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PubMed2026

Evaluation of Indigenous Bacillus Strains from Asian Fermented Foods for Probiotic Properties.

Bacillus species hold particular importance due to their versatile enzymatic repertoire and ability to synthesize diverse bioactive metabolites. In this study, two fermented food-derived strains, Bacillus siamensis BB3 (from douchi) and Bacillus velezensis TMA10 (from tapai) were evaluated for their probiotic, anti-microbial and functional potentials. Both strains exhibited desirable probiotic characteristics, including desirable tolerance to simulated gastric and intestinal conditions, with BB3 showing greater acid tolerance than TMA10. Safety assessments confirmed the absence of hemolytic activity, virulence factors and antibiotic resistance genes. Whole genome sequencing showed that the strains harbored genomic sequences for a wide range of metabolites, including non-ribosomal peptides and polyketides. Ethyl acetate (EtOAc) extracts from both strains demonstrated broad-spectrum anti-microbial activity against several indicator microorganisms, including Listeria grayi, Bacillus cereus, Serratia marcescens, Escherichia coli, and Pseudomonas aeruginosa, with TMA10 additionally inhibiting MRSA and Streptococcus mutans. Liquid Chromatography-Mass Spectrometry profiling identified key anti-microbial compounds, namely surfactins, macrolactins, bacillaene from BB3 and TMA10. In addition, difficidins were also detected from TMA10. Genomic analysis further indicated diverse carbohydrate utilization capacities; both strains encoded pathways for sucrose, raffinose-family oligosaccharides and lactose metabolism, while TMA10 possessed pathways for trehalose, glucomannan and arabinoxylan degradation. Both strains also showed anti-oxidant activity, with enhanced effects observed in their cell-free supernatants and heat-killed preparations. Overall, these findings highlight BB3 and TMA10 as promising candidates for the development of fermented food-derived Bacillus probiotics and functional cultures with anti-microbial, anti-oxidant, and broad carbohydrate-utilization capabilities.

Bacillus↗
PubMed2026

Probiotic supplementation increases fecal TLR4 agonists without improving disease activity in juvenile idiopathic arthritis: a randomized placebo-controlled trial.

Gut dysbiosis has been implicated in the pathogenesis of juvenile idiopathic arthritis (JIA), suggesting that microbiota-targeted interventions may influence immune signalling during early immune development. We conducted the PERMAJI multicentre randomized, double-blind, placebo-controlled trial to evaluate the effects of probiotic supplementation (VSL#3) on host-microbiota immune interactions and disease activity in children with oligoarticular or RF-negative polyarticular JIA. Participants were randomly assigned (1:1) to receive VSL#3 or placebo for 3 months in addition to standard therapy. Stool and serum samples collected at baseline and month 3 were used to assess gut microbiota composition, fecal innate immune agonists, intestinal permeability, and systemic cytokines. The primary clinical endpoint was the proportion achieving an ACR Pedi 30 response at 3 months. Forty-four children were enrolled between September 2017 and July 2022. Clinical responses did not differ between groups (ACR Pedi 30: 47% with VSL#3 vs 63% with placebo; p = 0.33), and conservative worst-case assumptions for missing data suggested lower response rates with VSL#3 (36% vs 68%; p = 0.03). Probiotic supplementation significantly increased fecal Toll-like receptor 4 (TLR4) agonist activity, whereas gut microbiota diversity, intestinal permeability, and systemic cytokine levels remained unchanged. These findings indicate that probiotic supplementation can modify microbial innate immune signalling without detectable changes in microbial community diversity and may increase exposure to pro-inflammatory microbial stimuli in early-life autoimmune disease. The results highlight the complexity of host-microbiota immune interactions and underscore the need for careful evaluation of microbiome-targeted therapies in paediatric autoimmune disorders.

Humans↗
PubMed2024

Engineering a probiotic Bacillus subtilis for acetaldehyde removal: A hag locus integration to robustly express acetaldehyde dehydrogenase.

