Journal Pre-proof

Effects of a flavonoid-rich blackcurrant beverage on markers of the gut-brain axis in

healthy females: Secondary findings from a 4-week randomized crossover control
trial.

Nicola A. Gillies, Brooke C. Wilson, Jessica R. Miller, Nicole C. Roy, Andrew Scholey,
Andrea J. Braakhuis
PII: S2475-2991(24)00092-1
DOI: https://doi.org/10.1016/j.cdnut.2024.102158
Reference: CDNUT 102158

To appear in: Current Developments in Nutrition

Received Date: 25 March 2024

Revised Date: 5 April 2024
Accepted Date: 9 April 2024

Please cite this article as: N.A. Gillies, B.C. Wilson, J.R Miller, N.C. Roy, A. Scholey, A.J. Braakhuis,
Effects of a flavonoid-rich blackcurrant beverage on markers of the gut-brain axis in healthy females:
Secondary findings from a 4-week randomized crossover control trial., Current Developments in
Nutrition, https://doi.org/10.1016/j.cdnut.2024.102158.

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© 2024 Published by Elsevier Inc. on behalf of American Society for Nutrition.

Effects of a flavonoid-rich blackcurrant beverage on
markers of the gut-brain axis in healthy females:
Secondary findings from a 4-week randomized crossover
control trial.
Nicola A. Gillies 1,*, Brooke C. Wilson 2, Jessica R Miller 1, Nicole C. Roy 3,4,5, Andrew
Scholey 6,7 and Andrea J. Braakhuis 1
1
Discipline of Nutrition and Dietetics, University of Auckland, Auckland New Zealand
2
The Liggins Institute, University of Auckland, Auckland, New Zealand
3
Department of Human Nutrition, University of Otago, New Zealand
4
The Riddet Institute, Palmerston North, New Zealand
5
The High-Value Nutrition National Science Challenge, New Zealand
6
Nutrition Dietetics and Food, School of Clinical Sciences, Monash University, Melbourne, Australia

f
7
Centre for Mental Health and Brain Sciences, Swinburne University, Melbourne, VIC Australia

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* Corresponding author:

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Dr Nicola Gillies, -p
Department of Nutrition and Dietetics, University of Auckland,
Private Bag 92019, Auckland 1142, New Zealand
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Email: n.gillies@auckland.ac.nz
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Sources of support: This research was funded by the New Zealand National Science Challenge
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(High Value Nutrition, #1968). The funders had no role in the study design, collection, analysis
or interpretation of data, or writing the manuscript. There were no restrictions regarding
submission of the manuscript for publication. All intervention and placebo beverages were
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contributed in-kind by AlphaGen NZ Ltd.

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Conflict of interest and funding disclosure: In the last three years, AS has received research
funding and/or consultancy/travel/speaker fees from: BioRevive, Coca Cola, Ekterra,
Frutarom, Givaudan, National Health and Medical Research Council, Nestlé, Nutricia
Research Foundation, PepsiCo, Reviv, Wörwag Pharma and Wrigley-Mars. AS is Chief
Scientific officer for Ārepa Nootropics. The other authors do not have any conflicts of interest
to declare.

Word count: 6417

Number of figures: 5
Number of tables: 4
Supplementary data: 2 supplementary tables, 1 supplementary figure.
Running title: Blackcurrant beverage and gut-brain axis.
List of abbreviations: BDNF, brain derived neurotrophic factor; DST, dietary screening tool;
FBB, flavonoid-rich blackcurrant beverage; MTF, multi-tasking framework; POMS, profile
of mood states; PSQI, Pittsburgh sleep quality index; STAI-S, state trait anxiety inventory
– state portion; VAMS, visual analogue mood scales.

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 1 Abstract: The microbiota-gut-brain axis is a promising target to alleviate the growing burden of

2 neurological and mental health disorders. Dietary polyphenols act on multiple components of the

3 microbiota-gut-brain axis, but this complex relationship requires further attention. This

4 randomized, placebo-controlled, double-blind, crossover trial (ACTRN12622000850774)

5 compared 4-weeks of a commercially-available flavonoid-rich blackcurrant beverage (FBB, 151

6 mg anthocyanins, 308 mg total polyphenols) versus placebo in 40 healthy females (18-45y). The

7 primary outcome of stress reactivity was assessed by change in present feelings of stress, mood

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8 and fatigue before and after completing a 20-minute cognitive stressor (Purple multi-tasking

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9 framework, MTF). Secondary endpoints included cognitive performance (MTF), mood (Profile of

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Mood States, POMS), sleep (Pittsburgh Sleep Quality Index), fecal microbiome composition and
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11 functional potential (shotgun sequencing) and blood biomarker concentrations (Brain Derived
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12 Neurotrophic Factor, tryptophan, kynurenine, interleukin-6). Statistical analyses were conducted

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13 on an intention-to-treat basis using linear mixed-effect models. Thirty-eight participants completed

14 both intervention arms. There was no significant treatment effect on the primary outcome of stress
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15 reactivity. Compared with placebo, working memory (letter retrieval scores, MTF), and
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16 ‘anxiety/tension’ and ‘anger/hostility’ domains of the POMS improved with FBB supplementation

17 (time x intervention interaction, p<0.05). There were no treatment effects on gut microbiome

18 composition or functional potential. Baseline abundances of Bifidobacterium genera and species

19 (B. longum, B. bifidum) tended to be higher in participants with the greatest improvements in letter

20 retrieval scores with FBB supplementation (nominally significant, p<0.05). In conclusion, 4-weeks

21 of FBB supplementation improved secondary outcomes of working memory performance during

22 multi-tasking and mood outcomes in healthy adult females. These results should be confirmed in

23 a larger cohort with a longer duration of follow-up.

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24 Keywords: blackcurrant; cognition; mood; gut microbiome; flavonoid

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25 Teaser text

26 Four weeks of supplementation with a flavonoid-rich blackcurrant beverage improved measures

27 of cognition and mood in healthy female adults, but had no effect on gut microbiome composition.

28 Introduction

29 The microbiota-gut-brain axis is a potential target to curb the growing social and economic burden

30 of mental health and neurological disorders (1–3). This dynamic network encompasses the

31 bidirectional communication between the central nervous system and gastrointestinal tract,

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32 mediated by immune, neurotransmitter, neuroendocrine, and metabolic pathways alongside the

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33 trillions of bacteria comprising the resident gut microbiota (4).

34
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Diverse aspects of diet can influence the microbiota-gut-brain axis, from whole diets to individual
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35 foods or bio-actives, including polyphenols (5). Polyphenols are a heterogenous group of bio-
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36 active compounds synthesized exclusively in plants, which share a common phenolic structure of
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37 hydroxyl groups on an aromatic ring (6). The flavonoid class of polyphenols have been extensively

38 researched, with their potential benefits for brain health attributed to antioxidant actions,
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39 neuroprotective effects, enhanced cerebrovascular blood flow, and stimulating neurogenesis (see
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40 Spencer et al (7) for a comprehensive review). Flavonoids have more recently been recognized as

41 prebiotic microbiota modulators, stimulating the proliferation of health-promoting species such as

42 Bifidobacterium spp., while inhibiting the growth of pathogenic species (8,9).

43 Despite the potential for flavonoids to enhance microbiota-gut-brain axis function through multiple

44 pathways, their efficacy on cognition and mood has varied greatly (10–12). This may be related to

45 low bioavailability (13,14), where an estimated 90-95% of dietary polyphenols are not absorbed

46 in the small intestine, reaching the colon where the gut microbiota transforms parent compounds

47 into bioactive secondary metabolites (9,13). The two-way interplay between polyphenols and the

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48 gut microbiota, including prebiotic effects of polyphenols and biotransformation from the gut

49 microbiota, is thought to underlie the ability of dietary polyphenols to produce their clinical effects

50 or lack thereof (15,16). This bidirectional relationship is highlighted in preclinical research, where

51 specific gut microbiota communities were associated with the bioavailability and memory

52 enhancing effects of grape-derived polyphenol supplementation in a sleep-induced model of

53 cognitive impairment, while clinical efficacy was attenuated by antibiotic-induced dysbiosis (17).

