Abstract
Stress significantly affects gastrointestinal and mental health, and the gut microbiota plays a pivotal role in this process. Enterococcus faecalis strain EC-12 (EC-12) is a lactic acid bacterium that has several health benefits. To investigate the impact of oral supplementation with heat-killed EC-12 on the discomfort caused by stress, a randomised, double-blind, placebo-controlled trial was conducted with students under academic stress taking EC-12 (n = 14) or a placebo (n = 13) daily for one week. Improvement in the students’ symptoms was assessed using the visual analogue scale. Faecal microbiota was characterised by next-generation sequencing of 16S rRNA genes, and faecal metabolites and short-chain fatty acids were analysed using a GC-MS metabolomics approach. Significant improvements in abdominal pain and rumbling of the stomach were found in the EC-12 group compared to the placebo group, but no changes were observed in mental symptoms or salivary cortisol levels. The relative abundance of E. faecalis significantly increased in the EC-12 group after the trial; however, the composition and diversity of the gut microbiota did not change significantly. Functional analysis of the gut microbiota suggested that EC-12 intake alters specific metabolic pathways. Although the levels of faecal short-chain fatty acids did not change between the groups before and after the trial, EC-12 intake altered the composition of faecal metabolites, with a significant increase in tryptamine levels. The ratio of students with improved symptoms to those with increased tryptamine levels was calculated based on the number of students with elevated faecal tryptamine levels who showed symptomatic improvements. The ratio of improved rumbling stomach was higher than that of other types of digestive discomfort. These results suggest that oral supplementation with EC-12 has a potentially beneficial effect on stress-induced gastrointestinal discomfort, which may occur through alterations in gut microbiota composition and metabolism.
This study was registered at the University Hospital Medical Information Network Center (UMIN) under the UMIN ID: UMIN000048184
1 Introduction
The effect of stress on psychological and gastrointestinal health has become a major public health concern. Stress is associated with mental disorders, such as depression, anxiety, and post-traumatic stress disorder (Bremner et al., 2020; Yang et al., 2015), and is closely related to the occurrence of gastrointestinal diseases, such as irritable bowel syndrome, gastroesophageal reflux disease, and inflammatory bowel disease (Bremner et al., 2020; Mayer, 2000; Schneider et al., 2023; Vona et al., 2021). Numerous pieces of evidence have demonstrated that bidirectional communication between the gut microbiota and the brain might play a role in the occurrence and interplay of psychological stress and gastrointestinal discomfort, and the gut microbiota may play a pivotal role in this context, which is also known as the ‘gut microbiota-brain axis’.
The gut microbiota constitutes a distinct gastrointestinal ecosystem and participates in host development, immunity, mental health, and numerous other physiological processes (Shoubridge et al., 2022; Tanaka and Nakayama, 2017; Zheng et al., 2020). Studies have demonstrated that the gut microbiota is critical for maintaining the mental health of the host. Moreover, the composition of the gut microbiota in patients with major depressive disorder differs from that in healthy individuals, and several specific microbial groups may be associated with symptoms such as insomnia and depression (Zhang et al., 2021). On the other hand, modifying the composition of specific gut microbial communities may lead to improvements. Recently, Roseburia, a gut bacterium, was shown to have the potential to modulate serotonin, a neurotransmitter derived from tryptophan, which could potentially improve adolescent depression (Zhou et al., 2023). However, the underlying mechanisms of the ‘gut microbiota-brain axis’ remain poorly understood.
