164.52) = 0.67, n.s.). Analysis of reaction time data revealed no significant interaction between group and emotion (main effect of group: F(2,42) = 0.53, n.s.; emotion × group interaction F(8.16,773.38) = 1.10, n.s.).
Emotional categorisation, recall and recognition
Participants responded faster to positive (mean reaction time = 1031 ms, SD = 228 ms) compared to negative (mean reaction time = 1083 ms, SD = 200 ms) self-referential personality words in the emotional categorisation task (valence × group ANOVA, main effect of valence: F(1,42) = 12.82, p < 0.01) and in the emotional word recognition task (mean reaction time to positive words = 1220.92, SD = 263.03; mean reaction time to negative words = 1381.07, SD = 348.13; main effect of valence: F(1,41) = 23.34, p < 0.001); however, there was no significant main effect of prebiotic treatment group (F(2,42) = 0.80, p > 0.1) and the relative speeding for positive words did not differ between groups (emotion × group F(2,42) = 0.35, p > 0.1). Positive words were also remembered more often than negative words in both the surprise recall task (mean accuracy, positive words = 7.41 (SD = 2.45), negative = 5.70 (2.29); F(1,41) = 16.16, p < 0.001) and in the recognition task (mean correct recognition, positive words = 25.67 (SD = 3.41), negative words = 22.36 (SD = 3.88); F(1,41) = 53.54, p < 0.001), but these effect did not differ between groups (p > 0.1).
There were no significant effects of group on self-report measures of state anxiety or perceived stress before or after prebiotic/placebo administration (see Table 2). Furthermore, there were no group differences in the overall cognitive status as assessed by digit span on the day of psychological testing.
To test the hypothesis that cortisol levels were associated with changes in attentional dot-probe performance in the B-GOS group, we correlated difference scores of positive versus negative reaction times in the unmasked condition with absolute cortisol levels upon waking on the day of testing and with the difference in pre- versus post-prebiotics cortisol values (day 0–day 21). There were no associations of cortisol with attentional performance (all p > 0.1).