Administration of bifidobacteria and dietary fiber improves cognitive function by increasing short-chain fatty acid-producing bacteria and reducing inflammation

Abstract

Bifidobacterium animalis subsp. lactis GCL2505 (GCL2505), commercially known as the “BifiX” strain in Japan, reaches the intestine alive, proliferates after a single intake, and is associated with several positive health effects. A randomized, double-blind, placebo-controlled, parallel-group clinical trial of this probiotic strain in combination with inulin (a prebiotic) reported an improvement of cognitive function in the elderly. In the present study, a follow-up analysis was performed to elucidate the underlying mechanism, using a multi-omics approach that integrated a high-throughput assay of blood inflammatory markers and metagenomic analysis of the fecal bacterial composition. After probiotic and prebiotic administration, short-chain fatty acid producers such as Faecalibacterium and Bifidobacterium were increased in the gut. Moreover, in the subgroup with greater improvement in cognitive function scores, the levels of inflammatory markers were decreased. Subgroup analysis revealed that the improvement of cognitive function was associated with a reduction of inflammation and an increase of Faecalibacterium. These results suggest that GCL2505 and inulin can improve cognitive function by alleviating inflammation via an increase of short-chain fatty acid-producing bacteria, which appears to elevate levels of short-chain fatty acids, particularly acetate and butyrate, in the gut. The present results contribute to a deeper comprehension of the gut-brain axis and propose new avenues for potential therapeutic intervention in cognitive disorders.

INTRODUCTION

Alzheimer’s disease (AD) is the most common form of dementia and is characterized by memory impairment that significantly impairs daily life. AD is a major public health problem, and its incidence and prevalence are predicted to reach epidemic proportions in the coming decades unless effective interventions aimed at preventing or mitigating disease progression are developed [1]. There remains a desperate need for treatment options for AD that can halt its progression and ameliorate symptoms.

A recent study suggested that neuroinflammation is closely associated with the etiology of AD [2], which is the most common form of dementia affecting the elderly. Neuroinflammation refers to an inflammatory response in the central nervous system related to neuronal damage [3]; cytokines play a particularly central role in neuroinflammation in AD [4]. Inflammatory cytokines produced in the brain and peripheral nervous system can cause pathological cell death [5], and the levels of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) are abnormally elevated in AD patients [6]. The Northern Manhattan Study reported that higher levels of interleukin 6 (IL6) are associated with an overall measure of cognitive function (total score on the Mini-Mental State Examination) and cognitive decline [7]. In other words, alleviating inflammation may help to improve cognitive function in patients with AD as well as metabolic diseases.

Previous studies have focused on the gut microbiota as one of the many factors influencing inflammation [1, 8]. Probiotics are recognized as having a high potential to alleviate inflammation, owing to the anti-inflammatory effects of short-chain fatty acids (SCFAs) produced in the gut by them [9]. More recently, animal studies have reported that butyrate improves neuroinflammation by reducing the secretion of inflammatory cytokines by microglia [10] and that acetate reduces cyclooxygenase-2 and interleukin 1β levels in AD models by inhibiting the extracellular signal-regulated kinase/c-jun-NH₂-terminal kinase/nuclear factor-κB (NF-κB) pathway [11].

Bifidobacterium animalis subsp. lactis GCL2505, commercially named the “BifiX” strain in Japan, is a probiotic bacterium originally isolated from the feces of healthy adults [12, 13]. SCFAs produced by GCL2505 in the gut exert anti-metabolic syndrome effects such as improved glucose tolerance and suppression of visceral fat accumulation [14]. Animal studies have demonstrated the effects of GCL2505 on host metabolic homeostasis, such as improved glucose tolerance and reduced adiposity, that are dependent on free fatty acid receptor 2, an acetate receptor previously known as G protein-coupled receptor 43 [15].

