The 18-week study consists of a 12-week intervention period and a 6-week follow-up period. Chinese older adults between 60 and 90 years of age and known to have Mild Cognitive Impairment and agree to participate in this study will be screened for eligibility and then randomized into 2 arms. Subjects will be recruited from current ongoing studies from NUS under NUS-IRB registration and the study posters at the National University Hospital clinic. Those participants who consented to be contacted by the study team to participate in further research activities and with a diagnosis of Mild Cognitive Impairment will be first contacted by research team members from that study. All of them will be briefly informed about this intervention study and asked if they agree to be contacted for more information. Written informed consent will be taken for the intervention groups. Subject screening and enrolment will be documented by the study team using a screening log. Drop-out and screen failures will be documented.

The probiotic and placebo drink bottles will be coded by the manufacturing company and delivered with a sealed envelope containing the code. Subjects who meet the study eligibility criteria will be randomly assigned using online study randomization software to one of the coded groups corresponding to the probiotic or placebo study groups. Both Study team members and subjects will be blinded to the intervention. The study product codes/blinding will be broken either: (1) at the end of the study for analysis, or (2) in the event of a serious adverse event as deemed necessary by the study team.

Subjects should drink the study product (active interventional probiotic or placebo depending on randomization) 1 bottle per day at breakfast for 12 weeks. Subjects will be given the study product at 2 weekly intervals till the study ends. Subjects who have successfully completed the product consumption period will enter the follow-up period, during which they will continue their normal diet with the exception of fermented dairy products (such as yogurt, Lactobacillus beverages and probiotic supplements). The safety and tolerability parameter will be reports of any adverse events related to the study intervention between the start at Week 0 till the end of the study at Week 18. These will include reports of mild events such as any discomforts (vomiting, bloated sensation, changes in defecation frequency, etc.). Compliance based on the study product consumed and intake of other dairy-fermented milk will be performed at each scheduled visit for all subjects. The study team will review the subject diaries together with the subjects in order to check compliance. Subjects will also be asked to bring empty bottles every 2 weeks when they come to collect another 2 weeks of the intervention product. Compliance will be reinforced at each study visit.

The primary parameter is the change in gut microbiota composition from before to after 12 weeks of probiotic consumption and after stopping probiotic use for 6 weeks (at Week 0, 6, 12 and 18 of the study period). The secondary study parameters will be the changes in cognition at 0, 12 and 18 weeks of the study period, and immune and inflammatory blood markers, and cortisol levels and changes in fecal short-chain fatty acid levels at 0, 6, 12 and 18 of the study period. Basic demographic data, medical history, medications and consumption of diets will be assessed before probiotic consumption.

The fecal sample collection by a subject will be in special containers at home. Two tubes will be required at each time point. The sampling kit will be provided by the study team. Instructions for fecal sample collection will be provided to the subjects by the study team on Visit 1 (screening) for the subsequent visits. Individual samples will be weighed and recorded. The fecal DNA extraction and 16s rRNA sequencing will proceed with the fecal samples from the OMNIgene GUT tube. The analysis will be performed by the NovaSeq 16srRNA amplicon sequencing method. Fecal SCFAs analysis will be performed by Gas chromatography-triple quadrupole mass spectrometer with the fecal samples from OMNI-MET tube.

Two tubes of blood will be collected from each subject at each time point: One tube with 3ml of blood for serum cortisol estimation, and 3mls of blood for the whole blood immune activation assays and for circulating inflammatory marker analysis. Fasting blood is required for cortisol estimation and so blood collection will be scheduled between 9:00 and 11:00 a.m. to minimize diurnal variation in cortisol. The serum cortisol level will be tested by the Electrochemiluminescence immunoassay. The whole blood samples will be centrifuged at 1650 × g for 25 min at room temperature to obtain plasma. The plasma samples will then be stored in aliquots at -80° C until further analyses. The whole blood will be stimulated by Toll-like receptors TLR2 and TLR4 agonists for 48 hours. The supernatant will be collected and aliquot and stored at -80°C. After sample collections from all the time points are completed, all samples for the same participants from different time points will be measured on the same day and on the same plates, to avoid batch effects. The cytokines analyzed will be IL-1β, IL-6, IL-10, IL-8 and TNF- alpha using commercially available enzyme-linked immunosorbent assay (ELISA) kits. All the experiments will be performed as per the instructions of the respective manufacturers of the kits. The stored plasma will be analyzed for 45 biomarkers (cytokines and chemokines) using the Proximity Extension Assay.

This neurocognitive battery of tests has been utilized and validated in the Singaporean population. The tests evaluate various cognitive functions, including attention, learning, memory, speed, and executive function. The tests included Rey Auditory Verbal Learning Test (RAVLT), Digit Span task, Color trails tests, Blcok design and Semantic Verbal fluency test, and Stroop test.

Data collected from demographics, questionnaires, Neuropsychological tests and collected samples will be summarized using descriptive techniques. Summary statistics (mean, standard deviation) will be presented for continuous variables (and their changes from baseline and absolute values at each time point). Counts and percentages will be presented for categorical variables. Where appropriate, the presentation of results will include plot and confidence intervals. Multivariate data analyses of cognitive and biomarker outcomes will be performed using SPSS and R.