Probiotics are a certain kind of bacteria that could provide various health advantages upon ingestion. They are commonly used as supplementary or alternative therapeutic approaches for various diseases, potentially functioning within the treatment process by ameliorating bacterial dysbiosis or modulating the immune system (Bjarnason et al., 2019; Deng et al., 2023). Lactobacillus and Bifidobacterium are well-known probiotic strains that ameliorate depression and anxiety (Liu et al., 2019). Recent studies have found that not only viable probiotics exhibit bioactivity and health benefits, but also non-viable probiotics (paraprobiotic) can provide health advantages (Cuevas-González et al., 2020). Additionally, the use of non-viable probiotics avoids the potential risks associated with viable bacteria (Liu et al., 2024). EC-12 is from heat-killed lactic acid bacterial material obtained from Enterococcus faecalis isolated from the human intestinal tract. In mice, EC-12 modulates gut immunity and prevents the development of intestinal mucositis by downregulating inflammatory responses (Inoue et al., 2011; Nobre et al., 2022). In addition, EC-12-fed mice show reduced anxiety-like behaviour and altered expression of anxiety-related genes in the brain (Kambe et al., 2020). However, studies on the effects of EC-12 in alleviating stress and anxiety in humans are limited.
In this trial, healthy university students subjected to exam-related stress, a classic model of chronic stress, were selected to participate in the experiments. This study aimed to determine whether EC-12 can reduce self-reported gastrointestinal symptoms and overall feelings of stress during the week of academic exams, and to investigate changes in the gut ecosystem by analysing the faecal microbiome and metabolome.
2 Materials and methods
Bacterial preparations
The EC-12 used in the experiment was a commercial product of the cell preparation of E. faecalis strain EC-12 (International Patent Organism Depositary in Japan number, FERM BP-10284; GenBank Accession number, AB154827; Combi Corporation, Saitama, Japan) isolated from healthy human faeces (Terada et al., 2004). The product was a dried powder of heat-killed bacteria sealed in an aluminium foil bag (200 mg containing 1 × 1012 cells). The exact amount of the placebo was packed in a bag similar to that of the supplement. The appearance and taste of the test and placebo groups could not be distinguished by sensory assessment.
Participant recruitment and randomisation
This double-blind randomised study was approved by the Ethics Committee of Tokyo University of Agriculture and Technology (#211105-0306) and was conducted in accordance with the Helsinki Declaration. Participants were recruited from the Tokyo University of Agriculture and Technology and provided informed consent; they were healthy adults aged 20-24 years, without a history of digestive system diseases, mental disorders, or medication use in the three months prior to enrolment. This study was registered at the University Hospital Medical Information Network Center (UMIN) under the UMIN ID: UMIN000048184.
Investigator 1 assigned 27 eligible participants to two groups with stratified randomisation based on sex. Investigator 2 assigned the cases to the control and EC-12 groups, and each product was prepared and given to Investigator 1. Investigator 1 distributed the products to the participants and obtained the survey results, saliva, and stool samples. Investigator 3 performed the data analysis.
Trial design
Students in the EC-12 and placebo groups were instructed to consume one pack of the EC-12 product or placebo daily for one week. During this period, the students experienced stress during examinations as a chronic stress model. Participants were asked to complete a 100 mm visual analogue scale (VAS) designed to assess their physical and mental experiences before and after the trial. Physical indicators included abdominal pain, stomach rumbling, and diarrhoea. The mental indicators included feelings of stress, nervousness, and anxiety. Students were asked to collect fecal and saliva samples on the morning of the first day and one week after the trial, using disposable collection tubes provided by the laboratory. The collected samples were stored at −80 ° until use.
Salivary cortisol measurement
Salivary cortisol was assayed using a Salivary Cortisol ELISA Kit according to the manufacturer’s instructions (Salimetrics Inc., Carlsbad, CA, USA).
Microbiome analysis
Microbiome analysis of faecal contents was conducted using 16S rRNA sequencing following established protocols (Liu et al., 2024). Genomic DNA was extracted using the QuickGene DNA Tissue Kit S (Kurabo, Osaka, Japan) following the manufacturer’s guidelines. The initial PCR targeted variable regions 3 and 4 (V3-4) of the 16S rRNA gene using the primers 341F (CCT ACG GGR BGC ASC AG) and 806R (GGA CTA CNN GGG TAT CTA AT). A subsequent PCR step was performed to incorporate the dual indices. The resulting library, combined with the phiX control, was sequenced using the MiSeq v3 kit (Illumina Inc., San Diego, CA, USA).