While the beneficial effects of GCL2505 alone, such as improvement of the intestinal environment [12, 13] and visceral fat reduction [16], were demonstrated by clinical trials, an approach that combines probiotics and prebiotics was investigated to achieve even greater benefits. Inulin, a soluble fructan-type dietary fiber [17] that has been shown to increase intestinal bifidobacteria [18, 19] and SCFAs [20], was selected for this purpose. The effects of the combination of GCL2505 and inulin were also investigated by clinical trials, and it has been reported that a synbiotic containing GCL2505 and inulin has a greater effect on the number of bifidobacteria [21] and is more effective at suppressing the accumulation of visceral fat than probiotics alone [22]. Furthermore, clinical studies have demonstrated that the combination increases resting energy expenditure [23] and improves vascular endothelial function [24]. These effects are thought to be due to the increased levels of SCFAs in the gut caused by the administration of GCL2505 and inulin [25]. We recently conducted a clinical trial using the Cognitrax test to evaluate the effect of GCL2505 and inulin on cognitive function [26]. Cognitrax is a cognitive function testing service designed for the Japanese population, based on cognitive function testing technology developed by CNS Vital Signs [27]. The Cognitrax test can be used to quickly test a wide range of cognitive functions (e.g., memory, attention, processing speed, executive function) online using a computer. In elderly subjects with mild cognitive impairment, 12 weeks of administration of GCL2505 and inulin significantly improved scores in the Neurocognitive Index domain, an assessment of overall cognitive function, in addition to the Complex Attention, Cognitive Flexibility, and Executive Function domains, and significantly increased the number of bifidobacteria in fecal material [26].

Although there have been several clinical interventions using probiotics that have shown beneficial effects on cognitive function associated with changes in systemic cytokine levels such as TNF-α and IL6 [28], to the best of our knowledge, no studies in humans have demonstrated that the cognitive-improving effects of probiotics and/or prebiotics are mediated through inflammation-reducing mechanisms. Therefore, in the present study, we performed a post-study follow-up analysis to determine whether there is a correlation between improved cognitive function and inflammatory status in subjects who consumed GCL2505 and inulin daily for 12 weeks, by using a multi-omics approach that integrated the proteomics of inflammatory markers in serum and shotgun sequencing of metagenomes from fecal samples. For this analysis, we performed a proteomic analysis of inflammatory markers in serum as well as shotgun sequencing of metagenomes from fecal samples. This study used a multi-omics approach that integrated proteomics and metagenomics to elucidate the interrelationships among the gut microbiota, inflammatory state, and cognitive function in humans for the first time, and it is expected to expand future treatment options for patients with AD.

MATERIALS AND METHODS

Study design

The details of the clinical trial design have been described previously [26]. Briefly, in this randomized, double-blind, placebo-controlled trial, older adults (40 men and women each) without AD, some of whom had mild cognitive impairment (MCI), were assigned to receive either a test drink that contained a probiotic (GCL2505, 1.0 × 10¹⁰ colony-forming units) and soluble fiber (inulin, 2.0 g; active group) or a placebo drink that did not contain either the probiotic or soluble fiber (placebo group) for 12 weeks. By the end of the study, one participant from the active group withdrew for personal reasons. After the completion of the entire study, one participant from the placebo group was dropped due to an extremely irregular lifestyle. One participant from the active group was dropped due to a confirmed illness unrelated to the study that may have affected the results. In addition, nine participants were also excluded because they were found to have consumed drugs or foods during the study period that might have affected the results (5 in the active group and 4 in the placebo group). Another participant in the active group was dropped due to partial missing primary-endpoint data. Moreover, the Cognitrax scores of four participants who scored below the cut-off for assigning a ‘screening diagnosis’ of depression were excluded from the analysis. For these reasons, 17 subjects were excluded during or after the study, so a total of 63 subjects (31 in the active group and 32 in the placebo group) were included in the analysis. The active group consisted of 16 men and 15 women, and the placebo group consisted of 15 men and 17 women. The primary endpoint was the Cognitrax test, the Japanese version of CNS Vital Signs. Cognitrax is a computer-based online cognitive assessment that comprehensively evaluates multiple domains, including memory, attention, processing speed, and executive function. Changes in fecal bifidobacterial quantification and inflammatory marker levels following the intervention were examined. Inflammatory markers were measured using Olink^® Target 96 Inflammation Panels (Olink Proteomics AB, Uppsala, Sweden). Expression levels of 92 inflammatory markers were examined, and 75 inflammatory markers that were deemed quantifiable were used in the analysis. The study was conducted at Nihonbashi Cardiology Clinic (Tokyo, Japan) from September to December 2022 by K.S.O. Corporation (Tokyo, Japan), a contract research organization, and was registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR; http://www.umin.ac.jp/ctr/index.htm) as UMIN000048386.

