The Acute and Chronic Effects of Ecologic Barrier© on Mood and Cognition
Phase: NA
Conditions: Cognitive Change, Mood
Sponsor: University of Reading

 
 
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Study title: Acute and Chronic Effects of Ecologic Barrier© on Mood and Cognition 
Acronym: ProCog 
Date: 29/12/2020 
 
 

 
 
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ProCog Study Design & Protocol 
 
Aims  
 
To investigate the acute (24 hours) and chronic (4/8 weeks) effects of probiotics (Ecologic 
Barrier) on cognitive performance and mood in ageing adults.  
 
Design  
 
Double-blind, cross-over, randomised control trial. All outcomes measured at baseline, 23 
hours, 4 weeks and 8 weeks (see page 3 for explanation). 
 
Intervention  
 
Ecologic Barrier© – daily intervention of 2g (5 billion CFU per day). Placebo (carrier) 
matched in appearance, smell and taste. 
 
Cognitive Outcomes  
 
Based on the literature review to-date, exact tasks to be confirmed (see page 10 for 
justifications). Parallel versions required for multiple test points.  
- 
An emotional recognition/decision task  
- 
Rey Auditory Verbal Learning Test (RAVLT) 
- 
Corsi blocks 
- 
Switching task 
 
Mood Outcomes 
 
Based on the literature review to-date, exact tasks to be confirmed (see page 10 for 
justifications). 
- 
Leiden Index of Depression Sensitivity revised (LEIDS-r) 
- 
Positive and Negative Affect Schedule expanded (PANAS-x) 
- 
State items from State Trait Anxiety Inventory 
 
Additional outcomes/possible covariates (see page 6 for justifications)  
 
- 
Dietary measures with a food frequency questionnaire (FFQ) 
- 
Two-day food diary to be completed over the acute arm 
- 
Perceived Stress Scale  
- 
Centre for Epidemiological Studies Depression Scale (CESD)  
- 
Faecal microbiota profile (baseline and 8 weeks. Samples will also be collected at 4 
weeks with analysis subject to cognitive results) 
 
No. of Participants: 30 based on previous studies and G*Power calculation, see page 11.  
 
Exclusion criteria:  
 

 
 
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Already a habitual user of probiotics, antibiotic treatment within last 3 months, no current 
diagnosis of and not currently receiving treatment for mental health disorder, gastrointestinal 
disorder, allergic to any ingredient of active or placebo treatment. Regular smoker. 
Participants must not consume probiotic or prebiotics supplements or live yoghurt throughout 
the course of study.  

 
 
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Schematic diagram of cross-over RCT 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening and 
familiarisation day 
 
T0 (baseline) 
measures 
(e.g. 09.00) 
Acute dose  
(e.g. 09.50) 
T23 measures 
(e.g. 09.00) 
W4 measures 
(e.g. 09.00) 
1 week apart 
W8 measures 
(e.g. 09.00) 
Chronic arm 
After the acute 
arm, participants 
immediately begin 
self-administration 
of Ecologic barrier 
(5x109/per day) for 
8 weeks. 
 
[Dose will be taken 
after testing on test 
days to avoid acute 
effects] 
Acute arm 
Outcome measures taken at baseline and 23 hours after administration 
of one dose of Ecologic Barrier to observe acute effects. 
Following a 4-week washout period, subjects will 
repeat this process (minus the familiarisation day) with 
the 2nd of the two interventions. Subjects will be 
counterbalanced to receive either the active or placebo 
treatment first and all treatments will be double blind. 

 
 
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[administer intervention immediately after baseline battery] 
 
 
 
 
Upon initial enquiry 
- 
A list of inclusion/exclusion criteria will be provided for participant to self-assess 
eligibility. 
 Familiarisation day 
- 
Double check that participants meet inclusion/ exclusion criteria  
- 
Take demographic information (see below) 
- 
Minimum 2 practice sessions of battery (one guided and one unsupervised) 
- 
Provide first sample kit with instructions 
T0 (baseline) 
- 
Collect faecal sample  
- 
Standardised breakfast  
- 
Cognitive/mood battery 
- 
Perceived Stress Scale  
- 
Provide 2-day food diary to complete 
T24 
- 
Standardised breakfast 
- 
Cognitive/mood battery 
- 
Supply with 4 weeks’ worth of treatment A and administration instructions 
- 
Provide FFQ to complete 
W4 
- 
Compliance check 
- 
Collect faecal sample 
- 
Standardised breakfast  
- 
Cognitive/mood battery 
- 
Perceived Stress Scale  
- 
Supply further 4 weeks’ worth of treatment A  
W8 
- 
Compliance check 
- 
Collect faecal sample 
- 
Standardised breakfast  
- 
Cognitive/mood battery 
- 
Perceived Stress Scale  