We have addressed critical challenges in probiotic design to develop a commercially viable bacterial strain capable of removing the intestinal toxin, acetaldehyde. In this study, we report the engineering of the hag locus, a σD-dependent flagellin expression site, as a stable location for robust enzyme production. We demonstrate constitutive gene expression in relevant conditions driven by the endogenous hag promoter, following a deletion of the gene encoding a post-translational regulator of σD, FlgM, and a point mutation to abrogate the binding of the translational inhibitor CsrA. Reporter constructs demonstrate activity at the hag locus after germination, with a steady increase in heterologous expression throughout outgrowth and vegetative growth. To evaluate the chassis as a spore-based probiotic solution, we identified the physiologically relevant ethanol metabolic pathway and the subsequent accumulation of gut-derived acetaldehyde following alcohol consumption. We integrated a Cupriavidus necator aldehyde dehydrogenase gene (acoD) into the hag locus under the control of the flagellin promoter and observed a rapid reduction in acetaldehyde levels in gut-simulated conditions post-germination. This work demonstrates a promising approach for the development of genetically engineered spore-based probiotics.

Acetaldehyde↗
PubMed2026

Metabolic Engineering of Probiotic Saccharomyces boulardii Enables Intestinal 3-Hydroxybutyrate Delivery and Alters Short-Chain Fatty Acid Profiles in Mice.

3-Hydroxybutyric acid (3-HB) is a bioactive ketone body involved in the regulation of intestinal inflammation and metabolic homeostasis. Although engineered bacterial probiotics have been developed for localized 3-HB delivery, their susceptibility to antibacterial antibiotics may limit their use during concurrent antibiotic treatment. The probiotic yeast Saccharomyces boulardii offers an alternative host for intestinal 3-HB delivery because of its compatibility with antibacterial antibiotics and the availability of well-established genetic engineering tools. Here, we engineered S. boulardii for 3-HB production using Cas9-mediated genome editing. A heterologous 3-HB biosynthetic pathway was introduced into S. boulardii MYA-797, and endogenous acetyl-CoA and ethanol metabolism was subsequently rewired by overexpressing ACS1, deleting ADH1, and overexpressing ADH7. The optimized strain, SbDY02, produced 1.7 g/L 3-HB under microaerobic conditions. Oral administration of SbDY02 to C57BL/6J mice increased fecal 3-HB and short-chain fatty acid (SCFA) concentrations by 1.89-fold and 1.68-fold, respectively, compared with mice receiving the parental strain. Repeated administration also increased fecal acetate and circulating total SCFAs, butyrate, and propionate. In human colonic epithelial cells, purified 3-HB attenuated lipopolysaccharide-induced p38 MAPK phosphorylation, supporting its direct activity toward inflammation-associated epithelial signaling. To our knowledge, this study provides the first demonstration of a 3-HB-producing probiotic yeast and links central metabolic engineering of S. boulardii with increased 3-HB availability, altered SCFA profiles, and a host-relevant epithelial response.

3-hydroxybutyrate↗
PubMed2026

Assessing the Safety and Probiotic Potential of Bifidobacterium longum subsp. infantis BI45: A Comprehensive Study from Genomic Analysis to Randomized Controlled Clinical Trial in Healthy Adults.