Gut microbiota analysis

For the present follow-up analysis, the intestinal microbiota of the subjects was examined by shotgun metagenomic analysis (i.e., constructing a shotgun library and performing sequencing; performing quality control of metagenomic reads; constructing a non-redundant gene set and functional annotations; and profiling the taxonomy of the samples) using fecal samples as previously described [24]. The present follow-up analysis was registered with the University Hospital Medical Information Network Clinical Trials Registry (http://www.umin.ac.jp/ctr/index.htm) as UMIN000052940.

Statistical analysis

All measurements are presented as the mean and standard deviation. All statistical analyses were performed using the open-source software program R (version 4.2.1) and Python 3.7.12. Missing data were treated as missing values, and no surrogate values were used. Shotgun metagenomic sequencing enables comprehensive profiling of the gut microbiota. In such analyses, microbial composition is commonly expressed as relative abundance, calculated by dividing the number of sequencing reads assigned to each microbial taxon by the total number of reads obtained. This metric reflects the proportional representation of each taxon within the sampled community. As these values do not represent absolute quantities, they are subject to compositional constraints that limit direct quantitative comparisons. To enhance both the interpretability and statistical robustness of the results, the data were subjected to normalization and transformation prior to analysis. Differential abundance analysis was conducted using the Linear Model for Differential Abundance (LinDA), a method that has been widely adopted in microbiome research and is recognized for its reliability [29]. Differences between two groups in terms of the relative abundance of the fecal microbiota measured using shotgun metagenomics were assessed using LinDA method, while the paired t-test was used to evaluate the changes in relative abundances in each of the high-response and low-response subgroups during the intervention period. Statistical significance was set to a p-value <0.05 with a false discovery rate (FDR) <0.1. P-values when the LinDA method was used were adjusted using the Benjamini and Hochberg [30] procedure to control the FDR based on relative abundance values, and they are presented as P_FDR. The unpaired t-test was used to evaluate inter-group differences in cognitive function scores and changes in inflammatory marker expression during the study period, with statistical significance set to a p-value <0.05.

Ethics statement

The studies involving humans were approved by the Research Ethics Committee of the Ezaki Glico Group (approval date: September 28, 2023; approval number: 2023-26). The studies were conducted in accordance with local legislation and institutional requirements. All participants provided written informed consent for their involvement in this study.

RESULTS

Effect of administration of GCL2505 and inulin on the gut microbiota

Alpha- (Shannon) and beta-diversity analyses were performed to compare the fecal microbiota in each group. Interestingly, no differences were observed between or within the two groups at week 0 and 12 (Fig. 1A, 1B). These results indicate that 12 weeks of administration of GCL2505 and inulin did not significantly affect the overall community structure of the gut microbiota.

Fig. 1.

Effects of GCL2505 and inulin intake on the fecal microbiota. Boxplots represent the interquartile range (25–75%), with the median shown in black. (A) Alpha-diversity (Shannon). (B) Beta-diversity (principal component analysis of the genus-level Bray–Curtis distance).

Analysis of changes in the gut microbiota due to the intervention

The relative abundances of the gut microbiota of each subject at weeks 0 and 12 are shown in Fig. 2A, while the dominant genera for the placebo group and the active group are shown in Fig. 2B and 2C, respectively.

Fig. 2.

Changes in gut microbiome composition following the 12-week intervention. Boxplots represent the interquartile range (25–75%), with the median shown in black. (A) Genus-level relative abundances of the fecal microbiota composition in the placebo and active groups. Comparison of relative abundances between weeks 0 and 12 for the placebo (B) and active (C) groups. The top 10 relative abundances are shown graphically. *P_FDR-value <0.10, intra-group difference (week 0 vs. week 12) in (B) and (C). Data were analyzed by Linear Model Differential Abundance.