 
 
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[administer intervention immediately after baseline battery] 
 
 
 
 
T0 (baseline arm 2) 
- 
Collect faecal sample  
- 
Standardised breakfast  
- 
Cognitive/mood battery 
- 
Perceived Stress Scale  
- 
Provide 2-day food diary to complete 
T23 (arm 2) 
- 
Standardised breakfast 
- 
Cognitive/mood battery 
- 
Supply with 4 weeks’ worth of treatment B and administration instructions 
W4 (arm 2) 
- 
Compliance check 
- 
Collect faecal sample 
- 
Standardised breakfast  
- 
Cognitive/mood battery 
- 
Perceived Stress Scale  
- 
Supply further 4 weeks’ worth of treatment B 
W8 (arm 2) 
- 
Compliance check 
- 
Collect faecal sample 
- 
Standardised breakfast  
- 
Cognitive/mood battery 
- 
Perceived Stress Scale  

 
 
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Demographic information to be collected 
 
 
Age 
Gender 
Ethnicity 
Education level 
Socio Economic Status 
Height/weight/BMI 
Regular medications 
Smoking status – smoking can affect both the gut microbiome¹ and cognitive function, 
therefore useful to know.  
 
Alcohol consumption – as above², although perhaps only an effect of chronic drinking rather 
than acute or low/moderate alcohol consumption³ à measured using AUDIT 
 
Alcohol Use Disorders Identification Test (AUDIT) – short 10 item questionnaire 
developed by the WHO to assess alcohol consumption and highlight potential alcohol 
problems. https://www.drugabuse.gov/sites/default/files/files/AUDIT.pdf 
 
 
Additional measures 
 
Perceived stress – previous studies suggest that probiotic intervention is effective at protecting 
cognitive function under acute stress4,5,6. Unclear whether this would apply to general 
perceived stress as opposed to physically induced stress. Use Perceived Stress Scale (PSS) to 
measure perceived stress and potentially include as a covariate. Some studies also used the PSS 
as an outcome measure to see if a probiotic intervention itself has an influence on perceived 
stress. This outcome will be measured at baseline and all chronic timepoints. 
 
Perceived Stress Scale – well validated measure of perceived stress drawing on thoughts 
and feelings from the past month. Has also been used previously as an outcome measure to 
examine whether a probiotic intervention can influence perceived stress, with mixed 
findings7,8.https://www.northottawawellnessfoundation.org/wp-
content/uploads/2018/04/PerceivedStressScale.pdf.  
 
 
A measure of habitual diet – useful to know the habitual diet of the participants.  Also, this 
may enable us to explore the idea that individual microbiome composition will influence 
response to probiotic intervention. Those with a naturally more diverse gut microbiota (and 
therefore possibly more diverse diet) may respond better to the treatment. Or vice versa?9,10,11,27 
 
There is therefore a trade-off between wanting to remove diet as a confound when looking at 
cognitive data and allowing participants to continue eating as normal in order to maintain 
normal gut composition. Therefore, our decision is to provide the standardised breakfast at 

 
 
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each test point to avoid acute effects of breakfast variation on cognitive performance. Food 
consumption throughout the rest of the study period won’t be controlled but should be recorded 
in a 2-day food diary during the acute phase, the rationale here being that it enables 
participants to maintain their normal diet.  
 
Epic Norfolk FFQ – will be used to gain an idea of habitual diet over last year. 
 
We may also want to include some questions after administration about tolerability of the 
intervention and any potential side effects.  
 
 
 

 
 
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Example test day timelines (based on testing no more than two participants at any one time to comply with COVID-19 rules)  
§ Check inclusion/ exclusion criteria 
§ Consent 
§ Complete all demographic info  
§ Complete first assisted cognitive/mood 
battery 
§ Short break if needed 
[experimenter to check data] 
§ Second run through of battery  
§ Confirm happy to continue  
§ Send home with sample kit and 
instructions 
09:00 
 
 
 
09:45 
 
 
10:45 
 
 
11:15 
 
 
12:15 
Familiarisation day 
Using 23 hours (rather than 24) allows us to keep in line with the time at which cognitive 
baseline measures were recorded. We see significant time of day effects on cognition, so 
keeping the times consistent within participants at all test points is important to allow 
more reliable and sensitive assessment of intervention effects 
§
Administer intervention 
08:30 | 09:15  
 
 
 
09:00 | 09:45 
 
10:00 | 10:45  
 
 
  