54 There have been remarkably few human intervention studies which have attempted to unravel this

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55 relationship (18,19), which theoretically could lead to targeted and enhanced intervention

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56 strategies.

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Blackcurrants (Ribes nigrum) have received little research attention compared to other berry fruits,
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58 yet are one of the richest and most variable sources of anthocyanins (15). The current study tested
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59 a commercially available blackcurrant-based beverage rich in anthocyanins (151 mg) and also
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60 containing 150 mg of pinus radiata extract (pine bark, predominantly comprised of

61 proanthocyanidin class of flavonoids) and 200mg of the amino acid L-theanine, herein referred to
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62 as a flavonoid-rich blackcurrant beverage (FBB).

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63 The objective of this research was to investigate the effects of 4 weeks daily FBB supplementation

64 compared to placebo in healthy female adults, on indicators of brain and mental health,

65 biochemical parameters, and gut microbiome composition and predicted function. We also aimed

66 to understand whether gut microbiome composition at baseline influenced cognitive or mood

67 responses to the intervention, or whether any changes in gut microbiome composition from

68 baseline were associated with changes in behavioral parameters.

69

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70 Methods

71 Study Design

72 The trial followed a randomized, double-blinded, placebo-controlled, cross-over study design. The

73 LINK (Polyphenol rich drink for gut and brain health) study was conducted between July 2022 –

74 January 2023 in Auckland, New Zealand, and was pre-registered with the Australian New Zealand

75 Clinical Trials Registry (ACTRN12622000850774). The protocol was developed in accordance

76 with the SPIRIT (Standard Protocol Items: Recommendations for Intervention Trials) statement

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77 (20), and reported according to the CONSORT statement (21). The procedures were approved by

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78 the Health and Disability Ethics Committee (2022 EXP 12513), and the study was conducted

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according to the guidelines laid down in the Declaration of Helsinki with all volunteers providing
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80 informed consent.
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81 This study measured multiple outcomes related to the microbiome-gut-brain axis following 4
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82 weeks each of FBB and placebo beverages. Stress reactivity to a cognitive stressor (20-minute

83 purple multi-tasking framework (MTF)) was selected as the primary outcome for this research,
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84 relating to both mental health and cognitive function (22,23). Pre-registered secondary outcomes
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85 include cognitive function measured by performance on the MTF, sleep measured by the

86 Pittsburgh sleep quality index (PSQI), mood measured by the profile of mood states questionnaire

87 (POMS), biochemical parameters (serum tryptophan, kynurenine, interleukin-6, brain derived

88 neurotrophic factor (BDNF)), and gut microbiome composition and functional potential. A

89 crossover design was selected to reduce inter-individual variation across these measures,

90 particularly the significant variation observed with gut microbiome outcomes (24).

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92 Participants

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 93 Participants were recruited between July 2022 – September 2022 through opportunity sampling,

94 including online advertising (social media and University of Auckland advertisements) and posters

95 around the University of Auckland. Following expression of interest, potential participants

96 completed an online screening questionnaire. The screening questionnaire included a modified

97 version of the Diet Screening Tool (DST) to categorize participants into balanced ‘optimal’ (n=20)

98 or ‘sub-optimal’ (n=20) diet groups during recruitment, described in more detail below.

99 Eligible participants were healthy females aged 18-45 years of age, with a BMI between 18-30

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100 kg/m2, who reported not using recreational/illicit drugs or to excessively consume alcohol (>15

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101 standard drinks per week), and had access to the internet and technology required to complete

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online questionnaires. Participants had not used antibiotics in the 4 weeks prior to beginning the
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103 intervention, and agreed to abstain from taking probiotics, prebiotics, or supplements/herbal
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104 extracts that might reasonably be expected to interfere with mood or cognition for 4 weeks prior
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105 to, and for the duration of, the study. Participants confirmed that they were not pregnant, nor

106 intending to become pregnant during the trial. Based on self-report, participants had not been
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107 treated for anxiety, depression, or psychiatric disorders within the last 2 years, and were free from
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108 neurological (e.g., Parkinson’s disease), gastrointestinal (e.g., inflammatory bowel disease), and

109 metabolic (e.g., cardiovascular disease) disorders or cognitive impairment. Participants were

110 excluded if they used medications likely to interfere with normal digestive processes (e.g.,

111 laxatives), or if they reported a sensitivity to any ingredient in the treatments.

112

113 Treatment

114 Participants received a 4-week supply each of the 300 mL FBB and placebo beverage, separated

115 by a 4-week washout period. Beverages were supplied and blinded by Ārepa IP Ltd. Treatment

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116 order was determined using a computer-generated randomizer (www.randomizer.org) prepared by

117 an independent researcher, and managed centrally on REDCap during the trial. Sequences were

118 stratified according to DST group, with a balance of ‘optimal’ and ‘sub-optimal’ diets allocated to

119 each treatment order. All research staff involved in the collection and analysis of data remained

120 blinded until all aspects of the study were complete, including statistical analysis.

121 The FBB contains blackcurrant juice and extracts, 150mg of Enzogenol (Pinus Radiata extract,

122 primarily comprised of proanthocyanidins) and 200mg of L-theanine. The placebo beverage was

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123 a taste- and appearance-matched control, which was also matched for macronutrient and vitamin

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124 C (Table 1). The placebo beverage has successfully been used in a prior RCT investigating the

125
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same active intervention (25). The phenolic content of the beverages was identified and quantified
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126 by an independent research laboratory (Cawthron Institute, New Zealand) using liquid
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127 chromatography methods coupled with photodiode array detection and mass spectrometry. The
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128 FBB provided 151mg anthocyanins, with a total polyphenol content of 308mg, while the placebo

129 beverage contained a negligible polyphenol content of 22mg (Table 1).

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130 The beverages were produced in a single batch in the week prior to the intervention starting, then
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131 stored in a dark, temperature-controlled room before distribution to participants. Participants were

132 directed to keep the beverages in their fridge or a cool, dark place at home to minimize polyphenol

133 degradation, and asked to consume their 300 mL beverage daily at a similar time of day for the 4-

134 week period.

135 Participants were asked to maintain their normal diet and lifestyle habits for the duration of the

136 study. Intervention compliance was assessed by weekly online questionnaires, as the return of

137 bottles was considered impractical. Compliance questionnaires included the number of beverages

138 the participant had consumed that week, the reason for not consuming all seven allocated

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139 beverages (if applicable), time of day the beverages were consumed, and if they had made any

140 changes to their diet or lifestyle over the last week. Compliance to the treatment was recorded as

141 a cumulative score (% of beverages consumed out of the total 28 provided), with minimum

142 compliance requirements defined a priori as 80%.