The results showed that the relative abundances of the SCFA-producing genera Bifidobacterium (P_FDR=0.059), Faecalibacterium (P_FDR=0.073), and Bacteroides (P_FDR=0.059) were significantly increased at week 12 compared with week 0 in the active group, while the relative abundance of the genus Collinsella (P_FDR=0.059) was significantly decreased. In contrast, there was no significant increase in these SCFA-producing bacteria in the placebo group, but the relative abundances of the genera Blautia (P_FDR=0.070) and Collinsella (P_FDR=0.087) were significantly decreased at week 12 compared with week 0. These results showed that administration of GCL2505 and inulin promoted the growth of SCFA-producing bacteria, such as Bifidobacterium, Faecalibacterium, and Bacteroides, in the gut.

Subgroup analysis by cognitive function score

For each of the Neurocognitive Index, Complex Attention, Cognitive Flexibility, and Executive Function domains, subgroups were created based on the changes in the cognitive domain scores of the Cognitrax test. Subjects in the active group with scores below the median were classified into low-response subgroups, and those with scores above the median were classified into high-response subgroups (Table 1). The high-response subgroups showed a significant improvement in cognitive scores compared with the placebo group. In contrast, there was a significant decrease in the Complex Attention domain score in the low-response subgroup compared with the placebo group, but there were no significant differences in the other domains. These results revealed that the active group consisted of high-response subgroups with improved cognitive function and low-response subgroups with no significant improvement.

Table 1. Changes in each cognitive function parameter at 12 weeks after the intervention.

		Active group			Placebo group			p-value
		n	Mean	SD	n	Mean	SD
ΔNeurocognitive Index	All	31	5.5	7.1	32	2.3	4	0.027*
	High-response	16	10.3	6.1				<0.001*
	Low-response	16	0.8	4.1				0.234
ΔComplex Attention	All	31	8.3	11.8	32	3.2	6.9	0.041*
	High-response	16	16.6	10.3				<0.001*
	Low-response	16	−0.3	4.4				0.044*
ΔCognitive Flexibility	All	31	9.8	11.5	32	4.8	6.7	0.038*
	High-response	16	17.9	9.8				<0.001*
	Low-response	16	1.6	4.9				0.068
ΔExecutive Function	All	31	9.5	11.8	32	4.5	6.9	0.044*
	High-response	16	17.8	10.1				<0.001*
	Low-response	16	1.2	5.4				0.079

Data are presented as the mean and standard deviation (SD) Cognitrax scores.

Comparisons between the active (all subjects, high-response subgroup, or low-response subgroup) and placebo groups were calculated as the change from week 0 to 12, indicated by a “Δ” symbol, and tested using an unpaired t-test (*p-value <0.05, inter-group difference).

Differences in inflammatory marker expression between the high- and low-response subgroups

To examine the effect of administration of GCL2505 and inulin on inflammation, changes in inflammatory markers after administration were compared between the active and placebo groups (Fig. 3). Olink^® Target 96 Inflammation Panels were used to measure inflammatory markers, and 75 markers were detected that had previously been described [26]. Notably, in the Neurocognitive Index domain, the high-response subgroup had significant decreases in two inflammatory markers and decreasing trends for 10 other markers (p-values ≥0.05 and <0.10) compared with the placebo group, whereas most of these markers did not decrease in the low-response subgroup (Fig. 3A, Supplementary Table 1). In the Complex Attention domain, the high-response subgroup had significant decreases in four inflammatory markers and decreasing trends for eight other markers compared with the placebo group, whereas most of these markers were not decreased in the low-response subgroup (Fig. 3B, Supplementary Table 1). In the Cognitive Flexibility domain, the high-response subgroup had significant decreases in six inflammatory markers and decreasing trends for 11 other markers compared with the placebo group, whereas most of these markers were not decreased in the low-response subgroup (Fig. 3C, Supplementary Table 1). In the Executive Function domain, the high-response subgroup had significant decreases in 14 inflammatory markers and decreasing trends in 11 other markers compared with the placebo group, whereas most of these markers were not decreased in the low-response subgroup (Fig. 3D, Supplementary Table 1). These results suggested that inflammation in the high-response subgroups of the active group was alleviated by administration of GCL2505 and inulin, based on the overall decrease in the expression of inflammatory markers compared with the low-response subgroups.