Sequence data were processed using QIIME2 (ver. 2021.11), and the reads were denoised using the DADA2 plugin to generate an ASV feature table. Singleton reads and ASVs assigned to the mitochondria and chloroplasts were excluded. Taxonomic assignments of the filtered ASVs were conducted using a pre-trained QIIME2-compatible SILVA version 138 database (99% full-length sequences). Microbial variance analysis was performed using the MicrobiomeAnalyst website (Chong et al., 2020) with the default settings. All 16S rRNA sequencing data were deposited and made accessible under the code PRJNA1106567 in May 2024.
Metabolomics analysis
Untargeted metabolomics analysis was performed using gas chromatography-mass spectrometry (GC-MS) as described previously, with some modifications (Liu et al., 2024). For the preparation of extracted faecal contents, 100 mg of faecal content, 15 mg of 0.2 mm glass beads (No. 02, Toshin Riko Co., Ltd., Tokyo, Japan), and 400 μl of ultrapure water were vigorously mixed and centrifuged for 5 min at 20,000
Measurement of short-chain fatty acids by GC-MS
Faecal contents (100 mg) were placed in 2 ml screw-cap tubes containing 15 mg of 0.2 mm glass beads (No. 02, Toshin Riko Co.). Subsequently, 1 ml of 10% isobutanol was added, and the samples were homogenised using a Micro Smash Cell Disrupter (MS-100, Tomy Seiko Co. Ltd., Tokyo, Japan) at 5,500 rpm for 20 s. This homogenisation process was repeated twice with a 30 s pause between cycles. After homogenisation, the samples were centrifuged at 21,000
An internal standard (20 μg 3-methyl pentanoate), 125 μl of 20 mM NaOH, and 400 μl of chloroform were added into each tube and thoroughly vortexed. After centrifugation at 21,000
The upper hexane/isobutanol phase was then transferred to a GC vial. Subsequently, 1 μl was injected into a GC-MS QP2020 NX (Shimadzu Corporation) using either splitless mode or a split ratio of 1:50. The GC column was an HP-5MS with dimensions of 30 m × 0.25 mm × 0.25 μm (Agilent Technologies, Santa Clara, CA, USA). The oven temperature gradient for the samples was as follows: after a 5-min isothermal period at 50 °C, the oven temperature was increased to 150 °C at a rate of 5 °C/min, then further raised to 330 °C at a rate of 40 °C/min, and held at 330 °C for 1 min. The GC-MS ion source and transfer line temperature were set to 250 °C. The scanning range of the mass spectrometer was 30-600 m/z.
Measurement of lactic acid using GC-MS
Faecal contents (100 mg) were combined with 100 μL of ultrapure water, 800 μl of acetonitrile, and 5 μl of 1 mg/ml 3-methyl valeric acid (internal standard). The mixture was agitated at 1,200 rpm and 37 °C for 30 min and then centrifuged at 21,000
Quantification of lactic acid in the samples was performed using a seven-point calibration curve (1.563, 3.125, 6.25, 12.5, 25, 50, and 100 mM) based on the ratio of 3-methyl valeric acid to the lactic acid peak. For GC-MS analysis, 1 μl of the derivatised sample was injected into a GC-MS QP2020 NX using a DB5 column (30 m × 0.25 mm i.d.; Agilent Technologies). The vaporising chamber temperature was set at 290 °C, and the gas flow rate through the column was maintained at 1.0 ml/min. The temperature programme involved 3 min of isothermal heating at 40 °C, followed by an increase to 80 °C at 10 °C/min, a further increase to 150 °C at 20 °C/min, a rise to 310 °C at 30 °C/min, and maintenance at 310 °C for 2 min. Target peaks were measured in SIM mode using ions (m/z) identified through standard measurements, with the ions (m/z) of 3-methyl valeric acid and lactic acid being 73, 75, 173, and 73, 147, 189, respectively.