While probiotics are increasingly consumed for health benefits, comprehensive safety assessments, particularly for novel strains, are imperative. This study aimed to conduct a holistic safety and efficacy assessment of Bifidobacterium longum subsp. infantis (B. infantis) BI45, spanning genomic analysis, in vitro tests, In vivo toxicity test, and a clinical trial. The safety of B. infantis BI45 was evaluated through: (1) whole-genome sequencing for antibiotic resistance and virulence genes; (2) in vitro phenotyping (hemolysis, cytotoxicity, gastrointestinal tolerance and antibiotic susceptibility); (3) an acute oral toxicity study in mice; and (4) a randomized, double-blind, placebo-controlled clinical trial. Forty-eight healthy adults were recruited and randomly assigned to receive either B. infantis BI45 or a placebo (n&#x2009;=&#x2009;24/group) for 8 weeks. Hematological, biochemical, immunological, and gut microbiota parameters were assessed. Genomic analysis identified no transferable antibiotic resistance or virulence genes. In vitro assays confirmed the absence of hemolytic and cytotoxic activity, alongside high gastrointestinal tolerance. Antibiotic susceptibility testing showed that B. infantis BI45 is sensitive to a range of antibiotics. No adverse effects were observed in the murine toxicity study at 2&#x2009;&#xd7;&#x2009;10&#xb9;&#x2070; CFU/kg. Importantly, the clinical intervention revealed no adverse events or significant alterations in hematological, hepatic, or renal function markers in the B. infantis BI45 group, demonstrating an excellent safety profile. Furthermore, B. infantis BI45 supplementation significantly increased serum levels of immunomodulatory markers Immunoglobulin A (IgA) and antimicrobial peptide LL-37 compared to the placebo (p&#x2009;<&#x2009;0.05) and modulated the gut microbiota by enriching beneficial short-chain fatty acid producers. The multi-tiered evidence demonstrates that B. infantis BI45 is a safe probiotic strain that does not induce adverse reactions in healthy adults. Its consumption positively modulates host immunity and the gut microbiota.Trial Registration Number: NCT06863415 (ClinicalTrials.gov).

Adult↗
PubMed2024

Gut microbial genomes with paired isolates from China illustrate probiotic and cardiometabolic effects.

The gut microbiome displays genetic differences among populations, and characterization of the genomic landscape of the gut microbiome in China remains limited. Here, we present the Chinese Gut Microbial Reference (CGMR) set, comprising 101,060 high-quality metagenomic assembled genomes (MAGs) of 3,707 nonredundant species from 3,234 fecal samples across primarily rural Chinese locations, 1,376 live isolates mainly from lactic acid bacteria, and 987 novel species relative to worldwide databases. We observed region-specific coexisting MAGs and MAGs with probiotic and cardiometabolic functionalities. Preliminary mouse experiments suggest a probiotic effect of two Faecalibacillus intestinalis isolates in alleviating constipation, cardiometabolic influences of three Bacteroides fragilis_A isolates in obesity, and isolates from the genera Parabacteroides and Lactobacillus in host lipid metabolism. Our study expands the current microbial genomes with paired isolates and demonstrates potential host effects, contributing to the mechanistic understanding of host-microbe interactions.

Probiotics↗
PubMed2026

Harnessing Probiotic LAB and Bacteriocins for Clean-Label Food Processing and Biopreservation: Omics, Molecular Innovations and Industrial Applications.

The persistence of microbial agents in foods, especially spore forming bacteria is one of the most significant challenges to food preservation and safety, undermining product quality, shelf life, and consumer health. The use of traditional control methods, including thermal processing and chemical preservatives, are increasingly limited by consumer demands for minimally processed foods, and the emergence of resistant microbial strains. Advances have been made in the use of probiotics like lactic acid bacteria (LAB) and their biometabolites like bacteriocins in food processing and preservation, particularly to control biofilm and endospore forming pathogens including Bacillus sp., Listeria sp., Staphylococcus sp., Clostridium sp., E. coli etc. in foods and food processing plants/surfaces. Given the ability of these organisms to cause foodborne illness and form resilient biofilms in the food processing ecosystem and their resistance to the conventional method of their elimination, the antimicrobial peptides (bacteriocins) are gaining increasing prominence as useful alternatives to synthetic antimicrobials in enhancing food safety and combating the threats of these pathogens. This review addresses current information on the inhibition of persistent microbial spoilage contaminants, biofilm-forming pathogens, and spore formers of interest to the food industry using LAB and their bacteriocins. Current developments in isolation, characterization, and mode of action of bacteriocins are explored, including synergistic activity with other preservative hurdle techniques such as encapsulation, and nanobiotechnology. Importantly, there is a focus on the utilization of molecular and omics-based approaches to enable a better understanding of bacteriocin biosynthesis, gene regulation, host-microbe interactions and gut microbiome regulation potential of probiotic LABs, permitting the rational development of targeted and strain-specific interventions. Developments in the incorporation of bacteriocin-producing LAB into functional starter cultures and bio-protective products, and challenges in stability, regulatory approval, and scalability for industrial use, are also discussed in the paper. Despite their considerable potential, broader translation remains constrained by regulatory requirements, production and formulation costs, variable efficacy in complex food matrices, and the limited validation of many candidate bacteriocins beyond laboratory and model-food systems. Collectively, these advances position LAB and their bacteriocins at the leading edge of developing sustainable, clean-label, and efficacious functional foods and food preservation systems. Their functionality can be expanded by integrating genomics, synthetic biology, and predictive modeling for the maximization of their biopreservative potential in diverse food matrices and in gut microbiota modulation.