Fig. 3.

Relationship of responsiveness to GCL2505 and inulin with changes in inflammatory markers. The change in each inflammatory marker from week 0 to 12 was set on the horizontal axis, and the logarithmically transformed p-value (−Log10 p-value) was set on the vertical axis. p-values were calculated by unpaired t-tests to compare the high- or low-response subgroups of the active group with the placebo group. A threshold of −Log10 p-value >1.30 (p-value < 0.05) was set to determine significant differences. Red plots indicate p-values <0.05, blue plots indicate p-values ≥0.05 and <0.10, and gray plots indicate p-values ≥0.10 for inter-group differences (change values for the high-response or low-response subgroups vs. placebo group; unpaired t-test).

Differences in the relative abundances of gut microbiota between the high- and low-response subgroups

The changes in the relative abundances of gut microbiota between weeks 0 and 12 in the high- and low-response subgroups for each of the cognitive function domains were examined and displayed using bubble plots (Fig. 4). The changes in relative abundances between weeks 0 and 12 were evaluated using a paired t-test, and only genera with significant differences were plotted. The changes in the relative abundances of genera were represented by the size of the bubble, and rates of change were indicated using a color scale. The relative abundances of Bifidobacterium, Sutterella, and Faecalibacterium in the high-response subgroups for the Neurocognitive Index, Complex Attention, Cognitive Flexibility, and Executive Function domains were significantly increased. The relative abundance of Bifidobacterium was significantly increased in the low-response subgroups, whereas there was no increase in Sutterella or Faecalibacterium. These results showed that the reduction of inflammation and improvement of cognitive function in the high-response groups were dependent on the increased relative abundance of Faecalibacterium, as only the high-response subgroups had increased levels of Faecalibacterium in addition to Bifidobacterium.

Fig. 4.

Changes between weeks 0 and 12 in the relative abundances of fecal microbiota in the high- and low-response subgroups for each cognitive function domain shown by bubble plots. The genera of bacteria with significant intra-group differences (week 0 vs. week 12; paired t-test) in the active or placebo groups are shown graphically.

DISCUSSION

Our follow-up analysis revealed that a decrease in the overall expression of inflammatory markers correlated with an improvement in cognitive function following the intervention with Bifidobacterium animalis subsp. lactis GCL2505 and inulin. The efficacy of GCL2505 and inulin in improving cognitive function in the elderly has been demonstrated [26]. The anti-inflammatory effect of Bifidobacterium breve MCC1274 was reported in an animal study [31], and a clinical trial demonstrated its efficacy in improving cognitive function [32]. A follow-up analysis showed a negative correlation between cognitive function scores and HbA1c levels, which is a marker of glucose metabolism [33]. In the present study, we aimed to comprehensively clarify how gut microbiota and inflammatory status affect cognitive function by using proteomics and metagenomics to analyze clinical trial samples. As a result, the relationship between cognitive improvement and inflammatory conditions was directly and clearly demonstrated.

Alpha- and beta-diversity analyses of the fecal microbiota showed that the intervention had no effect on the diversity of the gut microbiota (Fig. 1). It has been reported that dietary fiber intervention in healthy people increased the abundance of specific bacteria but had no effect on the diversity of the intestinal microbiota [34]. The lack of a change in the diversity of the fecal microbiota in the present study could be due to the fact that the subjects were healthy and had no abnormalities in their gut microbiota. The results of this study are consistent with those of another clinical trial of GCL2505 and inulin, which showed that the intervention did not change alpha- and beta-diversity in the gut but did affect intestinal bifidobacteria and blood low-density lipoprotein cholesterol levels and improve vascular endothelial function [24].

