This study explored the technological and probiotic potential of lactic acid bacteria (LAB) isolated from traditional Maltese Ġbejna and raw sheep milk. A total of 109 LAB isolates were isolated from 33 cheeses and 10 raw sheep milk samples and screened for acidification, coagulation, proteolytic and lipolytic activities, as well as tolerance to low pH, simulated gastric and pancreatic juices, autoaggregation, and surface hydrophobicity. LAB belonged to nine genera, predominated by Lactiplantibacillus , Enterococcus , and Lacticaseibacillus . Twenty-six isolates exhibited strong technological performance and survived simulated gastrointestinal conditions (50–88%). Several strains showed antimicrobial and antifungal activity against foodborne pathogens and spoilage fungi. Notably, selected L. plantarum and L. paracasei isolates combined desirable dairy functionality with probiotic-associated traits. These findings highlight the technological relevance of indigenous LAB in Ġbejna and support their potential use as adjunct cultures to enhance quality and safety of raw sheep milk cheeses. • 16S rRNA PCR identified 9 genera and 19 species of LAB. • 54 LAB isolates demonstrated strong traits essential for cheese production. • 26 LAB isolates demonstrated robust probiotic potential. • L. plantarum and L. paracasei exhibited superior technological and probiotic trait.

Pediococcus pentosaceus DF29-derived extracellular vesicles ameliorate EPEC-induced intestinal injury via M2 macrophage polarization.

Development and characterization of seriguela (Spondias purpurea) water kefir: metagenomic insights and functional potential of a spray-dried probiotic powder.

Phenotypic and whole-genome characterization of a novel Lysinibacillus fusiformis PWR01 isolated from rubber latex with multifunctional probiotic and antimicrobial potential.

Bacteriocin-producing Lactococcus lactis subsp. lactis A7 enhances health status, immunity and gut microbiota in Nile tilapia (Oreochromis niloticus)

Aquaculture farming often faces diseases, causing economic loss. Probiotics offers an eco-friendly alternatives antibiotic, improving fish health, immunity, and resistance, without adverse side effects. This study evaluated the probiotic potential of two enzyme producing probiotic strains, DAS-SCF02 and RKDAS1, isolated from the gut of Channa striata . Molecular identification using 16S rRNA gene sequencing confirmed their taxonomy and phylogenetic relationships. Juvenile Oreochromis niloticus were fed diets supplemented with 1×10 8 CFU/g of these isolates, either individually or in combination, for 60 days. Growth performance parameters including final average body weight (FABW), weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR) were significantly improved ( P < 0.05) in probiotic-treated groups, with the dual-strain diet (Diet 4) showing the most pronounced effects. Digestive enzyme activities (amylase, protease, and cellulase) were also markedly elevated in treated fish. Furthermore, antioxidant biomarkers such as total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were significantly increased, while malondialdehyde (MDA) levels were reduced, indicating enhanced oxidative stress resistance. These findings suggest that DAS-SCF02 and RKDAS1 are promising probiotic candidates for improving growth performance, digestive efficiency, and antioxidant defense in O. niloticus , supporting their application in sustainable aquaculture.

