The Pleurotus oyster species is a common edible mushroom rich in phytochemicals such as proteins, phenolic compounds, dietary fibre and is also particularly rich in ergothioneine, a bioactive known for its neurocognitive benefits. Current research suggests that a mushroom-rich diet may be associated with a lower incidence of age-related neurological disorders, as well as an improvement in the domains of memory and mood.

This randomized double-blind parallel trial investigates the chronic effect (12-weeks) of the equivalent 1 portion of ergothioneine-rich Pleurotus oyster mushrooms (in freeze-dried powdered form), consumed 4 times per week, on cognition and mood, with examination of the potential inflammatory, metabolic, and neurological related mechanisms that may underlie these effects.

The Pleurotus oyster species is a common edible mushroom rich in ergothioneine, a bioactive compound with known neurocognitive benefits. The aim of the OYSCOG trial is to investigate the chronic effect (12-weeks) of the equivalent 1 portion of ergothioneine-rich Pleurotus oyster mushrooms (in dried powdered form), consumed 4 times per week, on cognition and mood, with examination of the potential inflammatory, metabolic, and neurological related mechanisms that may underlie these effects.

A power calculation based on similar research investigating the chronic benefits of other mushroom interventions on cognitive function suggests that 72 participants should give sufficient statistical power (with a=0.05, beta=0.80, cohen's d=0.60). This calculation was based on the average cohen's d value obtained from 7 RCTs examining the effect of Lion's Mane, Reishi mushroom or vitamin D enriched mushroom on global cognitive performance. To allow for a 10% attrition rate, 80 healthy older adults aged 60-80 years old will be recruited. Given the parallel design of the RCT, the cohort will be half split to receive either the control maltodextrin intervention (N=40) or the oyster mushroom intervention (N=40).

During this 12-week RCT, participants will attend the University of Reading, Psychology department on three separate occasions. Specifically, in the pre-screening phase, to check for eligibility, the participants interested in our study will be sent a link to REDCap containing online versions of a Health and Lifestyle Questionnaire containing basic demographic questions and the Epic Norfolk Food Frequency Questionnaire (FFQ), to assess their habitual dietary intake. Then, participants, will be contacted to attend a 3-hour familiarisation session at our department, during which the participant's weight, height and blood pressure will be checked, along with a finger-prick, to ensure that the participants are not anaemic. Furthermore, participants will complete the Raven's Progressive Matrices (RPM) measure of fluid intelligence and will perform each of the cognitive battery tasks twice to control for practice effects in the run up to the test session days.

One week after the familiarisation visit, each participant will attend for the baseline test visit, where they will be asked to complete the battery of cognitive and mood tasks and to have their blood pressure and body weight measured. A few participants (n=40, n=20 from each treatment arm) will also be invited to wear an EEG cap using the Brain Products EEG system with 16 channel active electrodes in order to record EEG measurements while performing a simple computerised cognitive task (N-back task) and while resting with eyes open and eyes closed. EEG spectral activity and event-related potential (ERP) data relating to P300 and N200 peaks will be analysed. Finally, a blood test will be performed at the end of the visit. Following the blood draw, the serum will be separated via centrifuge and stored at -80°C until analysis is complete. Blood serum will be analysed for anti-inflammatory and metabolic ability by measuring the levels of fasting lipids (e.g. total cholesterol) and the markers of inflammation (eg cytokines, interleukins), as well as the levels of the brain derived neurotrophic factor (BDNF), a signalling protein known to be related to neuronal signalling and memory function.

At the end of the baseline visit, participants will receive a 12-week supply of sachets containing either maltodextrin or oyster mushroom, depending on their treatment group allocation. They will be asked to consume one sachet 4 times per week for 12-weeks, to be combined with their usual meals. On the 84th day (week 12), participants will be asked to attend the laboratory for their post-visit test day where they will complete the same test procedures as during the baseline visit. Also, at the end of the post-visit day, participants will be asked to complete two dietary surveys: the Epic Norfolk Food Frequency Questionnaire (FFQ), and a brief dietary questionnaire consisting of specialised questions relating to their habitual mushroom intake.

All test visits will take place in the morning, starting between 9am and 10am and finishing before lunchtime. Participants will be asked to follow a low flavonoid diet for 24 hours in advance of each testing visit. Also, they will be asked to consume a standardised breakfast of lightly buttered toast before attending each test visit.