We examined the changes in the gut microbiota at the genus level of the subjects during the study period to clarify the effects of GCL2505 and inulin on the gut microbiota. In the active group, there was an increase in the relative abundances of the genera Bifidobacterium, Faecalibacterium, and Bacteroides (Fig. 2C). GCL2505 and inulin administration probably caused an increase in the relative abundance of Bifidobacterium in the active group because GCL2505 can proliferate in the intestine [12, 13]. Many studies have reported that inulin administration increases the levels of specific intestinal bacteria, including the genera Bifidobacterium [20, 35, 36], Faecalibacterium [37, 38], and Bacteroides [35]. Therefore, GCL2505 and inulin intake appears to cooperatively increase these bacteria.

Several studies have reported that intake of probiotics and/or prebiotics modulates host immune status. Wastyk et al. showed that a high-fiber diet or fermented foods led to changes in the gut microbiota and a decrease in inflammatory biomarkers in the blood [39]. Another animal study showed that oral administration of Lactobacillus brevis OW38 to aged mice enhanced barrier tight junctions, decreased circulating lipopolysaccharide levels and inflammatory cytokine expression, and suppressed NF-κB activation [40]. Our previous study also showed that intake of GCL2505 and inulin affected several inflammatory markers, including LIF-R and ST1A1 [26]. Aoki et al. revealed in a mouse study that SCFAs in the gut, especially acetate, improve the phenotype of non-alcoholic liver disease/non-alcoholic steatohepatitis, e.g., fatty liver and fibrosis, by stimulating free fatty acid receptor 2 [41]. Given that the genera Bifidobacterium, Faecalibacterium, and Bacteroides were increased only in the active group, the gut microbial changes caused by administration of GCL2505 and inulin may reduce inflammation and decrease the expression of inflammatory markers.

Furthermore, we clarified the relationship between inflammatory status and cognitive function scores by examining the changes resulting from the intervention. More inflammatory markers were expressed at a lower level in the high-response subgroups for the cognitive function scores than in the low-response subgroups (Fig. 3). Eotaxin was shown in a study in mice to cause impaired learning and memory via its effect on neurogenesis [42]. C-C motif chemokine 23 is thought to be a candidate blood biomarker for detecting progression from MCI to AD, because it was suggested to be associated with neuroinflammation in the early stages of AD [43]. While the function of each of these inflammatory markers can be considered independently, cytokines, representative inflammatory markers, are known to interact with each other cooperatively or antagonistically. Therefore, it is suggested that a change in the overall profile of multiple inflammatory markers provides a clearer indication of the inflammatory state of the host than focusing on the changes in individual inflammatory markers [44, 45].

These results indicated that a reduction in inflammation was involved in the cognitive improvement caused by the administration of GCL2505 and inulin. Concomitantly, significant increases in Faecalibacterium after administration of GCL2505 and inulin were specifically observed in the high-response subgroups (Fig. 4). The genus Faecalibacterium is represented by Faecalibacterium prausnitzii, a Gram-negative anaerobic rod that accounts for 5% of the intestinal microbiota in healthy adults [46]. The genus is abundant in the large intestine because it is extremely sensitive to oxygen [47] and can utilize acetate to produce butyrate [48]. Furthermore, butyrate is taken up by the body and acts on the immune system, increasing the number of regulatory T cells responsible for suppressing inflammation and allergies and contributing to the suppression of colitis [49], activating macrophages and dendritic cells distributed in the intestinal tract, and increasing the number of regulatory T cells and IL10-producing T cells. Taken together, it is suggested that butyrate produced by Faecalibacterium plays a pivotal role in the reduction of inflammatory markers.

A significant increase in the genus Bacteroides was observed in some of the high- and low-response subgroups (Fig. 4). Most of the genus Bacteroides members produce acetate via the utilization of fructans, including inulin [41, 50], some of which have also been reported to secrete propionate by utilizing acetate [51]. The present study suggested that administration of bifidobacteria and inulin might support growth of the genus Bacteroides in the gut. While there was an increase in the genus Faecalibacterium in the high-response subgroups for all cognitive domains, the increase in the genus Bacteroides was not consistent across the cognitive domains. Further studies are needed to determine whether the genus Bacteroides contributes to improved cognitive function.

