PROMOTE: The Effect of a Six Week Prebiotic Supplementation on Wellbeing of Young Adults.

GSS-15 - Manners, J et al (2023) questionnaire A 15 item questionnaire validated in a population of Australian adults to give a self-reported indices of sleep on a scale of confidence of being a good sleeper.

10.1111/jsr.13717

GSS-15 ≥ 45: High confidence of being a Good Sleeper GSS-15 40 - 44: Moderate confidence of being a Good Sleeper GSS-15 < 40: Low confidence of being a Good Sleeper

Example outcome: 'There was no difference in the mean reported good sleeper score (43.25 43.25 (95% CI: -40.1, 46.2) of the prebiotic and placebo groups after 6 weeks of supplementation.'

Depression, Anxiety and Stress Scales Lovibond, P. F., \& Lovibond, S. H. (1995) 10.1016/0005-7967(94)00075-u

Scale Scores:

DASS (42) Scores are calculated for each subset of the survey with the scale as follows where lower scores indicate a better outcome, while higher scores indicate worse results:

Depression Anxiety Stress Normal 0-9 0-7 0-14 Mild 10-13 8-9 15-18 Moderate 14-20 10-14 19-25 Severe 21-27 15-19 26-33 Extremely Severe 28+ 20+ 34+

Example outcome: 'There was a significant reduction in the stress score observed in the prebiotic group (mean: -3.25 (p=0.004) after 6 weeks of supplementation when compared to the placebo group.'

16S ribosomal ribonucleic acid (rRNA) sequencing of stool samples measuring microbial composition change.

Units: Read counts, Operational Taxanomic Units (OTUs) and Relative abundance provide quantifications of the amount of bacterial genetic sequence present in a stool sample building an approximation of the faecal microbial composition to at least the genus level.

Results are typical reported in compositional scores like alpha and beta diversity.

Bray-Curtis dissimilarity is a scale of similarity where values closer to 1 are more distinct, while values close to 0 represent similar compositions.

Example outcome: 'The beta diversity (a measure of compositional change) of the prebiotic group using Bray-Curtis dissimilarity increased by 22.7%, suggesting a compositional change from the placebo group.'

Validated SFFQ for adults in the United Kingdom (UK) Cleghorn, CL. et al (2017) - University of Leeds

Scores per subgroup (Fruit, Vegetables, Oily Fish, Fat and Non-Milk Extrinsic Sugars (NMEs)) are converted to scores ranging from 1-3 based on consumption. Lower scores indicating less intake, while scores of 3 indicate high intake of particular food groups (except for fats and NMEs which is reverse scaled - lower score matching closer to nutritional guideline).

A final dietary quality score score is calculated from all subgroups with higher scores indicating better dietary quality.

Example outcome: 'Dietary quality scores did not significantly differ among the prebiotic and placebo groups after 6 weeks. This indicates that participants' dietary habits remained consistent throughout the intervention, thereby minimizing dietary intake as a confounding factor in the comparison of the interventional groups.'

Subsample analysis of biometric activity and sleep using FitBit Inspire 3.0. The Fitbit Inspire 2.0 can capture more objective measures of sleep including time spent in REM sleep phases, sleep duration, awakening and sleep latency.

Example outcome: 'Sleep latency, defined as the time taken to fall asleep, was significantly reduced by 5.5 minutes (p < 0.05) after 6 weeks of prebiotic supplementation compared to the placebo group.'

Untargeted Proton Nuclear Magnetic Resonance (1H NMR) metabolomic analysis of urine samples.

NMR generates a spectrum of metabolites by detecting hydrogen atoms' response to an electromagnetic pulse. The resulting peaks correspond to specific chemical shift ranges (δ shifts), allowing for the identification of metabolite compositions. It is untargeted as identification is based on what is returned in spectral form, as opposed to targeted which preselects specific metabolites to measure.

Example outcome: 'After 6 weeks of prebiotic supplementation, we observed significant increases in the urinary levels of dimethylglycine, indole-3-sulfate, and hippurate compared to the placebo group. These findings suggest that prebiotic supplementation may influence various metabolic pathways and gut microbiota activity.'

Untargeted Proton Nuclear Magnetic Resonance (1H NMR) metabolomic analysis of faeces.

NMR generates a spectrum of metabolites by detecting hydrogen atoms' response to an electromagnetic pulse. The resulting peaks correspond to specific chemical shift ranges (δ shifts), allowing for the identification of metabolite compositions. It is untargeted as identification is based on what is returned in spectral form, as opposed to targeted which preselects specific metabolites to measure.

Example outcome: 'After 6 weeks of prebiotic supplementation, we observed significant increases in the faecal levels of alanine and glutamate compared to the placebo group.

More targeted approaches would be needed to quantify and determine if these amino acids are from dietary, human or microbiome origins like Liquid Chromatography Mass Spectrometry LC-MS).'

Untargeted Proton Nuclear Magnetic Resonance (1H NMR) metabolomic analysis of plasma samples.

NMR generates a spectrum of metabolites by detecting hydrogen atoms' response to an electromagnetic pulse. The resulting peaks correspond to specific chemical shift ranges (δ shifts), allowing for the identification of metabolite compositions. It is untargeted as identification is based on what is returned in spectral form, as opposed to targeted which preselects specific metabolites to measure.

Example outcome: 'After 6 weeks, prebiotic supplementation significantly increased faecal levels of alanine and glutamate compared to placebo, while lysine levels remained unchanged. Further targeted analyses, such as LC-MS, are needed to determine the origins and concentrations of these amino acids'.

Height (cm), Weight (Kg), Waist Circumference (cm) and Body Mass Index (BMI (kg/m2)

Example outcome: 'Mean waist circumference (cm) did not differ significantly (p > 0.05) between the prebiotic and placebo groups after 6 weeks, suggesting that the prebiotic had no effect on body composition in terms of waist circumference.'

Monitoring or reporting of any adverse or severe adverse effects experienced during intervention.

Reported and logged in active survey responses / contacting the study team directly

Example outcome: 'There were two minor side effects reported early in the prebiotic supplementation, both instances self resolved within a few days and may be an effect of the initial introduction of a supplement to the individuals digestive system. No severe adverse effects were reported in either the prebiotic or the placebo control groups.'