Statistical analysis
Data were analysed and visualised using MicrobiomeAnalyst 2.0 (Chong et al., 2020), MetaboAnalyst 5.0 (Xia et al., 2009), R and GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). The chi-square test was used to compare the proportion of patients in each group with improvements in positive symptoms across different groups. In the microbiome analysis, the beta diversity of the faecal microbiome was plotted using principal coordinate analysis (PCoA) on the unweighted UniFrac distance and permutational multivariate analysis of variance (PERMANOVA). Linear discriminant analysis effect size (LEfSe) analysis was performed to discriminate microbial features among the groups. The Wilcoxon rank-sum test was performed by the R (v4.2.3) package of ‘stats’. In the metabolomics analysis, partial least-squares discriminant analysis was used to visualise the separation among the groups and to identify the essential metabolites responsible for the separation. A volcano plot was used to select significant metabolites with a fold-change threshold of 10 and a t-test threshold P-value < 0.05.
3 Results
Oral supplementation with EC-12 ameliorates gastrointestinal symptoms of students
From the results shown in Figure 1A, more than 90% of the participants taking EC-12 reported an improvement in abdominal pain and rumbling stomach symptoms, which was a significantly higher percentage than that in the placebo group. No differences in diarrhoea symptoms were observed between the two groups. Regarding mental symptoms, as shown in Figure 1B, 64%, 79%, and 43% of participants in the EC-12 group reported improvements in stress, nervousness, and anxiety, respectively. However, no significant improvements were observed in the placebo group. Salivary cortisol concentrations were measured to assess stress levels before and after experiencing chronic academic pressure. There were no significant differences between the two groups (Figure 1C).
Oral supplementation with EC-12 ameliorates gastrointestinal symptoms of students. Changes in VAS scores for gastrointestinal symptoms (A) and mental symptoms (B) were analysed following the EC-12 intervention. The y-axis represents the VAS questionnaire scores, with 0-10 indicating the severity of discomfort symptoms. Improvement rates and the number of students with improvement/total number of students per group are labelled below the x-axis. (C) Salivary cortisol concentration. Decreased cortisol levels and the number of students with reduced levels/total number of students per group are marked below the x-axis. * P-value < 0.05 indicates a significant difference between groups analysed by a chi-squared test.
Citation: Beneficial Microbes 2025; 10.1163/18762891-bja00046
Oral supplementation with EC-12 changed the gut microbiota composition
The 16S rRNA gene sequencing was conducted using faecal samples from the EC-12 and placebo groups to define variations in gut microbiota composition. In total, 1,465,274 (mean ± standard deviation: 27,341 ± 10996 reads per sample) sequence reads were obtained from 54 samples, and 1,950 features were present. Figure 2A shows the primary phylum composition and the top 10 abundant genus composition of the gut microbiota in the placebo and EC-12 groups, before and after the trial. In both groups, the five most abundant microbiota at the phylum level were Firmicutes, Actinobacteriota, Bacteroidota, Proteobacteria, and Desulfobacterota. The top 10 abundant microbiota at the genus are Blautia, Bifiobacterium, Faecalibacterium, Bacteroides, Subdoligranulum, Ruminococcus_gnavus_group, Anaerostipes, Escherichia_Shigella, Fusicatenibacter and Eubacterium halli group. Wilcoxon rank-sum test was performed to analyse the difference between EC-12 group and placebo group before and after the trial. No significant alterations in the main composition were observed at the phylum, family, or genus levels.
The alpha-diversity index was used to describe the dynamics of microbiome composition and was represented by the Chao1 and Shannon indices. Figure 2B illustrates that neither the EC-12 nor the placebo groups showed a statistically significant change in the alpha diversity of the faecal samples before or after the trial. Community composition was described by beta diversity through a PCoA plot and a PERMANOVA test. No significant difference was found in the beta diversity within the faecal samples between the EC-12 and placebo groups (Figure 2C). LEfSe analysis was employed to analyse the differential bacterial taxonomies between the groups and to assess the effect size of each taxonomic level. The results showed that participants who consumed EC-12 exhibited a significant increase in the relative abundance of the E. faecalis compared with pre-experiment levels (Figure 2D).