In all cognitive domains focused on in this study, the genera Bifidobacterium and Faecalibacterium were increased in the high-response subgroups, whereas the genus Bifidobacterium, but not Faecalibacterium, was increased in the low-response subgroups. This finding suggests that butyrate in addition to acetate in the gut are key substances for improving cognitive function. It is already known that Faecalibacterium assimilates acetate to produce butyrate [48]. GCL2505 grows in the intestine and increases intestinal acetate concentrations, so this intervention consisting of GCL2505 and inulin was considered to be a suitable combination for increasing intestinal acetate and butyrate concentrations. Through subgrouping of the active group, it was demonstrated for the first time that 12-week administration of GCL2505 combined with inulin is associated with an increase in the genus Faecalibacterium in the gut. The establishment of subgroups and analysis depending on the value of the cognitive scores were performed with reference to the report of Yulug et al. [52]. In the high-response subgroups, cognitive scores were improved significantly as expected compared with the placebo group. Contrary to expectations, only the low-response subgroup for the Complex Attention domain showed a statistically significant decrease in cognitive scores compared with the placebo group, but the amount of change was so small that it was deemed acceptable (Table 1).

A previous study using germ-free mice showed that acetate produced by Bifidobacterium prevents pathogenic Escherichia coli infections, which was attributed to the stimulatory effect of acetic acid on the anti-inflammatory response of the intestinal epithelium [9]. Inulin consumption also reduces the visceral fat area [53], and our previous clinical study showed that continuous consumption of a yogurt containing GCL2505 reduces the visceral fat area in the abdomen of humans [16]. Furthermore, animal studies have shown that the visceral fat-reducing effect of GCL2505 is caused by the acetic acid produced by GCL2505 [15]. Therefore, an increase in the genus Bifidobacterium in the intestine due to the consumption of GCL2505, inulin, or both may increase intestinal acetic acid levels and decrease the expression of inflammatory markers. Although there was no significant increase in the genus Bacteroides in the high-response subgroups for the Neurocognitive Index and Complex Attention domains, a significant increase was observed in the low-response subgroups for these domains. The inconsistency of these results suggests that further studies are needed, and the large increase, but low relative abundance, of the genus Sutterella suggests the limited impact of this genus.

In this study, we demonstrated in a clinical trial that the cognitive improvement following the administration of bifidobacteria and soluble dietary fiber may depend on an increase in the levels of Faecalibacterium, a representative SCFA-producing bacterium, in the gut. Continued intervention with GCL2505 and inulin improved cognitive function, and the subgroups with a greater improvement in cognitive scores had increased levels of SCFA-producing Bifidobacterium and Faecalibacterium in the gut and overall reduced levels of inflammatory markers. The results of this study suggest that the anti-inflammatory effect of increasing SCFA levels in the gut may have played an important role in improving cognitive function following GCL2505 and inulin administration. The SCFAs in the guts of the subjects were not examined in this study. However, since an increase intestinal SCFA levels in the gut has already been observed in a clinical trial that administered the same intervention [25], it is possible that SCFAs trigger and exert cognitive improvement as well. In the future, a comprehensive examination of the mechanisms underlying the cognitive improvement and anti-inflammatory effects associated with the administration of GCL2505 and inulin will provide insights into the gut-brain axis and may identify therapeutic targets for cognitive disorders, focusing on increasing SCFA-producing bacteria and reducing systemic inflammation.

AUTHOR CONTRIBUTIONS

N.W. and T.M.: conceptualization. N.W. and R.A.: methodology. N.W. and R.A.: validation. N.W., R.A., and M.S.: formal analysis. N.A., N.W., R.A., M.S., and Y.S.: investigation. N.A. and Y.S.: writing—original draft preparation. Y.S.: writing—review and editing. N.A., N.W., R.A., and M.S.: visualization. Y.S.: supervision. Y.S.: funding acquisition. Y.S.: project administration. All authors have read and agreed to the published version of the manuscript.

DATA AVAILABILITY

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

FUNDING

This research received no external funding.

CONFLICTS OF INTEREST

All authors are employees of Ezaki Glico Co., Ltd., Japan.