143

144 Procedure

145 Participants were required to visit the Clinical Research Centre (University of Auckland, New

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146 Zealand) on five separate occasions. An enrolment/familiarization visit took place approximately

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147 two weeks before the first testing visit. Following written informed consent, participants provided

148
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health and demographic data, and then completed the 2-minute built-in familiarization session of
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149 the MTF to minimize learning effects. Participants were given a 3-day food record to complete
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150 before their first testing visit, and also given a fecal sample collection kit to collect a fecal sample
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151 in the 24 hours before their first testing visit. Participants were randomly allocated to a treatment

152 order at the end of the enrolment/familiarization visit.

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153 Participants were asked to abstain from caffeine for 12 hours and alcohol for 24 hours before each
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154 testing visit. Participants attended their testing visits between 7am – 10am following an overnight

155 fast, with the time kept consistent across the four visits for each participant. Participants completed

156 stress reactivity and cognitive measures before providing a fasted blood sample. In-person contact

157 time was kept minimal due to ongoing Covid-19 requirements at the time this study was conducted,

158 and participants were instructed to complete online versions of the sleep and mood questionnaires

159 within 24 hours (see Figure 1). Participants were provided with their 28 beverages at the end of

160 visits 1 and 3, and were provided with another stool collection kit at the end of visits 1-3.

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162 Dietary assessment

163 Dietary screening tool

164 The Australian modification of the DST (26,27) was used, requiring only minimal adaptation to

165 the New Zealand context (e.g., updating the name of popular fast food restaurants). The DST is

166 comprised of 20 items, with 18 items related to the frequency of consumption of particular foods

167 (e.g., “How often do you usually eat wholegrain breads or crackers?”), and two items related to

168 the number of servings consumed (e.g., “How many different vegetable servings do you usually

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169 have at your main meal of the day?”). The DST has a maximum score of 104, with higher scores

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170 indicating better diet quality. Cut-off scores of ≤59 (sub-optimal), and ≥60 (optimal) were utilized,

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which have been previously validated in middle-aged adults (28), and shown to discriminate
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172 nutrient intake, blood nutrient status, mood, and cognition in middle-aged adults (27). The intent
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173 of a priori dietary screening was to capture a wider variety of dietary intakes than typically
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174 achieved with opportunity sampling in a healthy population, countering the issue of self-selection

175 bias (29).

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176
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177 Food records

178 Participants completed a 3-day food record before their first testing visit. Instructions on how to

179 complete the food record with as much detail as possible were provided by a Registered Dietitian

180 at the enrolment/familiarization study visit, along with a standardized template to use. All records

181 were checked for accuracy and completeness by a Registered Dietitian, with any concerns resolved

182 in person. One student dietitian entered the food records into FoodWorks software (Version 10,

183 Xyris, Australia), with 10% of the records randomly cross-checked by another student dietitian.

184 The average daily macronutrient and micronutrient composition of participants’ diets were

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185 calculated from the nutrient composition data primarily from the New Zealand FOOD files 2016

186 database, or the AusBrands 2019 and AusFoods 2019 databases if a suitable item was not available

187 in the New Zealand database. Energy over- and under-reporting was checked from the 3-day food

188 records, and all participants had plausible energy intakes (range 5281 kJ/d – 14180 kJ/d). Nutrient

189 intake was compared with age- and sex- specific Estimated Average Requirements (EAR) or

190 Adequate Intakes (AI, if EAR was unavailable) according to the joint Australia and New Zealand

191 Nutrient Reference Values (30).

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193 Stress reactivity and cognitive measures

194 Purple Multi-Tasking Framework

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195 The MTF (Purple Research Solutions, UK) was used for two purposes in this trial; acting as a
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196 cognitive stressor to allow for stress reactivity measurements, and assessment of cognitive function
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197 under increased cognitive demand. The MTF is comprised of four tasks presented in quadrants of

198 a split screen, and requires concurrent attention to all tasks (Figure 2). In the centre of the screen,
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199 a counter displaying the overall performance score based on accuracy and reaction time is
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200 presented. A 20-minute version of the MTF was utilized, with mental arithmetic, stroop, letter

201 retrieval, and visual tracking tasks set to the ‘medium’ intensity level. Participants were actively

202 monitored by research staff at regular intervals throughout the test to heighten performance

203 anxiety. The MTF has previously been shown to elicit cognitive demand, stress, negative mood,

204 and anxiety (31), and is suitable for use in cross-over trials with limited learning effects after

205 several repetitions (32).

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207 Cognitive performance

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208 The MTF produces a concomitant assessment of memory, psychomotor, and attentional

209 performance. A description of the four tasks and their respective scoring is summarized in Table

210 2. An aggregate total multi-tasking score was derived from the four individual tasks, reflective of

211 executive functioning.

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213 Stress reactivity

214 Participants were asked to complete questionnaires (STAI-S, Bond-Lader VAMS, stress and

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215 fatigue VAMS) asking them how they felt in the present moment immediately before and after

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216 completing the MTF. A change score (post MTF – pre MTF) was calculated to measure the

217
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subjective change in mood in response to the cognitive stressor. The questionnaire set has
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218 previously been used to measures stress reactivity in response to cognitive demand (26).
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219 Spielberger State-Trait Anxiety Inventory, State Portion (STAI-S)

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220 The STAI-S is comprised of 20 items (e.g., “I feel calm”) on a 4-point scale ranging from “not at

221 all” to “very much so”. Scores were combined to give a range from 20 – 80 with higher scores
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222 indicating greater levels of present-state anxiety.

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223 Bond-Lader Visual Analogue Mood Scales (Bond-Lader VAMS)

224 The Bond-Lader VAMS is comprised of 16 items, with a 100 mm line separating two antonyms

225 anchored at either end (e.g., tense-relaxed). Participants indicated their current subjective state

226 between the two antonyms on the line, with individual item scores calculated as the distance along

227 the line from the negative antonym. The item scores were combined to form the three domains of

228 alertness, calmness, and contentment, with higher scores indicating more positive mood states.

229 Stress and Fatigue Visual Analogue Scales

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230 Participants were asked to rate their current subjective experience of stress and fatigue on single

231 100 mm visual analogue scales with the words ‘extremely’ and ‘not at all’ anchored at either end.

232 Individual item scores were calculated as the distance along the line from the negative antonym,

233 with higher scores indicating higher levels of stress or fatigue.

234

235 Mood – Profile of Mood States (POMS)

236 The POMS questionnaire is comprised of 65 mood-related adjectives, and required participants to

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237 indicate the degree to which they identified with each item over the past week on a 5-point scale

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238 from “not at all” to “extremely”. Six dimensions were derived from the 65 items, including

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tension/anxiety, confusion/bewilderment, anger/hostility, depression/dejection, fatigue/inertia,
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240 and vigor/activity. A total mood disturbance was also computed as the sum of the first five domains
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241 minus the vigor/activity score. Lower scores indicate better mood states, except in the case of
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242 vigor/activity where a high score is favorable (33).

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243

Sleep – Pittsburgh Sleep Quality Index (PSQI)

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244

245 The PSQI questionnaire is comprised of 19 items which are grouped into seven component scores

246 including subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep

247 disturbance, use of sleep medications, and daytime dysfunction. Each component was scored from

248 0-3, with a composite global PSQI generated by summing the component scores. The global PSQI

249 score was reported in this study with a maximum score of 21, and higher scores indicating poorer

250 sleep quality (34).

