Study design and ethics

A triple-blind, randomized crossover trial with longitudinal follow-up was conducted to assess the association between cognitive performance in older adults and a synbiotic supplement. Participants, psychological assessors, and the statistical analyst were unaware of group assignments at all stages. The study was conducted at Universidad Anáhuac, México City, with recruitment beginning in mid-December 2022 and final baseline evaluations completed in mid-February 2023. Following completion of the baseline assessments, participants entered the intervention phase according to the study protocol. All procedures adhered to the Declaration of Helsinki and the Mexican standard NOM-012-SSA3-2012 for human research. Ethical approval was granted by the Universidad Anáhuac México, Campus Norte, Research Committee, Faculty of Health Sciences (Approval ID: CONBIOETICA-15-CEI-004-20160729). The trial was registered at ClinicalTrials.gov (NCT05688618).

Participants

Sample size

Nineteen participants completed all phases. Given the exploratory nature of the trial and the feasibility of close follow-up in a triple-blind crossover design, this sample size was deemed adequate to provide preliminary evidence of effect, allowing detection of an effect size of 0.1 and enabling both within-group and between-group comparisons across outcomes.

Recruitment and randomization

Participants were recruited via snowball sampling supported by community outreach, posters, and social media. Interested individuals telephoned the study team for brief screening, underwent baseline cognitive assessment, and were then randomized by simple lot to the control or experimental group. No age- or sex-stratification was performed.

Before recruiting patients, twenty numbers (from 1 to 20) were placed in a tombola. Afterwards, each number was randomly selected from the tombola and sequentially allocated to one of three groups, until reaching 10 numbers per group. Each number represented the time the patient was to be enrolled. This way, once the recruitment was initiated, the first patient was assigned number one; the second was number two, and so on. In this fashion, each patient was allocated (according to the time of enrollment) to the group to which their number corresponded. At the end of the study, one of the patients was eliminated because he did not follow the instructions for preparing the synbiotic.

Intervention

The synbiotic consisted of E. faecium (SF68; 1 × 10⁷ CFU) and agave inulin (8 g) provided as a tasteless, odorless, colorless powder dissolved in a powdered almond drink. The control (vehicle) was the almond drink without the synbiotic. Participants consumed one dose daily on an empty stomach for 8 weeks, followed by a 3-week washout. After washout, groups crossed over: the original control group received the synbiotic and the original experimental group received the vehicle for another 8 weeks, after which cognitive assessments were repeated (see Figure 1). Adherence was monitored by daily photographs submitted via WhatsApp and logged electronically; the same channel enabled real-time adverse-event reporting.

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Figure 1. Study intervention flow diagram. n: number; S: synbiotic; C: control.

E. faecium SF68 is a strain that has decades of clinical use as a probiotic. It does not belong to the hospital-associated clade (A1) responsible for vancomycin-resistant enterococci (VRE). It lacks major acquired antibiotic resistance genes characteristic of pathogenic hospital strains; does not carry typical virulence genes, and has an extensive history of tolerability in children, adults, and elderly patients, including vulnerable populations in controlled trials [32–35].

Supplement and placebo packaging

Both synbiotic and placebo powders were prepared and packaged under strict aseptic conditions in a laminar-flow hood. All materials were disinfected with 70% ethanol and exposed to ultraviolet light for 30 minutes. Each daily dose contained 25 g of unsweetened almond powder; the experimental sachets included 8 g of synbiotic plus 17 g of almond powder. Packages were visually identical and labeled only with the study logo. To maintain blinding, each daily sachet was supplemented with 20 g of unsweetened almond beverage powder, matching the vehicle used in the placebo. In contrast, the placebo consisted solely of 25 g of commercially sourced unsweetened almond drink powder without any probiotic or prebiotic components. Both the synbiotic and placebo were repackaged in identical plastic sachets under laminar-flow sterile conditions to standardize appearance, texture, and handling across groups, preserving the triple-blind design of the study.

The supporting literature assures that restoration of an altered intestinal microbiota may require up to 40 days, and therefore the intervention was extended to 8 weeks to ensure adequate exposure beyond the natural restitution period and to allow stabilization of the synbiotic’s biological effects. Additionally, because probiotic activity diminishes when dosing occurs less frequently than every three days, daily administration was required to maintain effective concentrations of 10⁷–10⁸ CFU, preventing natural washout of the probiotic strain and ensuring consistent colonization potential throughout the trial. As a result, all participants entered the study with their habitual microbiota composition and underwent a supplementation period long enough to surpass the natural microbial turnover cycle, thereby maximizing the likelihood of detecting clinically meaningful cognitive changes attributable to the synbiotic. The synbiotic consisted of E. faecium [1 × 10⁷ CFU; Alimentos Esenciales para la Humanidad (AEH) S.A. de C.V., Ciudad de México, México] and agave inulin (8 g; AEH S.A. de C.V., Ciudad de México, México) provided as a tasteless, odorless, colorless powder dissolved in a powdered almond drink (Almondlessa, Afenlessa Corporation S.A. de C.V., Ciudad de México, México).

