Effects of postbiotics on chronic diarrhea in young adults: a randomized, double-blind, placebo-controlled crossover trial assessing clinical symptoms, gut microbiota, and metabolite profiles

Abstract

Chronic diarrhea has a considerable impact on quality of life. This randomized, double-blind, placebo-controlled crossover intervention trial was conducted with 69 participants (36 in Group A, 33 in Group B), aiming to investigate the potential of postbiotics in alleviating diarrhea-associated symptoms. Participants received postbiotic Probio-Eco® and placebo for 21 days each in alternating order, with a 14-day washout period between interventions. The results showed that postbiotic intake resulted in significant improvements in Bristol stool scale score, defecation frequency, urgency, and anxiety. Moreover, the postbiotic intervention increased beneficial intestinal bacteria, including Dysosmobacter welbionis and Faecalibacterium prausnitzii, while reducing potential pathogens like Megamonas funiformis. The levels of gut Microviridae notably increased. Non-targeted metabolomics analysis revealed postbiotic-driven enrichment of beneficial metabolites, including α-linolenic acid and p-methoxycinnamic acid, and reduction of diarrhea-associated metabolites, including theophylline, piperine, capsaicin, and phenylalanine. Targeted metabolomics confirmed a significant increase in fecal butyric acid after postbiotic intervention. The levels of aromatic amino acids, phenylalanine and tryptophan, and their related metabolites, 5-hydroxytryptophan and kynurenine, decreased after the postbiotic intervention, suggesting diarrhea alleviation was through modulating the tryptophan-5-hydroxytryptamine and tryptophan-kynurenine pathways. Additionally, chenodeoxycholic acid, a diarrhea-linked primary bile acid, decreased substantially. In conclusion, postbiotics have shown promise in relieving chronic diarrhea.

Keywords: Chronic diarrhea; gut metabolomics; gut microbiota; postbiotics; quality of life.

Figure 1.

Study design and changes in psychological state and diarrhea symptoms during the intervention study. (a) The study was a randomized double-blind placebo-controlled crossover trial. Participants were randomly assigned to receive two 21-day treatments in a reciprocal sequence: the postbiotic Probio-Eco® and a placebo, with a 14-day washout period in between. Participants were randomly assigned to either group a (postbiotic-placebo) or group B (placebo-postbiotic), and 69 participants completed the study (n = 36 and 33 in groups A and B, respectively). Fecal samples were collected from each participant before and after Probio-Eco® and placebo treatments, resulting in a total of four fecal samples per participant. Participants also kept a daily diary, providing information for the weekly questionnaire. Longitudinal changes in (b) the distribution of participants reporting different degrees of diarrhea symptoms, including defecation frequency, the Bristol stool scale score, and defecation urgency (shown in the left, middle, and right panels, respectively) and (c) the psychological state of subjects reflected by anxiety, stress, and depression scores (shown in the left, middle, and right panels, respectively) in groups A and B. (d) The violin plots show the cross-sectional analysis results of diarrhea symptom and psychological state scores between groups A and B during the first phase of treatment when group a received the postbiotic and group B received the placebo. The Wilcoxon rank-sum test was used to evaluate statistical differences between groups, and the resultant p-values are shown.

Figure 2.

Fecal microbiota features of the subjects and their correlation with clinical indicators of diarrhea. (a) Shannon and Simpson diversity indexes and (b) non-metric multidimensional scaling (NMDS) analysis of the fecal microbiota of groups A and B. (c) K-means clustering analysis was conducted to investigate abundance changes in species-level genome bins (SGBs) in groups A and B during the intervention trial. The analysis grouped consistently changing SGBs into eight clusters (shown in the line charts). The x-axis represents the time points of the intervention trial, while the y-axis shows the standardized Z-scores of each SGB. The number of SGBs assigned to a particular cluster is written next to the corresponding cluster number. The standardized Z-score changes of an SGB are shown by the thin lines, while the thick lines represent the average change trends for all SGBs in that cluster. The red frames highlight postbiotic-responsive clusters of SGBs, which were exclusively influenced by the postbiotic intervention and not the placebo. (d, e) boxplots and heatmaps show the changes in abundance of postbiotic-responsive SGBs found by K-means cluster analysis and their correlations with the clinical indicators of diarrhea. The Wilcoxon rank-sum test was used to evaluate abundance differences in SGBs between time points, and significant p-values are shown (cut-off level: p < .05). The color scale in each heatmap represents correlation strength, positive and negative correlations are represented by red and purple, respectively. Significant correlations are represented by asterisks (*: p < .05; **: p < .01; ***: p < .001).

Figure 3.