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252 Biochemistry analysis

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253 Fasting venous blood samples were collected by a qualified phlebotomist into 10 mL serum

254 separator vacutainer tubes, and allowed to clot at room temperature for at least 15 minutes before

255 being centrifuged at 2000 x g for 15 minutes at 4oC. All samples were processed within 2 hours of

256 collection, and then stored at -80oC before delivery to a commercial laboratory for analysis

257 (Liggins Institute, Auckland, New Zealand). ELISA assays were used to measure serum

258 kynurenine and tryptophan (ImmuSmol, Paris, France) and BDNF (Invitrogen, USA)

259 concentrations. A Cobas e601 autoanalyser (Roche, Germany) was used to measure interleukin-6

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260 concentrations by immunoassay. Coefficients of variance were below the acceptable 20% cut-off

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261 for each assay, based on at least two sets of quality controls run alongside each plate. Over 95%

262
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of interleukin-6 samples were below the lowest detectable amount of 1.5 pg/mL, excluding
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263 interleukin-6 from any further reporting or statistical analysis.
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264
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265 Gut microbiome profiling and bioinformatic analysis

266 Fecal samples were self-collected by participants 24 hours before their study visits without
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267 preservative. Participants were instructed to store samples within their home freezer and transport
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268 them to their study visit with the provided ice packs. Upon receipt, samples were thawed and

269 aliquoted, combining 100mg of stool in 900 µl of DNA/RNA Shield (Zymo Research

270 International, USA, #R1100) by vortex. Samples were subsequently stored at -80°C until DNA

271 extraction was performed at the completion of the study. In total, DNA was extracted from 156

272 study samples, 3 ZymoBIOMICS Gut Microbiome Standards (Zymo Research International, USA,

273 # D6331), and 3 extraction blanks using the standard protocol for ZymoBIOMICS 96 MagBead

274 DNA Kit (Zymo Research International, USA, #D4308). Mechanical bead beating was performed

275 on a 1600 MiniG (SPEX SamplePrep), shaking at full speed (1500 rpm) for 5 minutes. Extract

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276 purity was assessed by spectrophotometry, using the Qubit dsDNA Broad Range Assay Kit

277 (Invitrogen, USA, #Q33266) to measure DNA concentration.

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279 Metagenomic sequencing data were processed by KneadData (version 0.10.0) to trim and remove

280 poor quality reads and those that mapped to the human genome (hg19). Taxonomic profiling was

281 subsequently performed with MetaPhlAn3 (version 3.1). Functional profiling was performed with

282 HUMAnN3 (version 3.6) with resulting gene families and pathway abundance tables renormalized

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283 to copies per million (CPM).

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Downstream analysis and plot generation were conducted in R statistical software (v 4.2.1). The
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286 vegan package was used to calculate α-diversity (Shannon index) and β-diversity (Bray-Curtis
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287 dissimilarity) based on species-level composition. Ordinations were performed by non-metric

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288 multidimensional scaling and differences in microbiome structure between intervention arms were

289 assessed by PERMANOVA. The Maaslin2 package was used to test for differentially abundant
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290 species, genera, and pathways utilizing both general linear models and linear mixed effects
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291 modeling designs. All models included a log transformation, minimum prevalence was set to 0.1,

292 and the default significance threshold following FDR correction was used (q < 0.25). To identify

293 microbiome features that changed after consumption of the active beverage, models were fitted

294 with fixed effects for intervention group (0 = placebo, 1 = active), time (0 = baseline, 1 = 4-weeks),

295 and their interaction (1 = active & 4-weeks, 0 = all other combinations), and subject ID was added

296 as a random effect.

297

298 Statistical analysis

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299 A power calculation was performed, based upon previous reported mean changes in stress

300 reactivity measures between an intervention containing similar doses of anthocyanins and a control

301 (35). Thirty-six participants would allow the detection of significant effects with a power of 0.8 at

302 α = 0.05, with 40 participants recruited to allow for drop-out.

303

304 Intention to treat (ITT) analyses were conducted including all participants who received at least

305 one treatment dose and satisfied the inclusion criteria at enrolment into the study. Statistical

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306 analyses were performed using R 4.2.1 Statistical Software (36), with p<0.05 considered

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307 significant unless otherwise specified. Before analysis, multiple imputations of missing data was

308
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conducted using chained equations in the MICE package, and distribution of continuous variables
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309 was graphically assessed for normality and outliers to ensure their appropriateness for parametric
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310 statistical tests.

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312 Linear mixed effect models were used to examine changes in stress reactivity, cognitive, mood,
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313 sleep, and blood measures from baseline to follow-up within and between intervention groups. The
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314 models included time (baseline, 4-weeks), intervention group (active, placebo), their interaction

315 (time x intervention group) and sequence allocation as fixed factors, and subject ID as a random

316 factor to account for repeated measures. Significant interaction effects were followed by multiple

317 pairwise comparisons with Tukey’s adjustment.

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319 Associations between gut microbiome features and clinical parameters were restricted to those

320 clinical parameters shown to improve in the analyses above. This included analyses to identify

321 differences in baseline gut microbiome features of clinical ‘responders’, using maaslin2’s linear

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322 mixed effect models fitted with a binary response category (0 = non-responder, 1 = responder).

323 Responders were defined as those participants with changes to clinical scores in the top quartile.

324

325 Results

326 Participants

327 Of 110 potential participants screened, 40 healthy females were enrolled into the trial. A total of

328 38 participants (95% retention) completed both intervention arms in this cross-over trial between

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329 July 2022 – January 2023, with 2 participants withdrawing before starting their second intervention

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330 arm for Covid-19-related reasons (Figure 3). As shown in Table 3, this was a healthy population

331
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of females with a mean age of 29.8 years, and most participants rating their own health as either
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332 excellent (37%) or very good (40%). Participants regularly engaged in moderate or high-intensity
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333 exercise, and the majority of participants were employed full time (63%) and had completed either
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334 undergraduate (47%) or postgraduate (45%) university degrees. Nutrient intake according to 3-

335 day food records is presented in Supplementary Table 1, with group averages within
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336 recommended ranges for most nutrients with the exception of calcium and sodium.
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338 All participants met minimum compliance requirements of 80% (Supplementary figure 1). A

339 summary of adverse events reported during the intervention period is provided in Supplementary

340 table 2. In summary, the frequency of reported adverse events was similar between active (18%)

341 and placebo (27%) beverages, with the majority of adverse events being categorized as mild (82%

342 of reported adverse events), and gastrointestinal symptoms were the most commonly reported

343 adverse event (82% of all reported adverse events).

344

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345 Primary outcome – stress reactivity

346 There was no difference in changes of stress reactivity measures (alertness, calmness,

347 contentedness, stress, fatigue) between intervention groups after 4 weeks of intervention with

348 active and placebo beverages, including scores from the Bond-Lader VAMS, fatigue and stress

349 visual analogue scales, and the STAI-S tools. Raw data and effect size estimates derived from

350 linear mixed effect models are presented in Table 4.

351

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352 Secondary outcomes

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353 Cognitive performance

354
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Changes (Δ = 4-week score – baseline score) in letter retrieval domain scores from the MTF, a
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355 measure of working memory, were significantly different between intervention groups (interaction
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356 effect p=0.034) (Table 4). Multiple pairwise comparisons with Tukey’s adjustment showed a 33%
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357 improvement in letter retrieval scores after 4-weeks of the FBB (Δ = 1026 point increase, p<0.001),

358 while no change was observed with the placebo beverage (Δ = 368 point increase, p=0.331)
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359 (Figure 4).

