This study developed a synbiotic rice yogurt cake with a medium glycemic index by combining resistant starch-enhanced rice with Lactobacillus acidophilus. The effects of substrate concentration (20-40%), pullulanase enzyme activity (20-40 U/g), and hydrolysis time (4-8 hr) on resistant starch content were investigated. At a substrate concentration of 30%, an enzyme activity of 30 U/g, and a hydrolysis time of 6 hr, the optimal resistant starch content was 14.54 ± 0.96%. After being treated to enhance its resistant starch content, IR504 rice was mixed with milk and processed to produce rice milk. The rice milk was fermented for 12 hr at 37°C with a 3% inoculum ratio, yielding the highest L. acidophilus count of 5.62 × 107 CFU/g. After freeze-drying, the synbiotic rice yogurt cake contained 3.8 × 107 CFU/g L. acidophilus and 3.55 g/100 g resistant starch. Nutritional analysis showed 82.4 g carbohydrates, 6.78 g lipids, 8.90 g proteins, and 1.05% moisture per 100 g. This product achieved a lower estimated glycemic index of 69.32 ± 0.534 compared with non-resistant starch (77.74 ± 0.369). Microbiological tests confirmed safety standards were met, with undetectable Enterobacteriaceae (<10 CFU/g), Listeria monocytogenes (undetectable/25 g), and undetectable total mold. The predicted shelf life of the synbiotic rice yogurt cake was approximately 198 days under typical storage conditions at 30°C and L. acidophilus count of 4.8 × 106 CFU/g. Therefore, the synbiotic rice yogurt cake can be used as a product supporting glycemic control while simultaneously providing a substrate for beneficial gut bacteria.
Urinary indoxyl sulfate (IS), a gut microbiota-derived uremic toxin, is associated with the adult gut microbiota and is elevated in children with autism spectrum disorder. However, its age-related trajectory and association with microbiota in healthy children remain uncharacterized. Therefore, we aimed to elucidate age-related changes in urinary IS in healthy children and to investigate the potential utility of urinary IS as a surrogate marker for their gut microbiota. We measured urinary IS/creatinine (Cr) ratios and analyzed the fecal gut microbiota composition (16S rRNA gene sequencing) in 102 healthy Japanese children aged 5 months to 12 years. Associations were assessed using Spearman's correlation. An empirical age-adjustment model (inverse proportional) was developed to derive an age-adjusted IS/Cr ratio (actual/predicted), thereby removing the strong age dependence before re-evaluating correlations with gut microbiota. The unadjusted IS/Cr ratio showed a strong negative correlation with age (rs=-0.47, p<0.001) and declined markedly during early childhood, stabilizing at approximately 5 years. Weak correlations with microbiota metrics were observed before adjustment; however, the age-adjusted IS/Cr ratio showed no correlation with age (rs=-0.03, p=0.768). Notably, age adjustment revealed significant positive correlations between the IS/Cr ratio and the phylum Bacteroidota (rs=0.26, p=0.008) and strengthened the correlation with the genus Bacteroides (rs=0.31, p=0.002), associations that were not apparent before adjustment. Correcting for this age-dependent variation is crucial, after which the IS/Cr ratio can serve as a potential non-invasive surrogate marker reflecting the abundance of Bacteroidota, particularly the genus Bacteroides, in the pediatric gut microbiota.
There is growing optimism regarding the potential therapeutic and preventive benefits of regulating intestinal microbiota for various diseases. Diet is one of the most straightforward and safest methods for modulating the intestinal microbiota; however, considerable individual differences have been observed in the microbiota response to dietary interventions. These individual differences pose substantial challenges in application, which are primarily attributed to variations in the commensal flora and bacterial competition for nutrients. Our previous research indicated that the microscopic localization of bacteria provides valuable insights into the mechanisms by which specific intestinal bacterial species acquire nutrients within a competitive gut environment. Furthermore, our analysis revealed that the combination of bifidobacterial species and the nutrient source found in the localization analysis determined individual differences in microbiota response. These findings suggest that bacterial colonization facilitates the efficient, preferential, and presumably exclusive utilization of solid nutrient sources in the human gut. Moreover, the impact of a single nutrient source on the gut and human body may vary depending on the presence or absence of the primary species colonizing that source. In this review, we examined the micrometer-scale localization of intestinal bacteria and individual variability in microbiota responses to diet, drawing upon the results of our previous studies.