Probiotics are increasingly recognized for their role in enhancing fish health, strengthening immune responses, and mitigating stress, particularly as rising inland water salinity continues to compromise fish health and reduce aquaculture productivity. To investigate the potential of probiotics as a countermeasure against the health and productivity challenges posed by brackish water, a 9-month feeding trial was conducted to evaluate the hematological and antioxidant responses of Labeo rohita (20.18 ± 0.22g; Mean ± SD) reared in inland brackish water earthen ponds (mean area: 1.0 acre; depth: 1.5m; salinity: 6.1 ppt) and fed diets with or without a multi-species probiotic (MSP) containing Bacillus subtilis (1.9 × 10¹⁰ "CFU/g"), Bacillus licheniformis (2 × 10¹⁰ "CFU/g"), and Clostridium butyricum (2.2 × 10⁸ "CFU/g"). A total of 900 fish were randomly distributed into three treatment groups (P I, P II, and P III), each with three replicate ponds. Fish in group pond P I (control) were fed a basal diet without probiotic supplementation, whereas fish in groups P II and P III received the same basal diet supplemented with MSP at 0.5% and 1%, respectively. A significant improvement (p < 0.05) in blood biomarkers was observed in the 1% MSP fed fish, even when reared at inland brackish water. Erythrocyte count, hemoglobin content, hematocrit count, total serum protein, albumin, and globulin were significantly higher (p < 0.05) in the 1% MSP fed fish compared to the non-probiotic-fed fish. A significant effect of rearing time on oxidative stress markers (ROS and TBARS) which increased over time and significant decline in antioxidant enzymes (SOD, CAT, POD, and GSH) were observed in brackish water ponds without probiotics (p < 0.05), however, dietary MSP supplementation significantly mitigated oxidative damage and enhanced antioxidant defenses in liver, kidney, and gills (p < 0.05). Frequency of erythrocytic alterations was also significantly reduced in MSP-supplemented groups compared to non-probiotic groups (p < 0.05). Marked recovery in histoarchitecture of liver, kidneys, and gills in MSP-supplemented groups compared to non-probiotics groups. In conclusion, dietary MSP supplementation effectively improved blood health and antioxidant capacity in L. rohita in inland brackish-water ponds, suggesting its practical potential to enhance health performance under brackish-water farming conditions.

Fermented foods: lessons learned from metagenomics.

The search for effective, sustainable alternatives to antibiotics in aquaculture has intensified interest in probiotics, yet the comparative efficacy of host-derived versus commercial probiotic strains remains poorly understood. This study evaluated the probiotic potential of two indigenous gut isolates including a putatively non-virulent strain of Pseudomonas, Pseudomonas aeruginosa PA4 and Bacillus sp.BA4P, previously screened for safety, against commercial Bacillus- and Pseudomonas-based formulations in red tilapia (Oreochromis sp.) over a 60-day feeding trial. Fish fed the combined indigenous probiotic (PA4 + BA4P) exhibited superior growth performance, feed efficiency, and digestive enzyme activities (protease, lipase, amylase) compared to control and commercial probiotic groups. Enhanced intestinal villus height, goblet cell density, and intraepithelial lymphocyte counts indicated improved gut health and mucosal immunity. Probiotic supplementation also modulated gut microbiota by increasing beneficial bacteria (Bacillus, Lactobacillus, Pseudomonas) and suppressing potential pathogens (Streptococcus, Aeromonas, Vibrio). Systemic immunity was strengthened, as evidenced by elevated leukocyte counts, serum IgM levels, and reduced hepatic stress markers. Following co-challenge with Aeromonas hydrophila and Streptococcus iniae, the PA4 + BA4P group showed the highest survival rate (75%), significantly outperforming the control (44% survival). These findings demonstrate that host-adapted, multi-strain probiotics confer more benefits in growth, digestive capacity, gut integrity, and disease resistance, offering a promising sustainable strategy for tilapia aquaculture.

Plant-based fermented foods are increasingly recognized as an underexplored niche for autochthonous and beneficial lactic acid bacteria (LAB) with multiple biotechnological and functional potential in health and agrifood systems. We isolated LAB from plant-based fermented foods and assessed their antimicrobial, functional, and probiotic potential using agar-well diffusion technique, in vitro colonization resistance and gastric-environment survivability assays, and 16S rRNA sequencing. Thirteen autochthonous LAB identified as Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Leuconostoc mesenteroides, Pediococcus acidilactici, Pediococcus pentosaceus, Weissella confusa, Weissella paramesenteroides, and Weissella cibaria demonstrated inhibitory activity against ≥ 3 pathogens, including Methicillin-Resistant Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, Enterococcus faecium, Enterococcus faecalis, and Pasteurella multocida. The selected LAB strains harbor at least 1 bacteriocin gene, including Plantaricin EF, Plantaricin K, Plantaricin J, Nisin, and Pediocin, and also competitively excluded most pathogens in co-culture experiments, with viable counts ranging from 3.0 to 6.0 log CFUmL-1. Interestingly, the LAB strains showed varying degrees of survivability to simulated gastric conditions, as well as auto- and coaggregation, hydrophobicity, hydroxyl scavenging, and exopolysaccharide production, and were universally non-hemolytic. Our study demonstrates plant-based fermented foods as notable sources of beneficial LAB with multifunctional biotechnological potential for application in health and agrifood systems.