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360

361 Mood

362 Changes in anger/hostility and tension/anxiety domains of the POMS questionnaire differed

363 between intervention groups (Anger interaction effect, p=0.043; Tension interaction effect,

364 p=0.030) (Table 4). Multiple pairwise comparisons with Tukey’s adjustment showed 37% less

365 anger (Δ = 1.9 point reduction, p=0.013) and 20% less tension (Δ = 1.7 point reduction, p=0.023)

366 in POMS scores after 4-weeks of the FBB, while no change was observed with the placebo

367 beverage (p>0.05) (Figure 4). The interaction effect neared significance for total mood

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368 disturbance scores (p=0.052), with multiple pairwise comparisons showing a 43% improvement

369 after 4-weeks of the active intervention (Δ = 8.9 point reduction, p=0.013) but not placebo (Δ = 0.9

370 point reduction, p=0.989).

371

372 Sleep and biochemical markers

373 There were no differences in changes to global PSQI scores or any of the biochemical markers

374 under investigation (BDNF, tryptophan, kynurenine, tryptophan/kynurenine ratio) between

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375 intervention groups (Table 4).

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377 Microbiome composition re
378 There were no differences in α-diversity metrics (Shannon index), nor in the number of detected
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379 species, gene families, or pathways between intervention arms (Figure 5A). There was also no

380 difference in microbiome composition shifts between the intervention arms, comparing
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381 participant’s baseline and 4-week samples (Wilcoxon Rank Sum Test, p=0.21, Figure 5B).
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382 Similarly, there were no intervention effects on β diversity (PERMANOVA, R2=0.45%, p=0.81),
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383 with microbiome samples instead clustering by participant (PERMANOVA, R2=79.5%, p=0.001)

384 (Figure 5C).

385

386 The next analysis tested whether there were any specific species, genera, or microbiome encoded

387 pathways whose relative abundance changed after supplementation with the FBB. Thirteen

388 species, 3 genera, and 7 pathways were differentially abundant based on a significance threshold

389 of p<0.05; however, none of these remained significant after adjusting for multiple testing (q>0.25)

390 (Supplementary File 2).

391

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392 Microbiome features associated with clinical response

393 We explored whether there were any baseline gut microbiome features that differentiated

394 participants who had greater improvements (responders defined as the top quartile) in behavioral

395 outcomes. This analysis was restricted to clinical variables shown to significantly improve with

396 FBB supplementation in prior analyses, including letter retrieval, tension/anxiety, and

397 anger/hostility scores.

398

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399 While there were no differences in α-diversity or richness (species-, and gene-levels), there was a

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400 nominally significant higher abundance of Bifidobacterium spp., at baseline who exhibited greater

401
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improvements in letter retrieval scores (Figure 5D), specifically B. longum and B. bifidum
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402 (maaslin2 model, p < 0.05, Figure 5E and 5F). A full summary of maaslin2 models identifying
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403 differences in species, genera, and pathways between responders and non-responders for letter
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404 retrieval, tension/anxiety, and anger/hostility outcomes is provided in Supplementary file 2.

405
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406 Partial bivariate Pearson correlation analyses were planned to assess associations between changes
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407 in microbiome features and clinical parameters, but this was not carried out given that there were

408 no significant change in species, genera, or pathways as a result of the FBB.

409
410

411 Discussion

412 The LINK study investigated the effects of a flavonoid-rich blackcurrant-based beverage on

413 markers of the microbiota-gut-brain axis. This randomized, double-blind, placebo-controlled,

414 crossover trial found that, compared with a placebo drink, daily consumption of the FBB for 4-

415 weeks improved secondary outcomes of cognitive performance during multi-tasking and mood in

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416 a group of healthy female adults. Although there were no changes to gut microbiome composition

417 or functional potential as a result of the FBB, there was a nominal difference in baseline relative

418 abundance of Bifidobacterium genera and species (B.longum, B. bifidum) participants who had

419 greater improvements in cognitive responses following the FBB supplementation.

420

421 Stress reactivity refers to the way in which people respond to stressful or demanding situations,

422 specifically in this case where cognitive resources are insufficient to meet ongoing task demands.

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423 It is a mechanism by which psychological stress is proposed to contribute to health or disease,

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424 including mental health and neurological disorders (22,23). Kimble et al (35) have shown that 3-

425
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months of supplementation with anthocyanin-rich tart cherries improves measures of stress
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426 reactivity in middle-aged adults, including fatigue and mental alertness, following a cognitive
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427 battery. Although no such improvements were found in our primary outcome of stress reactivity
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428 with the FBB, it is important to note that our participants were minimally responsive to the MTF

429 as a cognitive stressor. This is despite using the same MTF protocols that have reliably increased
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430 self-ratings of negative mood and anxiety as well as psychobiological stress responses (31). While
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431 the presence of learning effects is a possibility, previous research has shown that the MTF can be

432 repeated on multiple occasions without habituation of responses (32). Also given only trivial

433 changes in scores seen here, a learning effect is unlikely. Findings from this study therefore cannot

434 confirm or refute whether the FBB buffers psychological stress in response to situations perceived

435 as stressful or demanding, as any potential intervention effect would have been masked by the lack

436 of response to the MTF. Interestingly, acute blackcurrant supplementation has been shown to alter

437 electroencephalogram spectral power activity in healthy young adults during a 21-minute cognitive

438 battery despite no change to subjective mood questionnaires, including evidence of an anxiolytic

21
439 effect from increased slow wave δ and θ spectral powers (37). Using more objective measures of

440 stress (e.g., salivary cortisol or heart rate monitoring) would have been helpful to confirm

441 participants’ lack of subjective response reported here.

442

443 The improvements to cognitive performance observed after 4-weeks of daily FBB intake are

444 consistent with previous evidence describing cognitive benefits of supplementation with

445 anthocyanin-rich fruits (11,35,38,39) and flavonoid-rich foods or beverages (40,41). Memory

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446 appears to be a specific cognitive domain sensitive to flavonoid supplementation (42), though

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447 direct comparisons to this literature are somewhat limited by differences in cognitive testing

448
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paradigms. Our results do require interpretation in the context of how cognitive function was
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449 evaluated. Multi-tasking while being monitored is an ecological behaviour with clear parallels in
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450 day-to-day cognition. It requires integration of several complex processes across different
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451 cognitive domains (31). While improvements to the letter recall task do relate to working memory

452 function, this cannot be isolated from attention and executive functioning which are both required
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453 to perform throughout the multi-tasking paradigm. Regardless, it is promising to see benefits for
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454 performance in healthy, younger populations which is a critical time for brain health optimization

455 and prevention of disease (43).

456

457 There was also improvements in tension/anxiety, anger/hostility, and total mood disturbance

458 scores of the POMS questionnaire after 4-weeks of FBB supplementation, with a magnitude of

459 20–43% improvement across the domains. Although polyphenols target multiple

460 pathophysiological pathways relevant to both mental health and cognition (5,12), the evidence

461 base in human intervention trials is much more limited in the relatively nascent field of nutritional

22
462 psychiatry compared to that for cognitive function. Previous flavonoid interventions have typically

463 been conducted in disease states like depression (12) or in acute intervention settings (39,44),

464 although Fisk et al have shown improvements to mood with 4-weeks of wild blueberry

465 supplementation (253mg anthocyanins) in healthy adolescents aged 12-17 years (45). To the best

466 of our knowledge, this is the first time that improvements to mood have been observed in a healthy

467 adult population for any of the constituent ingredients or bio-actives. Although there appeared to

468 be consistency in changes across several domains of mood observed in the current study, cautious

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469 interpretation is needed given that these were secondary outcomes unadjusted for multiple testing.