Humanized mouse models are widely used to investigate host-microbiota interactions, yet the extent to which host background contributes to engraftment fidelity remains incompletely defined. In this study, we transplanted fecal microbiota from healthy human donors into NCG and SGM3 mice and characterized engraftment using 16S rRNA sequencing. Both models exhibited reduced diversity relative to donors, but their colonization trajectories diverged. NCG recipients appeared closer to donors in β-diversity space, a pattern largely associated with the expansion of a limited set of opportunistic Proteobacteria such as Escherichia-Shigella and Citrobacter. In contrast, SGM3 mice displayed modestly higher α-diversity and retained a broader set of donor-associated genera, with selective enrichment of Bacillus, yet exhibited greater predicted functional divergence, with reductions in pathways related to ABC transport and carbohydrate metabolism. Several strictly anaerobic commensals, including Faecalibacterium, failed to colonize in either genotype. Collectively, these findings suggest that host genotype is associated with selective colonization of human fecal microbiota and support the utility of integrating compositional and functional criteria when selecting experimental models for translational microbiome research.
Tongxie Yaofang (TXYF) is a classic formula for liver stagnation and spleen deficiency diarrhea, but its mechanisms involving the gut microbiota remain unclear. This study aimed to elucidate the mechanisms TXYF by integrating network pharmacology, molecular docking, and 16S rRNA gene sequencing. We employed network pharmacology and molecular docking to identify bioactive compounds, targets, and their associations with gut microbiota metabolites. Sequencing of the 16S rRNA gene revealed changes in the gut microbiota in a diarrheal mouse model after TXYF intervention. Thirty-nine active compounds (525 targets) were identified. Intersection analysis yielded 44 shared targets among TXYF, diarrhea, and gut microbiota metabolites. Molecular docking demonstrated favorable binding of the selected active compounds and representative gut microbiota-derived metabolites with the core targets GSK3B, TP53, and MAPK3. Sequencing of the 16S rRNA gene also showed that TXYF significantly modulated gut microbiota structure, notably altering abundances of Corynebacterium stationis, Proteus vulgaris, and Proteus mirabilis. TXYF treats diarrhea by modulating gut microbiota composition and maintaining microbial homeostasis via a multi-component, multi-target mechanism, supporting its potential as a microbiota-targeted therapy.
Lactococcus cremoris subsp. cremoris FC (L. cremoris FC) improves bowel movement; however, its associations with the intestinal microbiota remain unclear. To address this gap, this post hoc analysis of a four-week, placebo-controlled, parallel-group trial evaluates the associations between L. cremoris FC intake and gut microbiota profiles. We analyzed fecal samples from 83 healthy Japanese adults with mild constipation who received either placebo capsules containing corn starch (n=20) or L. cremoris FC capsules (50, 75, or 100 mg; n=21 each). Using 16S rRNA gene amplicon sequencing, we evaluated diversity and relative abundance and explored association patterns through Sparse InversE Covariance estimation for Ecological Association and Statistical Inference network analysis. Although no dose dependence was observed, alpha diversity decreased significantly in the L. cremoris FC 100 mg group, whereas beta diversity shifted significantly in the 50 mg group. Short-chain fatty acid producing genera, including Blautia, Anaerobutyricum, Anaerostipes, and Agathobaculum, showed higher relative abundances after intervention in both L. cremoris FC intake groups. Moreover, the relative abundance of Blautia was positively correlated with stool characteristics across post-intervention samples. Exploratory network analysis indicated altered statistical associations among bacterial taxa. Blautia and Roseburia showed higher centrality in the L. cremoris FC 100 mg group, whereas Bacteroides and Ruminococcus were more central in the placebo group. These findings suggest that L. cremoris FC intake was associated with coordinated alterations in microbial diversity, genus-level relative abundance, and association patterns, providing ecological insights into microbiota changes accompanying bowel improvement, though underlying mechanisms remain unclear.
Lactiplantibacillus plantarum and Lactiplantibacillus pentosus are the commonly found dominant species in post-fermented Japanese tea. The dominant species of lactic acid bacteria (LAB) depends on the production area, regardless of the production method. However, the selection mechanism of these bacteria remains unknown. Therefore, we investigated the mechanism by which these LAB species are selected. We investigated the effects of pH and catechins on the growth of L. plantarum and L. pentosus isolated from two types of post-fermented Japanese teas. When the pH of the medium was adjusted with hydrochloric acid, the strains derived from Ishizuchi-kurocha grew at pH 4 to 10, whereas the strains derived from Awa-bancha grew at pH 3 to 10. In contrast, when the pH was adjusted using lactic acid, no growth was observed at pH 4. The pH of the fermented fluid of Ishizuchi-kurocha was approximately 4, suggesting that the growth of the LAB in Ishizuchi-kurocha was inhibited by the accumulation of lactic acid. Furthermore, when a mixture of green tea-derived catechins containing epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate was added to the culture medium at a concentration of 1.0 mg/mL, Staphylococcus aureus growth was inhibited. However, the effect on L. plantarum or L. pentosus growth was negligible. Furthermore, when the catechins in the medium were analyzed using high-performance liquid chromatography after 24 hr incubation with LAB, the peak intensities of catechins, particularly catechin gallate, were decreased, and an unknown peak was observed. These results suggest that L. plantarum and L. pentosus metabolize catechins during tea leaf fermentation. During the fermentation of Ishizuchi-kurocha and Awa-bancha, at least, the catechins in them and decrease in pH due to mainly lactic acid accumulation are factors involved in LAB selection.