Oral supplementation with EC-12 changed the gut microbiota composition. (A) Left: gut microbiome composition of placebo and EC-12 groups before (Pre) and after (Post) the trial at the phylum level. Right: top 10 abundant gut microbiome composition of placebo and EC-12 groups before (Pre) and after (Post) the trial at the genus level. No significant bacteria were found by Wilcoxon rank-sum test. (B) The Chao1 alpha diversity of the placebo and EC-12 groups before and after the trial. (C) The beta diversity unweighted UniFrac distances of different groups. (D) The relative abundance of the Enterococcus faecalis. The line inside the box represents the median, and the dots represent individual samples.
Citation: Beneficial Microbes 2025; 10.1163/18762891-bja00046
Functional analysis of gut microbiota
To determine the impact of EC-12 intake on potential functional pathways and mechanisms, KEGG enrichment analysis based on 16S rRNA sequence data was conducted using STAMP software. Figure 3A illustrates the differential functional metabolic pathways between the EC-12 and placebo groups before the trial. Tyrosine metabolism, Staphylococcus aureus infection, phosphotransferase systems, and fructose and mannose metabolism pathways differed significantly. The same analysis was used to compare the differential pathways between the placebo and EC-12 groups after the trial, and 14 pathways showed significant differences (Figure 3B). Alanine, aspartate, and glutamate metabolism as well as galactose metabolism were found to be the characteristic metabolic pathways between the placebo and EC-12 groups after the trial.
Functional analysis of gut microbiota. KEGG enrichment analysis based on 16S rRNA sequence data was conducted using STAMP software. The comparison of gut microbiome abundance between the EC-12 and placebo groups revealed 4 significantly different metabolic functions before the trial (A) and 14 after the trial (B). Extended error bar plot indicating differences in functional profiles of the control and probiotic microbiota (at taxonomic level 3). All unclassified reads were removed, and q-values > 0.05 are displayed. Bar plots on the left side display the mean proportion of each KEGG pathway. The dot plots on the right show the differences in mean proportions between the two indicated groups using q-values.
Citation: Beneficial Microbes 2025; 10.1163/18762891-bja00046
Effect of EC-12 intake on faecal metabolic components
Non-targeted metabolomics analysis identified 157 metabolites. As shown in Figure 4A, a volcano plot delineated the metabolic differences between the EC-12 and placebo groups after the trial (
Effect of EC-12 intake on faecal metabolic components. (A) Left panel: The volcano plots highlight the faecal metabolites that were increased (red) and decreased (blue) in the EC-12 group compared to the placebo group after the trial;
Citation: Beneficial Microbes 2025; 10.1163/18762891-bja00046
The relationship between individual questionnaires on digestive symptoms and faecal tryptamine levels
To further investigate the relationship between digestive symptoms and faecal tryptamine levels, a table integrating faecal tryptamine levels and questionnaire results was created (Table 2). Based on the results of the non-targeted metabolomics analysis, students with an increased relative abundance of faecal tryptamine levels after the trial were labelled as ‘Increased’. Students with decreased VAS scores for abdominal pain, rumbling stomach, and diarrhoea after the trial were labelled as ‘Improved’. The ratio of students with improved symptoms to those with increased tryptamine levels was calculated based on the number of students with elevated faecal tryptamine levels who showed symptomatic improvements. The ratio of improved rumbling stomach, abdominal pain, and diarrhoea was 0.94 (15/16), 0.75 (12/16), and 0.56 (9/16), respectively.