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470 These findings warrant future investigation in a study adequately powered for such outcomes and

471
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with a longer-term follow-up to understand whether the effects are true or spurious.
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472
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473 We investigated blood biomarkers relevant to the gut-microbiota-brain axis in attempt to gain
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474 mechanistic insights, but found no changes to BDNF or the kynurenine/tryptophan ratio, and the

475 majority of our healthy participants had inflammatory markers below the detectable limit which
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476 prevented further analysis. BDNF is a critical modulator of synaptic plasticity, with previous
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477 literature showing increased BDNF concentrations following polyphenol supplementation in

478 human intervention trials (46,47) and preclinical research (48). Although no change was reported

479 in the current study, the testing conditions may have contributed to this. For example, stress

480 reactivity and cognitive measures were prioritized and performed before the blood draw which

481 may have itself resulted in changes in biomarkers. Additionally, the tryptophan/kynurenine ratio

482 may be more sensitive to changes in disease conditions, as previous research has shown an

483 attenuated increase in the kynurenine/tryptophan ratio in a mouse model of agitated depression

484 with flavonoid supplementation (49), although there are limited human intervention trials

23
485 investigating the markers of the kynurenine pathway in response to flavonoid supplementation

486 (12).

487

488 Modulating the gut microbiome is hypothesized as a mechanism by which mood (5) and cognitive

489 (50) benefits of polyphenols are mediated. Dietary polyphenols are proposed to have a prebiotic-

490 like effect, stimulating the abundance of health-promoting species such as Bifidobacteria spp.,

491 and Lactobacillus spp., and inhibit the growth of potentially pathogenic taxa such as Clostridium

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492 (51). Our data did not support a prebiotic effect of the FBB, although this is perhaps unsurprising

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493 given that this was a healthy population with generally high fibre intakes (29 ± 13 grams per day).

494
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Furthermore, the gut microbiome is highly variable between individuals which makes it difficult
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495 to find consistent changes associated with nutritional interventions, particularly with a small
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496 sample size (24,52). We might expect to see a more pronounced response in conditions
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497 characterized by dysbiosis. For example, supplementation with red wine polyphenols for 30 days

498 has been shown to increase relative abundance of Bifidobacteria and Lactobacillus spp., in patients
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499 with metabolic syndrome but not healthy controls (53).

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500

501 Although there was no significant changes to the relative abundance of microbial taxa or diversity

502 metrics in response to the FBB, this does not exclude the possibility that the cognitive- and mood-

503 benefits observed here are still mediated through the gut microbiome. For example, in vitro work

504 has shown the ability of anthocyanins to increase short chain fatty acid production (54,55), organic

505 acids which are known to influence cognition and mood (56). It may be that the underlying

506 structure and encoded functions of the microbiome remain stable, as seen here with microbial gene

507 abundance data, but its activity is altered. Future research should consider analyses of the actively

24
508 transcribed genes (meta-transcriptomics) or gut-derived metabolites and metabolome (standard

509 analytical chemistry methods and metabolomics) to better understand the role of the gut

510 microbiome in mediating the clinical benefits of polyphenol supplementation.

511

512 While research on cardiometabolic or cognitive effects of polyphenol supplementation are now

513 beginning to include gut microbiome measurements alongside clinical parameters (18,19), few

514 have considered baseline gut microbiome as a mechanistic key to understanding clinical effects.

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515 To the best of our knowledge, this is the first human intervention trial which has shown differences

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516 in baseline gut microbiome composition in cognitive ‘responders’. Specifically, we found that our

517
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clinical responder group (in the top quartile of response) for letter recall scores had nominally
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518 higher concentrations of Bifidobacterium spp., at baseline, including B. longum and B. bifidum.
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519 We propose these findings implicate a role for Bifidobacterium spp., in moderating how
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520 participants responded to the FBB. This aligns with previous in vitro (54) and human clinical

521 research (57) showing that bacteria from the Bifidobacterium genus have the capacity to
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522 metabolize anthocyanin extracts into small molecular phenolic acids. Indeed, microbial
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523 biotransformation has been proposed as a key step in the ability of polyphenols to exert clinical

524 benefits (15,16). The relevance of our findings for future research lies in more targeted (e.g., gut

525 microbiome profiling) intervention strategies to reveal the true potential of polyphenols in a field

526 with highly variable findings, or enhanced intervention strategies (e.g., co-supplementation with

527 relevant probiotic species). Larger-scale intervention trials with more targeted and diverse

528 polyphenol treatments are required to prove this concept.

529

25
530 This study was a rigorous, double-blinded, cross-over trial with excellent participant retention and

531 adherence, however the current study should be evaluated in the context of its limitations. First,

532 the commercially-available FBB investigated here contained a blend of bio-active ingredients.

533 This includes 151mg of anthocyanins from blackcurrant juice and extracts, 150mg of

534 proanthocyanidin-rich pinus radiata extract contributing towards the total polyphenol dose of

535 308mg, and 200mg of L-theanine. The observed effects can therefore not be attributed to any single

536 ingredient. This is also an issue for polyphenol-rich foods or beverages where other bioactive

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537 compounds are present, a good example being caffeine in cocoa or vitamin C content in fruits. We

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538 have discussed that our findings align with previous research on anthocyanin-rich fruits and the

539
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literature on flavonoids more broadly, but the fact that L-theanine is not an inert ingredient is an
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540 important limitation. The neuropharmacological effects of L-theanine are well-defined, including
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541 an influence on brain wave oscillations and neurotransmitter synthesis and degradation (58), with
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542 a net effect of reduced excitatory activity and enhanced performance under conditions of attention

543 or stress (59). Translation of efficacy to human clinical intervention trials have been much less
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544 clear however, and the majority of research has been performed in acute settings (60,61) compared
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545 to evidence from flavonoid intervention studies where more convincing effects are seen with

546 longer-term studies (10). Given that L-theanine peaks in plasma 50 minutes after ingestion and is

547 largely cleared from plasma within 24 hours, it is unlikely that L-theanine is driving the results

548 observed here. Analyzing urinary or fecal polyphenol metabolites and correlating with clinical

549 outcomes could have helped to overcome this issue. Having a third arm in the study (e.g.,

550 flavonoid-rich blackcurrant beverage with and without L-theanine) would also be an interesting

551 experiment to understand whether bio-actives like L-theanine can enhance or interact with

552 polyphenols (60), but was beyond the scope of the current research which was primarily focused

26
553 on understanding interactions with the microbiota-gut-brain axis where the influence of L-theanine

554 was considered negligible.

555

556 Another important limitation is that our dietary assessment methodology does not capture habitual

557 polyphenol intake, although this is widely acknowledged as a challenge in the research field due

558 to inadequate dietary assessment tools and limited locality-specific food content data (12,62).