§
Collect faecal sample 
§
Standardised breakfast 
§
Cognitive/mood battery 
Baseline  
All other test days (23 hours, 4 and 8 weeks) 
08:30 | 09:15 
 
 
09:00 | 09:45 
 
 
 
 
 
§
Cognitive/mood battery 
§
Collect faecal sample (at 4 & 8 weeks) 
§
Standardised breakfast 

 
 
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Justification for chosen outcome measures 
 
Outcome measure 
Supporting evidence 
An emotional recognition/decision task (TBC)  
Significant positive effect on both emotional 
recognition and decision tasks following 
probiotic intervention compared with placebo 4,12 
(both used Winclove multi-strain products) 
 
Findings also supported by fMRI data suggesting 
altered affective processing following 
supplementation with a fermented milk product13 
Ray Auditory Verbal Learning Task 
Some evidence to support positive effect on 
verbal memory using AVLTs in a depressed 
population14, volunteers with HIV-115 (although 
a few flaws in this study) and healthy adults16 
(although open-label).  
 
There is an overlap here of probiotics used in 
each study, including Lactobacillus plantarum, 
Bifidobacterium breve and Bifidobacterium 
longum. 
Corsi blocks 
 
 
Studies have found protective effect of 
probiotics on WM under stress4,6,8. Unclear 
whether an effect without the acute stressor?  
 
Switching task (from current battery) 
 
 
A couple of studies used the switching task from 
CANTAB on children but found no effect, while 
another reported improved performance in both 
active and placebo groups in healthy adults20, 
probably due to practice effects. One other study 
suggests a large effect size16 but is open-label 
and also likely to practice effects. So unclear 
from current literature but given potential 
improvements in attention could be an 
interesting one to include, as we know this 
switching task is, to a degree, an endurance test 
and is sensitive to nutritional interventions. 
Leiden Index of Depression Sensitivity revised 
(LEIDS-r) 
Has been used previously in studies 
supplementing with Ecologic barrier in healthy 
young adults21 and patients with MDD22, where 
chronic supplementation was associated with a 
significant reduction in reactivity to sad mood. 
Would therefore be good to see if these findings 
persist in an acute trial. 
Centre for Epidemiological Studies Depression 
Scale (CESD) 
Well validated measure of depressive symptoms. 
Positive and Negative Affect Schedule 
expanded (PANAS-x) 
As there is little evidence regarding how 
Ecologic barrier may influence general mood, 
particularly in an acute setup, the PANAS-x 

 
 
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Generally, these cognitive domains have been chosen as there is evidence from more than one 
paper using the same or similar tasks for a positive effect of probiotic supplementation on 
performance. Where possible, have also tried to use tasks that have been associated with 
improvement following Ecologic barrier or multi-strain interventions containing similar 
genera.  
 
 
 
Sample size power calculation 
 
Previous parallel RCTs looking at the effect of Ecologic Barrier on cognitive or mood 
outcomes have used between 20 and 36 subjects per group4, 21, 22, 24. There are some acute 
prebiotic studies looking at mood/cognitive outcomes – a positive effect of an intervention was 
reported using 30 participants in a cross-over RCT25 and 23 in a parallel groups RCT26.  
 
Previously, effect sizes between 0.4 and 0.6 have been reported for probiotic intervention on 
tasks of attention and executive function28. 
 
G*Power calculation based on a medium effect size of 0.5: 
 
 
t tests - Means: Difference between two dependent means (matched 
pairs) 
 
Analysis: 
A priori: Compute required sample size  
Input:  
Tail(s)                        = 
One 
 
 
 
Effect size dz                 = 
0.5 
 
 
 
α err prob                     = 
0.05 
 
 
 
Power (1-β err prob)           = 
0.8 
Output:  
Noncentrality parameter δ      = 
2.5980762 
 
 
 
Critical t                     = 
1.7056179 
 
 
 
Df                             = 
26 
 
 
 
Total sample size              = 
27 
 
 
 
Actual power                   
=
 
0.8118316 
 
Based on this information, we should aim for a sample of N=30.  
 
would allow us to cover 11 affective states (as 
opposed to the POMS which is longer and 
covers fewer affective states)  
State items from State Trait Anxiety Inventory 
Good to include an anxiety measure - chronic 
supplementation studies have reported 
reductions in anxiety23,24. Beck Anxiety 
Inventory and General Anxiety Disorder-7 
wouldn’t work for an acute study as they ask 
about feelings over the last 2 weeks/month. State 
items from the STAI therefore work better as 
they ask about how you feel in that moment.  

 
 
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