Cognitive assessment measures

Cognitive function was evaluated at baseline and post-intervention using three instruments:

• Modified Mini-Mental State Examination (MMSE): A 19-item screening test for cognitive impairment in older adults; scores ≤ 13 suggest impairment [36]. Clinical interpretation of the total score is as follows: scores of 17–19 points are considered within the normal range; scores of 14–16 points are consistent with MCI; and scores ≤ 13 points are suggestive of dementia.

• Rey-Osterrieth Complex Figure Test (RCFT): Assesses visuoconstructive ability and visual memory through copy and delayed recall; 18 figure elements are scored and converted to percentile ranks [37–39].

• Abbreviated Instrument for Expectations of Self-Efficacy for Daily Activities in Older Adults (AERAC): A 26-item measure of self-efficacy in independence, social interaction, and health-protection domains; global scores are classified as low (0–23), moderate (24–56), or high (57–100) [18].

Statistical analysis

Analyses were performed using GraphPad Prism version 10.5.0 (GraphPad Software, USA). Descriptive statistics are presented as mean ± standard deviation (SD). Normality was assessed using the Shapiro-Wilk test.

For the first intervention period, between-group comparisons were conducted using unpaired Student’s t-tests, and within-group pre-post comparisons were analyzed using paired Student’s t-tests.

Given the crossover design, post-crossover data were analyzed by comparing within-subject changes after each treatment condition (synbiotic vs. control). Paired Student’s t-tests were used to evaluate treatment effects within individuals across periods. Additionally, between-group comparisons during the crossover phase were performed using unpaired t-tests. To minimize potential carryover effects, a three-week washout period was implemented between intervention phases. Although formal testing for period and carryover effects was limited by the sample size, the crossover analysis primarily focused on within-subject comparisons to strengthen inference regarding treatment effects. All tests were two-sided, and a p value < 0.05 was considered statistically significant.

Participant demographics

The studied population consisted of 19 adults aged 64 to 85 years (mean ± SD: 77 ± 6.04 years), including 13 females and 6 males. Baseline psychometric assessments were conducted between December 2022 and February 2023 using the three validated instruments: the modified MMSE, the RCFT (memory and copy components), and the AERAC. After completion of the baseline assessments, participants proceeded to the intervention phase according to the study protocol. Table 1 summarizes the demographic characteristics of the participants and the baseline results of each test by group.

Baseline psychometric assessments

Before supplementation, all participants completed the three psychometric tests. Each test was scored individually, and results are presented as mean ± SD for both groups (Figures 2, 3, 4, 5). Independent t-tests were used for between-group comparisons, and paired t-tests for within-group analyses.

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Figure 2. Mini-Mental State Examination (MMSE) scores before, after intervention, and after crossover. This figure illustrates MMSE performance in the synbiotic (S) and control (C) groups across the three assessment phases. During crossover, the former control group showed significant improvement after receiving the synbiotic, while the initially treated group maintained cognitive gains. p values: **p < 0.01; ***p < 0.001. Tx: treatment.

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Figure 3. Rey-Osterrieth Complex Figure Test (RCFT)-copy test scores before, after intervention, and after crossover. Bar graph illustrating visuoconstructive performance across baseline, post-treatment, and crossover phases. Participants who switched to synbiotic supplementation during crossover exhibited significant improvement relative to their prior performance. p values: *p < 0.05; **p < 0.01. C: control; S: synbiotic; Tx: treatment.

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Figure 4. Rey-Osterrieth Complex Figure Test (RCFT)-memory test scores before, after intervention, and after crossover. Bar graph showing visual memory performance assessed through the RCFT-memory test in the synbiotic (S) and control (C) groups across baseline, post-treatment, and crossover phases. During the crossover phase, participants who switched from control to synbiotic demonstrated a marked and statistically significant improvement relative to their post-treatment values, achieving scores comparable to the group originally treated with the synbiotic. These results indicate that synbiotic supplementation consistently enhances visual memory recall, regardless of treatment order. p values: **p < 0.01; ***p < 0.001. Tx: treatment.

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Figure 5. Abbreviated Instrument for Expectations of Self-Efficacy for Daily Activities in Older Adults (AERAC) scores before, after intervention, and after crossover. This figure shows AERAC scores, which reflect perceived self-efficacy in daily functioning. During the crossover, both groups demonstrated further functional gains. The former control (C) group improved significantly relative to its own post-treatment performance, while the initially synbiotic (S)-treated group maintained high perceived functional capacity. p values: **p < 0.01; ***p < 0.001. Tx: treatment.