This study investigated the preventive effects of combination of Polygonatum sibiricum and Poria cocos on constipation due to pi deficiency and aimed to explore a potential early intervention strategy. The administered groups were gavaged with medicinal liquids containing different ratios of Polygonatum sibiricum to Poria cocos (A, 3:1; B, 1:1; and C, 1:3) for 14 days. Subsequently, a mouse model of constipation due to pi deficiency was established through a combination of Folium Sennae decoction gavage, diet regulation, water restriction, and abnormal hunger and satiety. The normal group was fed normally for 29 days. After 14 days of normal feeding, the model group mice were modeled according to the above modeling method. The study compared fecal characteristics, water content, serum D-xylose levels, intestinal microbial counts, and enzyme activities among the different groups. The results showed that mice with constipation due to pi deficiency exhibited altered body weights and behavior, along with altered fecal features and reduced fecal water content. Dysbiosis of the intestinal microbiota and disturbances in enzyme activities were observed. The mice in the administered groups showed less fluctuation in body weight and improved behavior. Fecal traits were improved, and the water content of feces was increased. Notably, ratio B (1:1) demonstrated the most significant effects, effectively restoring the intestinal flora balance and alleviating enzyme activity disorders. These findings suggest that the combination of Polygonatum sibiricum and Poria cocos at a 1:1 ratio exerts the most beneficial preventive effects on constipation due to pi deficiency by improving intestinal health and microbial homeostasis.
Periodontal disease is a prevalent inflammatory condition linked to tooth loss and systemic disorders. Plant-derived lactic acid bacteria (LAB) have emerged as promising probiotics for oral health. In this study, 80 LAB strains were isolated from tea leaves, young barley leaves, carrots, and fermented vegetables, and their antimicrobial effects against key periodontal pathogens were evaluated. Various assays, including biofilm inhibition, growth inhibition, disk diffusion, and RT-qPCR, were employed to assess activity. LAB strains from tea leaves and young barley leaves exhibited significantly stronger anti-biofilm activity and growth inhibition against Porphyromonas gingivalis than those from carrots and fermented vegetables. These strains also demonstrated notable antimicrobial activity against P. gingivalis and Fusobacterium nucleatum and significantly downregulated key virulence-related genes, including fimA, kgp, and rgpA in P. gingivalis. The results suggest that the antimicrobial efficacy of LAB is dependent on their plant source. Tea and barley-derived LAB strains may serve as potent candidates for probiotic development aimed at preventing periodontal disease. Further investigation is warranted.
This study evaluated the effects of Saccharomyces cerevisiae-fermentation-derived postbiotic (SCFP) on pathogenic and antimicrobial-resistant (AMR) bacteria in sows within a high biosecurity system. Thirty sows were divided into three groups: a standard basal diet (CON group), CON with 1.0 kg/MT beta-glucan 50% (BG-50 group), and CON with 2.0 kg/MT SCFP (SCFP group). Fecal samples were collected at day 0, 60 days of gestation, and farrowing for enumeration of Escherichia coli and isolation of Salmonella. Isolates were assessed for AMR and antimicrobial resistance genes (ARGs) using disk diffusion and polymerase chain reaction (PCR) methods. The results showed that E. coli counts (log10 CFU/g) were significantly reduced in the SCFP group compared with the CON group (p=0.03). The SCFP decreased the frequency of E. coli isolates with resistance to chloramphenicol and tetracycline (p<0.05), while BG-50 reduced resistance to chloramphenicol, doxycycline, erythromycin, trimethoprim-sulfamethoxazole, and tetracycline (p<0.05). The mean inhibition zone for most antimicrobials against E. coli increased with gestation (p<0.05). Overall, dietary SCFP supplementation alleviated E. coli counts and the frequency of Salmonella. In addition, the effect of dietary SCFP and BG-50 supplementation on the level of AMR in E. coli and Salmonella was observed.
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.
KK-Ay mice spontaneously develop obesity-associated type 2 diabetes. We compared the gut microbiota structure between 4-week-old (4W) and 11-week-old (11W) female KK-Ay mice. Amplicon sequencing of the 16S rRNA gene revealed a different β-diversity of gut microbiota between the 4W and 11W mice and a higher Bacillota/Bacteroidota ratio in the 11W mice than in the 4W mice. The relative abundance of genus Blautia positively correlated with body weight, gonadal white adipose tissue weight, inguinal adipose tissue weight, and urine glucose level. We propose that genus Blautia represents the most consistent microbial signature associated with metabolic deterioration in KK-Ay mice.