Fecal phageome features of the subjects and their correlation with the bacterial microbiota. (a) Non-metric multidimensional scaling (NMDS) analysis and (b) the Shannon diversity index of the fecal phageome of groups A and B at different time points during the intervention study. (c) Cross-sectional comparison of the Shannon diversity index of the fecal phageome of groups A and B at days 0 and 21. Group a received the postbiotic between days 0 and 21, while group B received the placebo between days 0 and 21. (d) Family-level taxonomic distribution of the phage metagenome. (e) Longitudinal and cross-sectional comparisons of the post-interventional changes in the abundance of fecal Microviridae. (f) A strong positive Spearman’s rank correlation was observed between the Shannon diversity index of the fecal bacterial microbiota and phageome in both groups. (g) Procrustes analysis of the fecal bacterial species-level genome bins (SGBs) and bacteriophages of the groups A and B at different time points revealed a positive cooperativity between the gut bacteria and bacteriophages.

Figure 4.

Identification of differential metabolites and metabolite biomarkers of diarrhea. (a) Boxplots show the results of targeted metabolite analysis for butyric acid. P-values were generated by the Wilcoxon rank-sum test. (b) Orthogonal partial least squares-discriminant analysis (OPLS-DA) of fecal metabolomes after the postbiotic intervention. The upper and middle panels show the results of longitudinal comparison between day 0 and day 21 in groups A and B, respectively, while the lower panel shows the intergroup comparison at day 21. Group a received the postbiotic from days 0 to 21 and the placebo from days 35 to 56, while group B underwent the two interventions in the reverse order. Chromatography was performed in both positive and negative ion modes. Heatmaps show (c) the changes in the six significant postbiotic-responsive metabolites in groups A and B and (d) their associations with diarrhea-related clinical indicators. The color scales in (c) represent the metabolite abundance, from more (green) to less (pink); (d) represent the correlation strength, from positive (red) to negative (purple). Asterisks represent significant correlations (*: p < .05; **: p < .01; ***: p < .001). (e) The Venn network plot illustrates the common and unique postbiotic-responsive metabolites identified in longitudinal (intragroup but at different time points) and cross-sectional (intergroup, at day 21) comparisons of pre-/post- or with/without postbiotic intervention. Each dot represents one metabolite. Pink dots represent metabolites exclusively identified in one pairwise comparison. Tan, light sea green, and purple dots represent metabolites common to two pairwise comparisons. Slate gray dots represent metabolites commonly shared by the three pairwise comparisons. (f) Boxplots show the results of targeted metabolite analysis of α-linolenic acid and p-methoxycinnamic acid. P-values were generated by the Wilcoxon rank-sum test. (g) Receiver operating characteristic curves evaluate the predictive accuracy of six different metabolites (left panel) and their combined analysis as a biomarker set using multiple logistic regression analysis (right panel). The area under the curve (AUC) value reflects the discriminatory ability of the model in distinguishing between normal individuals and subjects suffering from chronic diarrhea (AUC <0.7 and above 0.8 represent poor and good discriminatory ability, respectively).

Figure 5.

Changes in fecal amino acid and bile acid levels after postbiotic intervention. Group a received the postbiotic intervention from day 0 to 21, while group B received it from day 35 to 56. The boxplots illustrate changes in (a) significant postbiotic-responsive amino acids; (b) 5-hydroxytryptophan and 5-hydroxytryptamine levels; and (c) significant postbiotic-responsive bile acids. The Wilcoxon rank-sum test was used to evaluate significant differences between time points, with the corresponding p-values shown (cut-off: p < .05).

Figure 6.

Proposed role of postbiotics-associated host gut microbiota/metabolomic changes in relieving diarrhea. The gut microbiome produces short-chain fatty acids, such as butyrate, directly stimulating trp hydroxylase 1 and resulting in the synthesis and secretion of 5-HT by intestinal enterochromaffin cells. 5-HT interacts with receptors from neurons in the enteric nervous system to modulate gastrointestinal (GI) motility. The vagus nerve senses 5-HT and connects the GI tract to the nucleus of the solitary tract and the dorsal raphe nucleus, where most 5-HT neurons reside. These areas then interact with brain networks that regulate emotions and mood. Trp and Kyn can cross the blood-brain barrier, with Kyn potentially inducing oxidative stress, anxiety, and other neurotoxic effects. Butyrate has also been associated with enhanced neuronal excitability in the enteric nervous system and contractile responses of intestinal smooth muscle. Excessive production of CDCA, phenylalanine, piperine, theophylline, and capsaicin in the intestinal tract may stimulate GI and colonic motility. Dysosmobacter welbionis, p-methoxycinnamic acid, and α-linolenic acid appear to have a less direct link with diarrhea, but they generally play beneficial roles in promoting colonic health. CDCA: chenodeoxycholic acid; 5-HT: 5-hydroxytryptamine; 5-HTP: 5-hydroxytryptophan; Kyn: kynurenine; try: tryptophan.