Concentration of short-chain fatty acid in faeces
Citation: Beneficial Microbes 2025; 10.1163/18762891-bja00046
Results of individual questionnaires on abdominal pain, rumbling stomach, diarrhea and changes in faecal tryptamine levels
Citation: Beneficial Microbes 2025; 10.1163/18762891-bja00046
4 Discussion and conclusions
This study was a double-blind, placebo-controlled trial investigating whether oral supplementation with a heat-killed E. faecalis strain EC-12 product affected the digestive and mental states of healthy undergraduate students experiencing exam-related stress. We designed a questionnaire to investigate changes in students’ symptoms and feelings of discomfort. Additionally, we studied whether the EC-12 oral supplement could alter the composition of the gut microbiota under stress conditions and affect metabolic status. This study suggests that supplementation with EC-12 products can reduce gastrointestinal discomfort under stress conditions by altering the gut ecosystem.
Stress stimulates the hypothalamus-pituitary-adrenal axis and triggers cortisol secretion, and salivary cortisol levels are reliable stress indicators (Hellhammer et al., 2009). Long-term exposure to external stressors may induce an organism to produce additional cortisol (Gerding and Wang, 2022). In this study, an academic stress model was applied to evaluate the effects of the daily intake of placebo and EC-12 on stress-related gastrointestinal and mental symptoms. This experiment did not find significant fluctuations in salivary cortisol levels before or after the trial. Similarly, the VAS questionnaire results indicated no significant improvement in students’ mental health discomfort. These results suggest that the stress experienced by students may not significantly impact mental health to precipitate severe symptoms but may, instead, primarily manifest as gastrointestinal discomfort.
The VAS results showed that EC-12 intake ameliorated gastrointestinal symptoms, particularly abdominal pain and rumbling stomach. Several studies have shown that certain probiotics alleviate digestive discomfort and contribute to the treatment and recovery of patients with gastrointestinal diseases. Studies have already revealed the role of EC-12 as a paraprobiotic agent in enhancing health, and its efficacy in improving gastrointestinal function is well established; the mechanism revolves around reducing the levels of gastrointestinal inflammatory markers and modulating innate immune function (Nobre et al., 2022; Sakai et al., 2007). The results of next-generation sequencing indicated that in both the EC-12 and placebo groups, the primary composition of the gut microbiota at the phylum level included Firmicutes, Actinobacteriota, Bacteroidetes, and Proteobacteria before and after the experiment. These findings are consistent with previous studies’ findings (Jackson et al., 2023; Kato-Kataoka et al., 2016). The composition of the gut microbiota showed no significant changes in both alpha and beta diversity, and E. faecalis was the only bacterium that showed statistically significant changes in the LEfSe analysis, indicating that EC12 intake in this study did not significantly alter the composition of the gut microbiota. Enterococcus faecalis is a well-known probiotic that is widely used in clinical therapy for irritable bowel syndrome (Hong et al., 2023). Substantial research has suggested that products containing dead beneficial bacterial cells and their metabolites can elicit biological responses similar to those of live cells (Piqué et al., 2019).
To investigate the potential functional pathway changes relevant to EC-12 intake, KEGG enrichment analysis was conducted based on the next-generation sequencing results. This analysis revealed that alanine, aspartate, glutamate, and galactose metabolism were among the main metabolic pathways. Alanine, aspartate, and glutamate belong to the glutamate group (glutamic acid, gamma-aminobutyric acid, glutamine, aspartic acid, and alanine), and are critical components of amino acid metabolism. Alanine is crucial for intestinal inflammation (Xu et al., 2005). These amino acids are essential signalling molecules in the intestine and are precursors of many critical gastrointestinal hormones, playing crucial regulatory roles in metabolism, immunity, and function (Baj et al., 2019). Galactose metabolism is also thought to be related to inflammation, and its metabolites are associated with endothelial damage and an increase in inflammatory factors (Hobbs et al., 2014; Mhd Omar et al., 2021).