Probiotic supplements are marketed for diverse health benefits, yet species inclusion often lacks functional rationale. Our survey of 352 over-the-counter probiotic products available in the USA revealed 36 unique microbial species. However, there is no clear link between species inclusion and the intended health benefit. Here, to address this gap, we developed HaPaPro, a collection of 1,012 genome-scale metabolic models spanning pathogenic, probiotic and host-associated bacteria, constructed from publicly available genome sequences. Flux balance analysis revealed that probiotic species fail to capture the metabolic diversity of host-associated microbes. Focusing on vaginal health, we computationally identified vaginal microbes with metabolic profiles overlapping Gardnerella vaginalis. In vitro spent media assays using 11 vaginal isolates showed variable inhibition of G. vaginalis, primarily driven by D-lactic acid production, which was also produced by non-Lactobacillus species. These findings highlight the need for function-based probiotic design and demonstrate a scalable framework integrating metabolic modelling with experimental validation.

Fermented bamboo shoot (FBS) products are widely consumed traditional foods across the Northeast region (NER) of India, yet their microbiome structure, functional capacity, biosynthetic potential, and safety attributes remain insufficiently explored. Here, comparative shotgun metagenomics of ten traditional FBS products from six NER states was used to address these gaps integrating previously generated metagenomic data from Tripura with newly generated datasets from Manipur, Meghalaya, Arunachal Pradesh, Nagaland, and Sikkim thereby bringing the total number of samples to 24. Taxonomic profiling revealed a predominance of lactic acid bacteria, primarily members of Lactiplantibacillus, Levilactobacillus, Lactobacillus, Lactococcus, and Pediococcus, with pronounced product- and region-specific community signatures. Functional annotation demonstrated predominance of genes involved in carbohydrate metabolism, stress response, quorum sensing, ABC transporters, vitamin biosynthesis, and energy metabolism, supporting strong probiotic-associated functional potential across FBS types. AntiSMASH analysis enabled the identification of diverse biosynthetic gene clusters (BGCs) responsible for the production of various secondary metabolites, including bacteriocins, non-ribosomal peptides, terpenes, and siderophores, with higher biosynthetic diversity observed in Mesu (Sikkim), Tuaithar (Manipur), Lung-Seij (Meghalaya), and Bastenga (Nagaland). Antimicrobial resistance (AMR) profiling revealed a generally low resistome burden, dominated by intrinsic resistance determinants, with FBS Sikkim and Tripura exhibiting the lowest AMR prevalence among all products. High-quality metagenome-assembled genomes affiliated with Lactiplantibacillus plantarum, Lactobacillus acetotolerans, and Pediococcus pentosaceus exhibited conserved probiotic traits, carbohydrate-active enzymes, biosynthetic pathways, and a limited presence of mobile genetic elements. Overall, the microbiome-based comparative analysis provides a framework for understanding the microbial community structure and functional potential across the NER, demonstrating broad probiotic potential and biosynthetic richness, with mesu samples from Sikkim showed a comparatively consistent distribution of functional pathways, biosynthetic gene clusters, and AMR-related features relative to the other FBS samples analysed.

Hypertension is a prevalent chronic disease related to multiorgan injury. Lactic acid bacteria (LAB) produce angiotensin-converting enzyme-inhibitory peptides (ACEIPs) to regulate blood pressure. This study aimed to screen LAB with high ACE inhibitory activity and investigate their protective effects against high-salt diet (HSD)-induced hypertension and organ injury. From diverse sample sources, Lactiplantibacillus plantarum T4-1 exhibited the highest ACE inhibitory activity (98%). Four highly active ACEIPs were identified via peptidomics analysis, molecular docking, and in vitro ACE inhibition assays. Among them, Peptide 1 showed the strongest inhibition (IC50 = 0.8622 μg/mL). Whole genome sequencing of Lactiplantibacillus plantarum T4-1 revealed related functional gene loci. Subsequently, animal experiments demonstrated that oral administration of T4-1 suspension significantly reduced mean arterial pressure by about 18 mmHg and mitigated multiorgan injury in HSD mice. This work enriches antihypertensive probiotic resources and provides theoretical support for developing functional food-derived ACEIPs.