In terms of the mood and cognitive tests, there will be two cognitive test sessions taking place at the familiarisation visit, one at the baseline visit, and then one at the 12-week post-intervention visit, to assess participants' episodic and working memory, psychomotor and executive function and mood domains. Different versions of the cognitive battery will be used at each presentation to minimise learning, and all versions will be matched for difficulty. The mood and cognitive tests will be completed on a computer and should take no longer than 50-60 minutes to complete. The battery will contain the following tasks:

• Positive and Negative Affect Schedule (PANAS-X)- In this mood task, participants are asked to rate their emotion on a 5-point Likert scale, in response to negative emotions (e.g., guilt, fear), positive emotions (e.g., attentiveness, joy) and other affective states (e.g., surprise, shyness).
• Depression, Anxiety and Stress Scale - 21 items (DASS-21)- This 21-item measure assesses symptoms of depression, anxiety and stress. Each item is scored from 0-3 where higher scores indicate higher levels of distress in anxiety (scores range from 0 to >20), depression (0 to >28) and/or stress (0 to >34).
• Rey Auditory Verbal Learning Task (RAVLT)- This episodic memory task consists of 5 consecutive free recalls of the same 15 nouns presented as an auditory list (list A), followed by recall of a further 15 nouns presented as an interference list (list B) which is recalled only once. List A is subsequently recalled straight after list B and then again after a longer delay of around 30 minutes.
• Task Switching Task (TST) - In this executive function task, participants view 8 spaced radii of a circle above and below a bold line and a stimulus digit selected from 1-9 (except 5) appears clockwise in each segment, either above or below the bold line. Depending on the stimulus position in the segments, participants perform different tasks with each being switched every 4 trials.
• Corsi block tapping task (CBTT)- In this spatial working memory task, participants are shown 9 arranged blocks positioned on a computer screen and are asked to reproduce a given sequence by clicking on the blocks (using the mouse) in the same sequence as they see them.
• Simple and complex finger tapping task (SFT & CFT)- In this psychomotor function task, participants tap on a key as quickly as possible with the index finger of their dominant hand for 1 minute. They then tap out a specific sequence using 4 fingers, again for 1 minute.
• RAVLT word recognition- In this delayed memory task, participants are shown a sequential list of 50 nouns containing the words from lists A and B from the previously described RAVLT task plus 20 additional words not previously heard, and are asked to indicate those from list A.
• N-back (0-Back & 1-Back versions)- In this working memory and attention task, participants are shown a series of stimuli and they have to respond whether the stimuli match or not the target presented at the beginning of the task. For a subset of participants (n=40), this task will be performed while also taking EEG measurements (using Brain Products software and 16 active electrodes), to examine changes in brain activity in response to the target and non-target trials.
• Subjective mental fatigue- At the end of the cognitive battery participants are asked to rate their mental fatigue level on a 9-point Likert scale.

Inclusion Criteria:

• Aged between 60-80 years old
• Have normal vision and hearing
• Have a Body mass index (BMI<=30)
• Have healthy status

Exclusion Criteria:

• Smokers
• Vegans/vegetarians
• Being diagnosed with a psychiatric/neurological condition (eg., stroke, schizophrenia, depression, cognitive impairement, dementia)
• Being diagnosed with a learning/behavioural disorder (e.g. dyslexia, autism, ADHD)
• Being diagnosed with a metabolic disease (eg., type I/II diabetes and cardiovascular disease), or suffer from unmediated hypertension or thrombosis related disorders, or suffer from cancer, kidney or liver disease
• Being anaemic
• Taking disease medication such as anticoagulants, antiplatelet medication, dementia medication, antidepressants, antiepileptic medication, thyroid medication
• Refusing to stop taking vitamin supplements (including prebiotics/probiotics) during the 12-week testing period
• Have food allergies
• Having a difficulty in completing computer-based cognitive tasks

All personally related collected information will be kept in locked filing cabinets in the Nutritional Unit of the School of Psychology. The digital health questionnaires used for the purpose of pre-screening participants as well as any digital research data will be stored in the Research Electronic Data Capture (REDCap) database, stored on the University secure network cloud.

All relevant study outcomes described in the Ethics Review Application Form (including the demographic and anthropometric data (such as age, sex, BMI, blood pressure) as well as the performance scores of the battery tests assessing cognitive function and mood and the results of the blood tests measuring immunological and metabolic function) will be preserved and made available in anonymised form.

Double-blind