Bioactive Peptides↗
PubMed2026

A combinatorial construct library enables an expanded expression range of secreted therapeutic proteins by probiotic yeast.

Orally administered engineered probiotics, including Saccharomyces cerevisiae var. boulardii (Sb), are of emerging interest as protein therapeutic delivery platforms to treat gastrointestinal diseases. Tools to readily optimize protein output are required to optimize the therapeutic index of Sb-produced therapies. In this study, a 125-plex Sb secretion construct library was developed consisting of all possible combinations of five promoters, five secretion signals, and five terminators, which enabled a greater than 1800-fold range in Sb expression of a Gaussia luciferase (GLuc) reporter. Secretion signal and promoter identities had significant effects on secretion output. This library further enabled a 28-fold improvement of binding activity of Sb-secreted haPD-1, an established anti-tumor immunotherapeutic, and improved haPD-1 detection in mouse stool samples following oral gavage of Sb_haPD-1. Sb secretion trends of both GLuc and haPD-1 in vitro mirrored payload expression in vivo. This protein secretion library toolkit will serve as a valuable resource to rapidly optimize protein therapeutic output from engineered Sb.

Saccharomyces boulardii↗
PubMed2026

Draft genome sequence of Bacillus thuringiensis GIFSPR-111, a putative probiotic isolated from shrimp farm soil in Bangladesh.

Bacillus thuringiensis GIFSPR-111, isolated from shrimp farm soil in Shamnagar, Bangladesh, inhibits Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease in shrimp and exhibits probiotic potential. The 5,608,442 bp draft genome contains multiple biosynthetic gene clusters, with potential to synthesize diverse bioactive metabolites.

Bacillus thuringiensis↗
PubMed2026

Complete genome sequence of Bacillus subtilis strain S-LA1, a potential plant probiotic endophyte from the medicinal plant Leucas aspera.

Bacillus subtilis strain S-LA1 is an endophytic bacterium isolated from Leucas aspera roots that harbors a 4.2 Mbp genome predicted to encode several traits for nutrient acquisition, plant growth promotion, and plant probiotic efficacy. Genomic characterization underscores its potential as a microbial resource supporting sustainable agriculture and crop disease management strategies.

Bacillus↗
PubMed2026

BLIS study: a feasibility randomised controlled trial assessing compliance, acceptability and colonisation with different dosing regimens of the probiotic supplement Streptococcus salivarius K12 (Bactoblis) in adults in England.