Development and application of a fast and efficient CRISPR/Cas12f -based genetic toolkit in Bacillus cereus GW-01.

Probiotics provide an efficient and relatively safe method for preventing disease and increasing production in aquaculture. During the screening of beneficial bacteria in the economically important mud crab (Scylla paramamosain), an indigenous gut strain, Cetobacterium somerae R9, was isolated for the first time as a butyrate producer. This study aimed to evaluate the probiotic potential of C. somerae R9 both in vitro and in vivo. In vitro assays revealed that C. somerae R9 exhibited grow at pH 7-9, NaCl concentrations of 0.5-2.5%, and bile salt concentrations of 0.4-1.0%, and displayed susceptibility to most of the antibiotics tested. In the in vivo study, dietary supplementation with C. somerae R9, either alone or in combination with prebiotics (resistant starch and galactooligosaccharides), enhanced growth, improved antioxidative status (evidenced by elevated SOD and CAT activity and reduced MDA content), reduced hepatopancreatic damage (reduced AST activity), and maintained intestinal integrity. Supplementation also selectively enriched beneficial gut microbiota (e.g., members of Fusobacteriota). Transcriptome analysis showed that C. somerae R9 appeared to activate the PI3K-Akt signaling pathway, focal adhesion, and ECM-receptor interaction, while the combination of C. somerae R9 and prebiotics activates complement and coagulation cascades, amino sugar and nucleotide sugar metabolism, and protein digestion and absorption. Furthermore, mud crabs fed diets supplemented with C. somerae R9 exhibited significantly higher survival after challenge with Vibrio parahaemolyticus, with the synbiotic providing greater benefits than the probiotic alone. These findings collectively suggest that C. somerae R9 is a promising probiotic candidate (used either alone or in combination with prebiotics) for mud crab aquaculture.

This study investigated the probiotic potential of Bacillus subtilis strain S1, isolated from fermented chilli pickle. Genome sequencing revealed a genome size of 4.48Mb, 42.6% GC content, and 4,969 protein-coding sequences, including genes linked to probiotic activity. In vitro assays confirmed that strain S1 displayed strong aggregation, antioxidant activity, hydrophobicity, and tolerance to acid, bile, and simulated gastrointestinal conditions. It showed antibiotic resistance within safe limits and passed hemolytic and fibrinolytic safety assessments. Notably, strain S1 remained stable at alkaline pH and in up to 6.0% saline. Functional characterization showed that the strain produces antimicrobial metabolites with strong inhibitory activity against a wide range of food-borne pathogens. The metabolite remained stable up to pH 8.0 and 50℃. Enzymatic treatment efficiently identified the substance as protein-based. Purification and mass spectrometry identified the metabolite as Subtilosin A, which demonstrated promising antibacterial activity against Listeria monocytogenes. FE-SEM analysis provided insight into the mechanism of action of Subtilosin A. B. subtilis S1 whole genome sequence analysis confirms its uniqueness in possessing three different bacteriocins, such as Subtilin, Subtilosin A, and Bacillibactin. Subtilosin A and previously reported sequence of Subtilosin A showed no genetic variations. These findings indicate that the isolated S1 strain possesses genetic traits associated with probiotic properties, as confirmed by experimental evidence. This study is novel for isolating, identifying, and sequencing bacteriocins from a traditional fermented chilli pickle, thereby reporting the first Bacillus spp. This represents a major advance in microbial biopreservation, enabling safer, germ-free foods.

Lactococcus lactis subsp. lactis strains have been widely recognized for their probiotic potential, including their immunomodulatory and anti-obesity effects. This study aims to determine the genomic basis of the health-promoting properties of L. lactis subsp. lactis CAB701 and WiKim0124 using whole-genome sequencing and comparative genomic analysis. Taxonomic classification using average nucleotide identity and digital DNA-DNA hybridization confirms both strains as L. lactis subsp. lactis. Genome alignment reveals high synteny among CAB701, WiKim0124, and the reference strains, with minor structural variations. Functional annotation identifies genes associated with carbohydrate metabolism, biofilm formation, and immune modulation. Pan-genome analysis shows a substantial proportion of strain-specific genes, suggesting niche-specific adaptation. Notably, L. lactis subsp. lactis CAB701 and WiKim0124 harbor unique biosynthetic gene clusters for nisin Z and sactipeptides, which may contribute to their antimicrobial and immunomodulatory functions. Additionally, genes involved in short-chain fatty acid metabolism are identified, highlighting their potential role in metabolic regulation. These findings provide valuable insights into the functional genetics of L. lactis subsp. lactis and their applicability as probiotics for immune modulation and obesity management. KEY POINTS: • Comparative genomics revealed strain-specific immune- and metabolism-linked genes •CAB701 and WiKim0124 uniquely harbor nisin Z and sactipeptide biosynthetic clusters •Genomic features support L. lactis application as functional probiotics.

