Participants

Forty-five volunteers (22 males, 23 females) recruited through online and poster adverts completed the study. Inclusion criteria were aged 18–45 years, fluent English speaker and BMI range 18–25. Exclusion criteria were previous or current neurological, psychiatric, gastrointestinal or endocrine disorders, or other relevant medical history; current or recent (<3 months) regular medication use; previous or current substance/alcohol dependence or abuse within the last 3 months; regular tobacco use (>5 cigarettes/day); and participation in research studies involving medication intake (within 3 months) or prior completion of the Emotional Test Battery (ETB). To ensure that the enteric environment was consistent in all volunteers, additional exclusion criteria were: no antibiotic use 3 months prior to the study, no regular use of pre- and probiotics (and within 3 months prior to the study) and no vegan diets. Finally, participants were asked to adhere to their regular diets and avoid supplements or special diets. No significant dietary variations were noted.

Participants were assessed with the Structured Clinical Interview for DSM-IV (SCID; First et al. 1997) to confirm the absence of DSM-IV axis I psychiatric conditions. The study was approved by the Oxford Central University Research Ethics Committee. All participants provided written informed consent and were reimbursed for their time and expenses.

Materials

Demographic and questionnaire measures

Participants completed the National Adult Reading Test (NART; Nelson 1982) to provide an estimate of verbal IQ. Self-report questionnaires assessing trait measures of personality (Eysenck Personality Questionnaire, EPQ; Eysenck and Eysenck 1975), stress responsivity (Perceived Stress Reactivity Scale, PSRS; Schlotz et al. 2011), subclinical symptoms of depression (Beck Depression Inventory, BDI; Beck et al. 1961) and anxiety (State-Trait Anxiety Inventory, STAI-trait; Spielberger et al. 1970) were also completed. Before and after prebiotic/placebo intake (days 0 and 21, respectively), anxiety (STAI-state) and measures of perceived stress (Perceived Stress Scale, PSS; Cohen et al. 1983) and mood (Visual Analogue Scales, VAS; Bond and Lader 1974; Positive and Negative Affect Schedules, PANAS; Watson et al. 1988) were measured using self-report questionnaires. The digit span index of verbal working memory was used in order to monitor group differences in executive functioning on the final day of treatment.

Prebiotic supplements

The study was placebo controlled, and male/female participants were randomised to receive one of two prebiotics (fructooligosaccharides [N = 15; 8 males, 7 females] or Bimuno®-galactooligosaccharides [N = 15; 7 males, 8 females]) or a placebo (maltodextrin [N = 15; 7 males, 8 females]). The use of maltodextrin as a placebo compound in prebiotic trials is well established (Vulevic et al. 2008). Preparations were provided by Clasado Research Services Ltd., Reading, UK. Participants took the supplements (at 5.5 g per day) in powder form orally with breakfast for 3 weeks. The study used a double-blind randomised design with both the participant and the experimenter being unaware of the group they had been allocated to.

Salivary cortisol

HPA axis activity was assessed on the day before (day 0) and on the final day of prebiotic/placebo administration (day 21), using the salivary cortisol awakening response (CAR; Pruessner et al. 1997). For each CAR measurement, participants were instructed to provide five saliva samples (using Salivettes, Sarstedt Ltd., Nümbrecht, Germany) taken in their own home immediately upon waking and subsequently every 15 min until 1 h post-waking. Saliva samples were stored at 4 °C prior to analysis. Cortisol was measured using a commercial ELISA (Salimetrics Europe Ltd., Newmarket, UK), within 7 days of sample collection.

Emotional processing tasks

On the final day of prebiotic/placebo intake (day 21), participants completed a validated computerised test battery assessing the processing of emotional stimuli (the ETB; Harmer et al. 2004).

Attentional dot-probe task

Sixty negative and 60 positive words were paired with neutral words matched for length. On each trial, a fixation cross was presented for 500 ms in the centre of the screen, followed by two words presented at the top and bottom of the screen. In the unmasked condition, the words were presented for 500 ms. In the masked condition, word pairs were presented for 17 ms after which a mask was displayed for 483 ms. Masks were constructed from digits, letters and non-letter symbols and were matched for word position and length. Words or masks were replaced by a probe of either one or two stars in the location of one of the preceding stimuli (probes were presented at the top or bottom of the screen with equal frequency). Participants were instructed to indicate the number of stars as quickly and accurately as possible using two labelled keys. A key press terminated the probe presentation and trial. There were 180 trials in total (30 positive-neutral, 30 negative-neutral, 30 neutral-neutral word pairs each for masked and unmasked conditions), and emotional words were presented at the top and the bottom location with equal frequency. Masked and unmasked trials were presented in random order. Reaction time and accuracy scores were recorded, and attentional vigilance scores were calculated for each participant by subtracting the reaction time from trials when probes appeared in the same position as the emotional word (congruent trials) from those trials when probes appeared in the opposite position to the emotional word (incongruent trials).

Facial expression recognition task

In the facial expression recognition task (FERT), the perception of six basic emotions (happiness, surprise, sadness, fear, anger, disgust) or a neutral expression (taken from the Pictures of Affect Series; Ekman and Friesen 1976) was assessed. Each emotion was shown at 10 morphed intensity levels from neutral to maximum emotional expression (Young et al. 1997) leading to a total of 250 randomly presented stimuli. Each stimulus was presented for 500 ms and replaced by a grey screen until the participant responded (as quickly and as accurately as possible) by selecting a key corresponding to one of the basic emotions. The outcome measures were classification accuracy, number of misclassifications and reaction times.

Emotional categorisation and memory

Sixty words representing either disagreeable (N = 30) or agreeable (N = 30) personality characteristics (from Anderson 1968) and matched for meaningfulness, word frequency and word length were presented on a computer screen for 500 ms each. Participants were asked to categorise each word as quickly and accurately as possible according to whether they would like or dislike to be described by it. After completion, participants were instructed to recall and write down as many words as they could within a 2-min time limit. Subsequently, participants were instructed to categorise words presented on the screen into those which were previously presented (60 target words) or those which were novel words (60 matched distracter words). Outcome measures for the emotional categorisation were classifications and reaction times. For the memory recall, the number of correct responses and false positives was recorded, and participants’ memory recognition was assessed using correct responses, false positives and reaction times.

Statistical analysis

Demographic and questionnaire values were analysed using one-way ANOVA with group as factor. Salivary cortisol values (in nmol/l) were square root transformed and analysed in a mixed design ANOVA with time point of sampling (0, 15, 30, 45 and 60 min after waking) and day of sampling (pre- vs. post-treatment) as repeated-measures variables and prebiotic treatment group (placebo, FOS or B-GOS) as a between-subjects variable. Raw cortisol values are presented for clarity. The behavioural outcome variables of the ETB were also analysed with mixed design ANOVAs, with prebiotic treatment group as between-subjects factor and emotion and task condition as within-subjects factors. Significant interactions were followed up using main effects analyses. Greenhouse-Geisser corrections were used where assumptions of sphericity were not met.