At baseline, cognitive and functional performance were comparable between the synbiotic and control groups across all administered assessments. MMSE scores did not differ significantly between groups (16.78 ± 0.97 vs. 16.10 ± 2.08, p = 0.1923), indicating similar global cognitive status at study entry. Likewise, visuospatial memory measured by the RCFT-memory test showed no significant group differences (11.06 ± 3.45 vs. 10.90 ± 2.75, p = 0.9142), and visuoconstructive ability assessed through the RCFT-copy test was nearly identical (24.74 ± 7.22 vs. 24.70 ± 6.71, p = 0.9891). Baseline functional self-efficacy, evaluated with the AERAC scale, also showed no significant differences between the synbiotic and control groups (68.52 ± 5.06 vs. 67.69 ± 6.62, p = 0.4626). These results confirm that both groups began the intervention under equivalent cognitive and functional conditions.

Post-treatment psychometric assessments

After eight weeks of supplementation—during which the control group received almond powder alone and the synbiotic group received the same formulation with E. faecium and agave inulin, the same tests were administered to evaluate cognitive changes.

Modified MMSE

At baseline, global cognitive performance measured by the MMSE was comparable between the synbiotic and control groups (16.78 ± 0.97 vs. 16.10 ± 2.08, respectively; p = 0.1923), indicating similar initial cognitive status.

After eight weeks, participants receiving the synbiotic demonstrated a significant within-group improvement, increasing their MMSE score to 18.11 ± 1.05 (p = 0.0066 vs. baseline). In contrast, the control group showed lower post-treatment scores (14.90 ± 1.59). The between-group comparison at this time point strongly favored the synbiotic (p < 0.001) (Figure 2).

During the crossover phase, participants who initially received the control formulation exhibited a marked cognitive improvement after switching to the synbiotic, reaching 18.20 ± 0.63 (p < 0.001 vs. their own post-treatment values). Meanwhile, participants originally treated with the synbiotic maintained stable cognitive performance during crossover (17.89 ± 1.45). These findings indicate that synbiotic supplementation exerted a reproducible and sustained positive effect on global cognition.

RCFT-copy

At baseline, visuoconstructive performance assessed through the RCFT-copy task was comparable between groups (24.74 ± 7.22 vs. 24.70 ± 6.71; p = 0.9891).

After eight weeks, the synbiotic group showed significant within-group improvement (30.14 ± 4.22; p = 0.0354 vs. baseline), whereas the control group exhibited lower post-treatment scores (22.00 ± 8.52). The between-group comparison at this time point favored the synbiotic (p = 0.0095). During crossover, participants who switched from control to synbiotic demonstrated further improvement (p = 0.0157 vs. their post-treatment values), achieving scores comparable to the initially treated group (29.52 ± 5.60 vs. 29.83 ± 4.18). These results indicate a robust and reproducible enhancement in visuoconstructive ability following synbiotic supplementation (Figure 3).

RCFT-memory

Baseline visual memory performance was similar between groups (11.06 ± 3.45 vs. 10.90 ± 2.75; p = 0.9142). Following intervention, the synbiotic group demonstrated significant within-group improvement (17.56 ± 5.96; p = 0.0060 vs. baseline), whereas the control group remained at lower post-treatment values (9.35 ± 3.70). The between-group comparison strongly favored the synbiotic (p = 0.001). During crossover, individuals who switched from control to synbiotic showed marked improvement (p = 0.0005 vs. post-treatment), reaching scores comparable to the initially treated group (17.20 ± 3.29 vs. 17.56 ± 6.73). These findings demonstrate consistent and reproducible benefits of synbiotic supplementation on visual memory (Figure 4).

AERAC

Baseline AERAC scores were similar between groups (68.52 ± 5.06 vs. 67.69 ± 6.62; p = 0.4626). After eight weeks, the synbiotic group demonstrated a significant within-group improvement (85.70 ± 11.63; p < 0.001 vs. baseline). The control group showed lower post-treatment scores (71.48 ± 5.66), and the between-group comparison favored the synbiotic (p = 0.0015).

Following crossover, participants who switched to synbiotic supplementation showed further improvement (85.46 ± 8.28; p < 0.001 vs. post-treatment), while the initially treated group maintained high functional performance (82.60 ± 10.49). These results confirm sustained and reproducible improvements in perceived functional self-efficacy (Figure 5).

Conclusion

The results of this study suggest that supplementation with the synbiotic formulation containing E. faecium and agave inulin exerts beneficial effects on cognitive function in older adults with MCI. This finding highlights the potential of synbiotic as a promising intervention for the prevention and management of age-related cognitive decline. Future studies with larger sample sizes and longer follow-up periods are recommended to confirm and extend these findings.