The aim of this study was to isolate and select biologically active local strains of Bacillus subtilis from the rumen contents of Mongolian pasture-grazing sheep and to evaluate their potential probiotic characteristics for application in ruminant health. Samples of rumen contents were collected from 15 sheep grazing in Bayan-Unjuul soum, Tuv province, Mongolia, and 62 primary isolates were obtained through culturing on selective solid and liquid media. The primary isolates were evaluated based on their morphological, biochemical, and physiological characteristics, enzymatic activities (amylase, protease, catalase), ability to produce acetoin, milk-coagulating ability, antagonistic activity, antibiotic susceptibility, and growth conditions. Phenotypic and genotypic analyses confirmed that these isolates belonged to the Bacillus subtilis species. Among the isolates, Bacillus subtilis c27c demonstrated superior probiotic properties for animal use, including strong enzymatic activity, antagonistic effects, and favorable antibiotic resistance profiles. The results show that the Bacillus subtilis c2-7c strain isolated and selected in this study has the most active probiotic characteristics, making it a promising candidate for applications in animal husbandry and veterinary medicine.
The human gut microbiota plays a crucial role in overall health, impacting various diseases, including autism spectrum disorder (ASD). This study explores the relationship between gut microbiota changes and ASD by analyzing microbial compositions and abundances of 692 gut microbiota samples using public 16S rRNA sequencing datasets. Data preprocessing included normalization and redundancy reduction, retaining 367 microbial features. A machine learning model was then developed to predict ASD, utilizing feature-selected random forest algorithms that showed superior performance in both training and independent test sets. Identified microbial features with high correlation to ASD included Clostridiales bacterium VE202-08, Solobacterium moorei gene, and other features. The findings suggest that modulating the gut microbiota composition could mitigate ASD risk or alleviate symptoms. These insights pave the way for novel ASD diagnostic methods through microbiota analysis, although further research is required to validate these possibilities. This study offers a new perspective on the etiology and progression of ASD and proposes potential predictive tools for its diagnosis.
Breath hydrogen concentration measurement is a valuable tool for assessing the intestinal environment; however, few epidemiological studies have investigated the relationship between exhaled hydrogen and gut microbiota in healthy subjects. This study aimed to epidemiologically elucidate the relationships between exhaled hydrogen, gut microbiota, and nutrient intake in healthy residents of the Iwaki area of Hirosaki City, Aomori Prefecture, including those who exhaled methane. We categorized participants into low- and high-exhaled hydrogen groups based on the median exhaled hydrogen concentration of 6.13 ppm and matched background factors by propensity score matching for age, body mass index, and defecation habits. In the high exhaled hydrogen group, intestinal butyrate-producing bacteria such as Faecalibacterium, Anaerostipes, and Roseburia increased, and Bacteroides strains decreased. In addition, the group with high exhaled hydrogen concentrations had a high dietary fiber intake, and positive correlation was observed between dietary fiber intake and butyrate-producing bacteria. This trend was particularly pronounced for soluble dietary fiber. The exhaled methane concentration decreased in the higher exhaled hydrogen concentration group, and intestinal Methanobrevibacter was positively correlated with the exhaled methane concentration, although in extremely small amounts. No significant relationship was found between each nutrient intake and Methanobrevibacter strain. Measurement of the exhaled hydrogen concentration is useful for assessing the intestinal environment associated with nutritional intake. However, methane gas production was not changed by dietary intake, suggesting that intervention with prebiotics may be necessary.
Soybean-related pollen-food allergy syndrome (PFAS) is a food allergy triggered by birch or alder pollen-specific immunoglobulin E, which cross-reacts with soybean proteins homologous to pollen proteins. Although soybean-related PFAS generally causes mild symptoms, some individuals develop severe symptoms, including anaphylaxis, especially after consuming soymilk. This study attempted to reduce the risk of allergy by fermenting soymilk with Lacticaseibacillus paracasei strain Shirota (YIT 9029, LcS). The levels of Gly m 4, which causes soybean-related PFAS, in soymilk and fermented soymilk were analyzed using enzyme-linked immunosorbent assay (ELISA) and western blotting. Reactions to soymilk and fermented soymilk were evaluated in patients with soybean-related PFAS using skin prick tests to compare allergic risks. Gly m 4 levels in the soluble fraction of fermented soymilk were significantly lower than those in soymilk. The reduction in Gly m 4 levels was associated with alterations in the fermentation process, including pH reduction. Additionally, compared with soymilk, fermented soymilk reduced skin prick test reactions in patients with soybean-related PFAS. In conclusion, fermenting soymilk with LcS reduced soluble Gly m 4 levels and alleviated allergic reactions associated with soybean-related PFAS. This study demonstrates the potential of fermented soymilk as a safe and effective alternative for individuals with soybean-related PFAS.