OBJECTIVES: Streptococcus salivarius K12 (SsK12) is a bacterium used as a probiotic with some evidence for preventing acute sore throat/tonsillitis, but the optimal dosing strategy is unclear. This study aimed to evaluate two dosing regimens of SsK12 to establish (a) the prevalence and duration of colonisation with SsK12 and (b) the acceptability and feasibility of these regimens. DESIGN: A randomised, non-blinded feasibility trial. SETTING: Primary care in the south of England. PARTICIPANTS: Adults with &#x2265;2 episodes of sore throat within the 3 years before recruitment. INTERVENTIONS: Participants were randomised to take SsK12 lozenges once weekly or daily over 14 days. MAIN OUTCOME MEASURES: The primary outcome was the prevalence and duration of colonisation with SsK12. This was determined by real-time PCR conducted on self-taken whole-mouth swabs provided by participants at baseline and on days 2, 7, 14, 21 and 35. Secondary outcomes were the compliance and acceptability of the two SsK12 dosing regimens, based on questionnaire data. RESULTS: 53 participants were recruited (26 randomised to weekly and 27 to daily dosing) between 26 April and 5 December 2023. All swabs were returned by 65.4% (17/26) and 70.4% (19/27) of the weekly and daily groups respectively. Swabs at all timepoints from participants with PCR positives observed at baseline (two and four from the weekly and daily group, respectively) were excluded. Therefore, swabs from 23 participants in the weekly and 22 in the daily group did not have evidence of baseline colonisation and were included in the microbiology analysis. SsK12 colonisation was found in 59.1% (13/22, 95% CI 38.6% to 79.6%) of samples in both groups on day 2; in 13.6% (3/22, 95%&#x2009;CI 0.0% to 28.0%) and 85.7% (18/21, 95%&#x2009;CI 70.8% to 100.0%) of the weekly and daily groups, respectively, on day 7; in 19.1% (4/21, 95%&#x2009;CI 2.3% to 35.8%) and 59.1% (13/22, 95%&#x2009;CI 38.6% to 79.6%), respectively, on day 14; and in only one participant (weekly group) on days 21 and 35. The dosing regimen was reported as easy to follow by 85.0% (17/20, 95%&#x2009;CI 69.4% to 100.0%) of the weekly and 96.2% (25/26, 95%&#x2009;CI 88.8% to 100.0%) of the daily dosing groups. CONCLUSIONS: SsK12 colonisation was more prevalent with daily dosing; however, colonisation was not maintained when dosing stopped. Both dosing regimens were acceptable to participants. These findings support the use of daily dosing in a future trial to evaluate the efficacy of SsK12 in preventing recurrence of sore throat/tonsillitis. TRIAL REGISTRATION NUMBER: NCT04297878.

Humans↗
PubMed1978

Comparison of stationary phases in reversed-phase TLC for correlation between structure and biological response of probiotics.

A series of C16,C18 and C20 fatty acids and their ethyl esters and alcohols were investigated as possible stationary phases in reversed-phase TLC for the correlation between structure and biological response (antistaphylococcal activity). Ten probiotics (omego-amino acids and their L-histidine dipeptides) were used as the biologically active compounds. The mobile phase was 70% acetone in water. The best correlations were obtained with hexadecanoic acid (palmitic acid) or cis9,cis-12,cis-15-octadecatrienoic acid, 1-hexadecanol or cis-9-octadeconol, and ethyl hexadecanoate for the fatty acids, their alcohols, and their ethyl esters, respectively. Among all compounds, the following relation was obtained: fatty acids = alcohols greater than ethyl esters greater than white paraffin oil.

Anti-Bacterial Agents↗
PubMed2026

A black soybean yoghurt fermented with a reusable probiotic cellulose gel: beneficial effects and metabolic characteristics.