559 Some studies do attempt to isolate intervention effects by restricting background polyphenol intake

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560 (35). We also did not conduct follow-up dietary assessment, and while participants reported that

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561 they had not made any changes to diet/lifestyle which was cross-checked with researchers, we

562
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cannot be absolutely certain that dietary changes may have contributed to intervention effects.
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563 Finally, females were selected as the study population given their greater risk of mental health
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564 disorders (63) and conditions relevant to microbiota-gut-brain axis dysfunction like irritable bowel
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565 syndrome (64,65) but this does of course limit generalizability of findings. The clinical relevance

566 of our findings is also limited by the younger age range (18-45y) and health of our participants,
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567 and relatively short 4-week intervention period. We also acknowledge the inflated risk of type 1
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568 error because of the number of outcomes measured and statistical tests conducted, but these were

569 pre-defined and conducted with appropriate correction in the case of multiple pair-wise

570 comparisons. It is pertinent to highlight that we have not adjusted to a more conservative level of

571 significance for the number of secondary outcomes tested. Although this approach does generate

572 pathways for future research such as in the case of mood effects observed here, this does

573 necessitate cautious interpretation of the current study.

574

575 Conclusion

27
576 This study is a novel investigation into the effects of a flavonoid-rich blackcurrant-based

577 beverage on markers of the microbiota-gut-brain axis in healthy female adults. We found that

578 daily consumption of the FBB for 4-weeks improved secondary outcomes of cognitive function

579 and mood. Although the FBB did not alter gut microbiome composition or diversity, participants

580 with the greatest cognitive responses had a greater relative abundance of Bifidobacterium spp., at

581 baseline which implicates the gut microbiome in mediating the benefits of the flavonoid-rich

582 intervention. This research adds to a growing body of evidence which supports flavonoids as a

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583 plausible preventative strategy to tackle the burden of inter-related brain and mental health

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584 disorders, but improvements to secondary outcomes observed should be confirmed in larger

585
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sample sizes with a longer intervention period. Future research is needed to understand whether
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586 enhanced intervention effects can be observed with a more targeted approach through gut
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587 microbiome profiling at baseline, or perhaps with concurrent probiotic supplementation.

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588
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28
Acknowledgements

NAG, NCR, AS, and AJB designed the research, NAG and BCW conducted research, NAG, BCW,
and JRM analyzed data, NAG and BCW wrote the paper, NAG had primary responsibility for final
content. All authors read and approved the final manuscript.

We are grateful to all participants for their contribution to the study, and thank Eric Thorstensen,
Christine Keven, and Tommi Vatanen for their technical help and expertise.

Data Availability:
Deidentified data described in the manuscript, code book, and analytic code will be made available
upon reasonable request via e-mail to the corresponding author.

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Tables

Table 1. Phenolic and nutritional content of treatment beverages.

Active intervention beverage Placebo control beverage
Total Polyphenols (mg) 308 22
Anthocyanins (mg) 151 7
Energy (kj) 155 155
Protein (g) 0.4 0.4
Fat total (g) 0 0
Fat Saturated (g) 0 0
Carbohydrate (g) 23 23
Sugars (g) 14.8 14.8
Sodium (g) 8 8
Vitamin C (mg) 90 90

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Phenolic content was independently analyzed by a research laboratory (Cawthron Institute, New Zealand) using liquid
chromatography methods coupled with photodiode array detection and mass spectrometry.

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Table 2. Multi-tasking framework tasks and scoring
MTF task Description Scoring
Presents an addition equation of two rows of
Mental
three-digit numbers, requiring participants to
arithmetic
enter their answer using the number pad.
Displays colour words (“blue”, “green”, “red”,
or “yellow”) in text which are presented in one
Stroop of the four corresponding colors. Within 20 +10 points are allocated for
seconds, participants are required to indicate correct responses and -10
the font colour by selecting the correct colour points for incorrect
displayed in the panel. responses, timeouts, or
Displays a set of four letters which disappear prompts.

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after four seconds at the start of the test. A
Letter
series of single probe letters are then presented.

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recall
Participants are required to indicate whether the

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probe letter appeared in the original set of four
letters.
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A maximum of +10 points if
the reset button is selected
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while the red dot is within the

outermost circle, through to
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A red dot moves outwards through a series of +2 points if within the

Visual concentric circles. Participants are required to innermost circle.
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tracking select the reset button before the red dot move
beyond the outermost circle. Points are lost at a rate of -10
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points for every 0.5 seconds

where the red dot has reached
the boundary of the
outermost circle .

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Table 3. Characteristics of participants who completed both intervention arms in the LINK
trial.
Total sample Active > Placebo Placebo > Active
(n=38) sequence (n=20) sequence (n=18)
Age, years 29.8 ± 7.1 30.2 ± 8.0 29.5 ± 6.0
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BMI, kg/m 22.8 ± 3.3 23.3 ± 4.1 22.1 ± 1.9
Alcohol (drinks/week) 2.6 ± 2.8 3.2 ± 3.0 1.9 ± 2.4
Supplement use
Yes 8 (21.1%) 5 (25%) 6 (33%)
No 30 (78.9%) 15 (75%) 12 (67%)
Education
Secondary school 3 (7.9%) 2 (10%) 1 (6%)

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University - undergraduate 18 (47.4%) 10 (50%) 8 (44%)
University - postgraduate 17 (44.7%) 8 (40%) 9 (50%)

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Exercise
High 21 (56.8%) 9 (45%) 12 (67%)
Moderate
Low
14 (36.8%)
3 (8.1%)
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1 (5%)
4 (22%)
2 (11%)
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Self-rated health
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Excellent 14 (36.8%) 7 (35%) 7 (39%)

Very good 15 (39.5%) 7 (35%) 8 (44%)
Good 8 (21.1%) 5 (25%) 3 (17%)
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Fair/poor 1 (2.6%) 1 (5%) 0 (0%)

Dietary screening tool (DST)
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Total score 65.8 ± 12.6 65.7 ± 12.7 65.9 ± 12.9

Optimal group (DST > 60) 19 (50%) 9 (50%) 10 (50%)
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Suboptimal group (DST ≤ 60) 19 (50%) 9 (50%) 10 (50%)

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Exercise intensity was estimated from the International Physical Activity Questionnaire short
form (IPAQ-SF).

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Table 4. Change in clinical parameters rom baseline to follow-up according to intervention group.

Difference in change between

Outcome Measure/Domain Active intervention Placebo control
active and placebo
Baseline 4-weeks Δ Baseline 4-weeks Δ Effect size P-value
Stress reactivity Bond Lader VAMS
Alertness 0.3 ± 1.1 0.5 ± 1.0 0.2 0.1 ± 1.4 0.2 ± 1.3 0.1 0.1 0.723
Calmness -1.5 ± 2.3 -1. 1 ± 1.8 0.4 -1.6 ± 1.7 -1.3 ± 1.9 0.3 0.1 0.893
Contentedness 0.3 ± 0.9 0.0 ± 0.8 -0.2 0.3 ± 0.8 0.2 ± 1.0 -0.1 0.1 0.821
Fatigue VAS 0.4 ± 2.0 0.2 ± 1.5 -0.2 0.6 ± 1.6 0.3 ± 1.6 -0.3 0.0 0.999

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Stress VAS 0.3 ± 1.9 0.2 ± 1.8 -0.1 0.8 ± 2.1 0.5 ± 1.9 -0.3 0.2 0.749
STAI-S 1.6 ± 6.7 1.7 ± 5.2 0.1 2.6 ± 5.1 1.8 ± 4.9 0.8 0.9 0.539

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Cognition Mental arithmetic 440 ± 311 462 ±331 22 423 ±353 505 ± 341 82 60 0.383