Bacillus velezensis has emerged as a promising probiotic alternative to antibiotics in the livestock industry. This study aimed to comprehensively evaluate the probiotic potential and safety of B. velezensis CH3-2, a strain isolated from the feces of Chahua chicken, using a combined phenotypic and genomic strategy. Whole-genome sequencing yielded a 3.91Mb circular chromosome with no plasmids. Strain CH3-2 exhibited high tolerance to simulated gastrointestinal fluids, after sequential exposure to simulated gastric fluid (4h) and intestinal fluid (4h), the viable cell count remained at 6.69 log CFU/mL. These phenotypes were corroborated by the genomic identification of acid tolerance genes (e.g., atpA-atpH) and bile salt efflux transporter genes (e.g., dppB1-E). The strain demonstrated high cell surface hydrophobicity (> 79%) and time-dependent auto-aggregation (77.68% at 20h), indicating strong adhesion potential supported by genes encoding moonlighting proteins and lipoteichoic acid synthase. Furthermore, CH3-2 displayed significant broad-spectrum antimicrobial activity against Escherichia coli K88, Shigella sonnei CMCC51592, and Listeria monocytogenes BNCC33687. Genome mining revealed 13 secondary metabolite biosynthetic gene clusters, such as those for macrolactin H and fengycin, providing a molecular basis for its antagonism. Additionally, the strain produced exopolysaccharides (0.70mg/mL at 30h) and exhibited strong extracellular antioxidant activity. Safety assessments confirmed that CH3-2 is non-hemolytic, does not produce biogenic amines, and lacks virulence genes. Six intrinsic, chromosomally encoded antibiotic resistance genes were identified, posing no risk of horizontal transfer. Acute oral toxicity tests in mice further indicated its preliminary in vivo safety, with no adverse effects on growth or histopathology. Collectively, these findings confirm that B. velezensis CH3-2 is a safe and robust candidate for development as a functional probiotic.

Acute and sub-chronic oral toxicity evaluation and gut microbiota modulation of indigenous Lactobacillus helveticus strains from Indonesia in rats

This study evaluated the probiotic potential of Marichromatium gracile DH-1 for improving the performance of juvenile Hippocampus abdominalis in aquaculture. A 40-day trial was conducted to evaluate the effects of the marine photosynthetic bacterium M. gracile DH-1 on early juvenile seahorses (H. abdominalis), using four treatments with three replicate tanks per treatment (n = 3) and an initial stocking density of 200 individuals per tank. Juveniles were exposed to M. gracile DH-1 at 25, 50, and 100 ppm (corresponding to 2.5 × 102, 5.0 × 102, and 1.0 × 103 CFU/mL, respectively) in the rearing water. Compared with the control, water-borne M. gracile DH-1 supplementation significantly improved growth performance, including body length, body weight gain, specific growth rate, and condition factor. The highest survival rate was recorded in the 100 ppm (1.0 × 103 CFU/mL) group (12.0%) versus 1.33% in the control group. M. gracile DH-1 also enhanced digestive enzyme activities, particularly α-amylase and total protease, and increased antioxidant enzyme activities, including catalase and glutathione peroxidase. In addition, supplementation improved intestinal microbial composition by increasing beneficial taxa and reducing potentially harmful bacteria. Water quality was also improved, as indicated by reduced ammonia nitrogen and nitrite, together with increased dissolved oxygen. These results suggest that M. gracile DH-1 is a promising probiotic candidate for juvenile seahorse culture by enhancing growth performance, antioxidant defense, intestinal microbial balance, and water quality.