Based on the predictions derived from the microbial functional analysis, we focused on changes in faecal metabolomics. Faecal metabolite analysis revealed elevated tryptamine levels in the EC-12 group after the trial. In addition, the ratio of students with symptom improvement to those with elevated faecal tryptamine levels was high for rumbling stomach. Tryptamine is a tryptophan metabolite produced by the gut microbiota. It is also a neurotransmitter that exerts various effects on the gastrointestinal tract by interacting with 5-HT4 receptors on colonic cells, including the promotion of gastrointestinal motility and mucus secretion (Bhattarai et al., 2018). Research has found that other bacteria identified in the human gut microbiome, such as Clostridium sporogenes, can decarboxylate tryptophan to produce tryptamine and affect digestive functions (Cryan et al., 2018; Williams et al., 2014). Highly selective 5-HT4 agonists are also promising for the treatment of chronic constipation (Cole and Rabasseda, 2004; Shin et al., 2011). In contrast, exposure to certain concentrations of tryptamine can induce changes in the metabolism of short-chain fatty acids produced by the intestinal microbiota (Otaru et al., 2024). Short-chain fatty acids can improve health by regulating inflammatory factors, particularly IL-22, an immune factor crucial for maintaining intestinal barrier function (Gao et al., 2022; Hao et al., 2021; Yang et al., 2020). Studies have found that psychological stress impairs IL-22’s role in the gut barrier and induces digestive dysfunction (Zha et al., 2019). This suggests that EC-12 products may exert their effects by modulating the composition of the gut microbiota through tryptamine, although we did not observe significant intergroup differences in short-chain fatty acid levels.
Hydroxyphenyllactic acid was also identified as the differential metabolite. It is a respective aromatic lactic acid transferred from tyrosine. The function of aromatic lactic acids remains to be fully described, several studies have confirmed that 4-hydroxyphenyllactic acid is present in various body fluids and plays a potential role in predicting the risk and progression of clinical scenarios, such as predicting postoperative meningitis after neurosurgery, identifying critically ill patients and breast cancer screening and (Pautova et al., 2022; Sobolev et al., 2023; Yang et al., 2017). The production of aromatic lactic acids has been shown to be associated with gut microbes, such as Bifidobacterium species (Laursen et al., 2021; Sakurai et al., 2021). The relationship between hydroxyphenyllactic acid and other gut microbiomes is still under investigation.
This study has some limitations. Academic stress is a stress model widely used in various studies of chronic psychological stress (Venkataraman et al., 2021). We recruited students who were healthy adults aged 20-24 years without a history of digestive system diseases, mental disorders, or medication use in the three months before trial. However, there is no assurance that all students are exposed to the same stress or how they cope with their own stress. As for women, abdominal pain caused by menstruation should also be taken into consideration. In future studies, it will be necessary to select students based on these considerations. In addition, saliva samples were collected at the beginning and end of this trial. Considering the duration of the trial and the mechanisms of chronic stress, the measurement of salivary cortisol may not accurately reflect the continuous fluctuations in cortisol levels during periods of chronic stress. Salivary cortisol levels are influenced by circadian rhythms (Shin et al., 2011). Regarding the improvements in metabolic states induced by EC-12 intake, the specific mechanisms still require further experimental exploration.
In conclusion, this study demonstrated the positive impact of oral supplementation with heat-killed EC-12 on the health of students under academic stress. Following EC-12 intake, the students experienced relief from digestive symptoms, such as abdominal pain and rumbling stomach. Moreover, alterations in both the gut microbiota composition and faecal metabolomic profiles were observed, suggesting that these effects may be mediated through the regulation of intestinal metabolic function. However, owing to the limited sample size and lack of in-depth investigations into the specific mechanisms of action of EC-12, further studies are needed to validate these findings.
Corresponding author; e-mail: nagaokak@cc.tuat.ac.jp
Authors’ contribution
Conceptualisation, JL, YM-I, and KN; methodology, SM and JK; validation, YY and KN; formal analysis, JL, TT, HL, and RI; writing-original draft preparation, JL; writing-review and editing, YY and KN; funding acquisition, YM-I and KN. All the authors have read and agreed to the published version of the manuscript.
Conflict of interest
The authors have no conflicts of interest to disclose.
Data availability
All 16S rRNA sequencing data were deposited and made available under accession number PRJNA1106567 in May 2024.
Funding
This study was supported by a research grant from JST-OPERA (JPMJOP1833).
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