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Letter retrieval 3098 ± 1924 4124 ± 2036 1026 3485 ± 1665 3853 ± 1985 368 658 0.0341

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Stroop 2750 ± 1995 3158 ± 2178 408 2933 ± 1632 3221 ± 1463 288 121 0.773
Visual tracking 406 ± 255 448 ± 56 42 441 ± 88 429 ± 131 12 53 0.551

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Total MTF score 6696 ± 2782 8205 ± 2702 1509 7281 ± 2455 8004 ± 2106 723 785 0.182
Mood Anger 5.4 ± 4.8 3.4 ±3.2 -2.0 4.2 ±3.5 4.0 ± 4.2 -0.2 1.8 0.0432

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Confusion 7.0 ± 2.9 5.8 ± 2.1 -1.2 6.7 ±2.7 6.4 ±2.7 -0.3 0.9 0.126
Depression 5.8 ±6.1 3.8 ±4.4 -2.0 6.5 ± 6.0 6.0 ± 7.2 -0.5 1.5 0.293
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Fatigue 9.0 ± 4.4 7.1 ± 4.9 -1.9 8.9 ± 5.5 8.7 ±5.1 -0.2 1.8 0.100
Tension 8.6 ± 4.0 6.9 ± 3.6 -1.7 8.0 ± 4.0 8.1 ±4.5 0.1 1.9 0.0303
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Vigour 15.1 ±5.5 15.3 ± 5.5 0.2 14.1 ±5.7 14.1 ± 6.7 0.0 0.1 0.920
Total mood disturbance 20.6 ± 20.5 11.7 ± 16.3 -8.9 20.1 ± 20.7 19.1 ±23.7 -1.0 8.0 0.0524
Sleep PSQI 5.8 ±.9 2.9 5.0 ± 2.9 -0.8 6.0 ±2 5.6 ± 3.0 -0.4 0.4 0.441
Biochemical marker BDNF, pg/mL 25676 ± 6885 25959 ± 6661 283 27068 ± 8820 26716 ± 7235 -352 636 0.727
Tryptophan, µg/mL 11.3 ± 2.8 11.0 ± 2.6 -0.3 11.7 ± 2.4 11.2 ± 2.6 -0.5 0.2 0.715
Kynurenine, ng/mL 457 ± 86 480 ± 160 23 469 ± 98 457 ± 95 -12 36 0.265
Tryptophan/Kynurenine 0.03 ± 0.01 0.02 ± 0.01 0.0 0.03 ± 0.01 0.03 ± 0.01 0.0 0.0 0.683
Abbreviations: BDNF, brain derived neurotrophic factor; PSQI, Pittsburgh sleep quality index; STAI-S, State trait anxiety inventory – Stress subscale; VAMS,
Visual analogue mood scale; VAS, Visual analogue scale. Effect sizes (estimated marginal means of differences between groups) and p-values are derived from
linear mixed effect models. According to post-hoc analyses with Tukey adjustment for multiple comparisons on variables with significant interaction effects:
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Letter retrieval scores increased with the active intervention (p<0.001) but not placebo (p=0.331); 2Perceived anger levels improved with the active intervention
(p=0.013) but not placebo (p=0.997); 3Perceived tension levels improved with the active intervention (p=0.023) but not placebo (p=0.998). 4Given the closeness to

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significance threshold, post-hoc analyses were performed and revealed that total mood disturbance improved with the active intervention (p=0.013) but not placebo
(p=0.989).

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Figure Legends

Figure 1. LINK Study schematic

Figure 2. Screen layout of the Purple Multi-Tasking Framework (MTF). The MTF requires

participants to simultaneously perform four cognitive tasks which were (clockwise from top left)

mental arithmetic, stroop, letter recall, visual tracking).

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Figure 3. Consolidated standards of reporting trials (CONSORT) flow diagram of the LINK

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study.

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Figure 4. Change in cognitive and mood measures differ between intervention groups. Post-
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hoc analyses of significant time x intervention group interactions revealed significant

improvements following 4-weeks of the active intervention for A) Letter retrieval scores: Δ = 1026
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point increase, p<0.001; B but not placebo (p>0.05); B) Tension/anxiety: Δ = 1.7 point reduction,
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p=0.023; C) Anger/hostility: Δ = 1.9 point reduction, p=0.013; D) Total mood disturbance: Δ =

8.9 point reduction, p=0.013, though note a near-significant interaction effect for this domain

(p=0.052).

Figure 5. Effect of the PBB intervention on gut microbiome composition.

(A) Shannon diversity index based on species composition at baseline and 4-weeks after

supplementation with active and placebo beverages. (B) Bray-Curtis dissimilarity in species

composition between participant’s baseline and 4-week sample for both intervention arms. (C)

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Non-metric multi-dimensional scaling (MDS) plot showing that variability in species composition

was largely driven by subject rather than by intervention. Relative abundance of the genus

Bifidobacterium (D) and species Bifidobacterium longum (E) and Bifidobacterium bifidum (F)

was higher at baseline in the top quartile of participants whose letter retrieval scores improved (i.e.

the “Top responders”).

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 Assessed for eligibility (n=110)

Excluded (n=70)
Did not meet inclusion criteria (n = 27)
▪ BMI >30 (n = 3)
▪ Medication (n = 5)
▪ Supplement use (n = 19)
Declined to participate (n = 17)
Other reasons (n = 26)
▪ Dietary subgroup already full (n=26)

Randomized (n=40)

Allocated to placebo > active sequence (n = 20) Allocated to active > placebo sequence (n = 20)
20 Received allocated placebo beverage (4 weeks) 20 Received allocated active beverage (4 weeks)

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4-week washout period 4-week washout period
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Lost to follow-up (n=2) Lost to follow-up (n=0)
▪ Covid-19 illness (n=2)
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Allocated to active beverage (n = 18) Allocated to placebo beverage (n=20)

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20 Received allocated active beverage (4 weeks) 20 Received allocated placebo beverage (4 weeks)
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Lost to follow-up (n=0) Lost to follow-up (n=0)

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Data analysed from n = 18 Data analysed from n=20

• Stress reactivity (n = 18) • Stress reactivity (n = 20)
• Cognitive function (n = 17) • Cognitive function (n = 19)
▪ Software corrupted (n = 1) ▪ Software corrupted (n = 1)
• Mood and sleep (n = 18) • Mood and sleep (n = 20)
• Microbiome composition (n = 18) • Microbiome composition (n = 20)
• Blood biomarkers (n = 14) • Blood biomarkers (n = 17)
▪ Technical failure (n = 2) ▪ Technical failure (n = 3)
▪ Declined to participate (n = 1)
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Declaration of interests

☐ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☒ The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

Nicola A Gillies reports financial support was provided by High Value Nutrition National Science
Challenge. Nicola A Gillies reports equipment, drugs, or supplies was provided by AlphaGen NZ Ltd. In
the last three years, AS has received research funding and/or consultancy/travel/speaker fees from:
BioRevive, Coca Cola, Ekterra, Frutarom, Givaudan, National Health and Medical Research Council,

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Nestlé, Nutricia Research Foundation, PepsiCo, Reviv, Wörwag Pharma and Wrigley-Mars. AS is Chief
Scientific officer for Ārepa Nootropics. If there are other authors, they declare that they have no

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known competing financial interests or personal relationships that could have appeared to influence
the work reported in this paper.

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