The demand for plant-based yoghurts is continuously increasing. However, achieving stable physicochemical properties and acceptable flavor of the plant-based yoghurts remains challenging. In our previous work, we encapsulated Lactiplantibacillus plantarum (L. plantarum) LCC-605 biofilm into bacterial cellulose (BC), obtaining a LP605@BC gel. LP605@BC gel exhibited excellent harsh-environment resistance abilities and storage stability, and is very suitable as a starter culture. In this work, we used LP605@BC as a starter culture to prepare the fermented plant-based yoghurt (e.g., black soybean yoghurt, BSY-LP605@BC). After fermentation, the inverted nonflowing yoghurt was formed due to the strong interaction between protein and exopolysaccharide (EPS) produced by LCC-605 during fermentation. In addition, the water holding capacity (67.2%) of BSY-LP605@BC was also greatly improved. The viable bacterial counts in BSY-LP605@BC reached 11.2 log CFU/mL after 21&#xa0;days of storage. BSY-LP605@BC showed increased antioxidant, cholesterol-lowering abilities, and hypoglycemic potential compared with the unfermented black soybean milk. Interestingly, LP605@BC could be reused at least 5 times, demonstrating excellent sustainability. Significant metabolomic differences between BSY-LP605@BC and the black soybean milk were observed via untargeted metabolomic analysis, further proving the beneficial effects of BSY-LP605@BC. Overall, our work developed an effective reusable starter culture for preparing the plant-based yoghurt in a sustainable manner, providing a new design direction and form of starter culture.

Yogurt↗
PubMed2026

Probiotic potential of Parabacteroides johnsonii in mitigating age-related ovarian functional decline.

The gut microbiota is increasingly recognized as a regulator of reproductive health, yet its role in ovarian aging remains unclear. Here, we combine Mendelian randomization (MR) analysis with experimental validation to investigate the causal relationship between gut microbiota and ovarian aging. MR analysis identifies four microbial taxa significantly associated with age at natural menopause. In mouse models, germ-free mice exhibit accelerated ovarian functional decline, including reduced ovarian reserve and impaired folliculogenesis. Fecal microbiota transplantation (FMT) from young donors alleviates ovarian aging phenotypes, whereas FMT from aged donors exacerbates functional decline. Metagenomic analysis reveals species-level differences between young and ovarian-aging mice, with Parabacteroides johnsonii (P. johnsonii) enriched in young mice. Administration of P. johnsonii to middle-aged mice improves ovarian reserve, reduces follicular atresia, enhances granulosa cell proliferation, and decreases systemic inflammation. These findings highlight a causal role of the gut microbiota in ovarian aging and support microbiota-targeted interventions as a potential strategy to preserve ovarian function.

Female↗
PubMed2026

A geroprotective probiotic and its functional metabolite counteract inflammaging to extend healthspan.

The gut microbiome profoundly influences host aging, yet the specific microbes and mechanisms governing divergent aging trajectories remain elusive. In this study, we delineated enterotype-specific gut microbial remodeling during aging and developed a microbiome-based aging clock (MicroAge) to track biological aging trajectories. We identified Bifidobacterium pseudocatenulatum (B. pseudocatenulatum) as a candidate geroprotective species consistently depleted during aging across both sexes and multiple Chinese cohorts. In naturally aged mice, oral B. pseudocatenulatum monotherapy rescued intestinal homeostasis, mitigated multiorgan inflammaging, enhanced cognitive-motor performance and extended healthspan. Mechanistically, we characterized 5-aminovaleric acid betaine (5-AVAB) as a key B. pseudocatenulatum-derived metabolite whose levels decline physiologically in aging humans. 5-AVAB supplementation partially recapitulated a broad spectrum of the systemic benefits observed with B. pseudocatenulatum treatment, including improved cognitive and motor function and suppressed multiorgan inflammaging. Our findings identify the B. pseudocatenulatum-5-AVAB axis as a promising target for microbiome-based interventions to promote healthy aging.

Animals↗
PubMed2026

Draft genome sequence of Pantoea sp. strain S-LA4, a potential plant probiotic endophyte isolated from the medicinal plant Leucas aspera.

Pantoea sp. strain S-LA4 is an endophytic bacterium isolated from the leaf tissue of the medicinal plant Leucas aspera. The 4.93-Mbp draft genome of S-LA4 is predictive to encode several enzymes and secondary metabolites of plant growth promotion and bio-pesticidal activity, underscoring its potential for sustainable disease management in agriculture.

Oxford Nanopore sequencing↗