Search Results (75)

Bile salt hydrolase (BSH) enables microbial-mediated deconjugation of bile acids (BAs) in the gastrointestinal tract. BSH enzymes initiate bile acid metabolism by catalyzing the first, essential deconjugation step. Due to the strict connection between dysregulations of the BA pool and human or animal diseases, identification and characterization of strains with BSH activity are relevant for both healthcare and agroindustry. However, current methods are expensive, poorly sensitive, or require complex procedures. Here, a BSH screening assay for cultivated microbes is proposed, based on a bacterial biosensor that reports the concentration of different BA types via fluorescence. Although the biosensor is broadly responsive to various bile acids, the assay was designed to guarantee specificity by testing individual primary BAs within controlled concentration ranges. The assay was evaluated on two recombinant Escherichia coli strains bearing BSH genes from Lactobacillus johnsonii PF01 and a BSH-positive probiotic strain (Lactobacillus rhamnosus GG). Data showed a consistent activity pattern with previous assays on these enzymes. A crucial aspect addressed was the matrix effect, i.e., the impact of the growth media of the BSH-containing strains on biosensor output. This assay is expected to be a reproducible and accessible option, compatible with automated protocols. Full article

Despite growing recognition of the role of the gut microbiome in host health and in modulating pathogen activity, the dynamic and reciprocal relationship between enteric viruses and the gut microbial ecosystem remains insufficiently defined and requires further exploration. This comprehensive review examines the bidirectional interplay between the gut microbiome and enteric viral infections by addressing (i) viruses associated with gastrointestinal alterations, (ii) how enteric viral infections alter the composition and function of the gut microbiome, (iii) how the gut microbiome modulates viral infectivity and host susceptibility, and (iv) current microbial-based approaches for preventing or treating enteric viral infections. Gastrointestinal viral infections induce gut microbiome dysbiosis, marked by reductions in beneficial bacteria and increases in potentially pathogenic populations. Specific gut microorganisms can modulate host susceptibility, with certain bacterial genera increasing or decreasing infection risk and disease severity. Pattern recognition receptors in the intestinal epithelium detect microbial signals and trigger antimicrobial peptides, mucus, and interferon responses to control viral replication while maintaining tolerance to commensal bacteria. The gut microbiome can indirectly facilitate viral infections by creating a tolerogenic environment, suppressing antiviral antibody responses, and modulating interferon signaling, or directly enhance viral replication by stabilizing virions, promoting host cell attachment, and facilitating coinfection and viral recombination. In turn, commensal gut bacteria can inhibit viral entry, enhance host antiviral responses, and strengthen mucosal barrier function, contributing to protection against gastrointestinal viral infections. Probiotics and fecal microbiota transplantation constitute potential microbial-based therapeutics that support antiviral defenses, preserve epithelial integrity, and restore microbial balance. In conclusion, the role of the gut microbiome in modulating enteric viral infections represents a promising area of future investigation. Therefore, integrating microbiome insights with virology and immunology could enable predictive and personalized strategies for prevention and treatment. Full article

Background: African swine fever (ASF) is a highly contagious and often deadly disease that poses a major threat to swine production worldwide. The lack of a commercially available vaccine underscores the critical need for innovative immunization strategies to combat ASF. Methods: Six ASFV antigenic proteins (K78R, A104R, E120R, E183L, D117L, and H171R) were fused with the Lactiplantibacillus plantarum WCFS1 surface anchor LP3065 (LPxTG motif) to generate recombinant Lactiplantibacillus plantarum NC8 (rNC8) strains. The surface expression was confirmed using immunofluorescence and Western blotting assays. Additionally, the dendritic cell-targeting peptides (DCpep) were co-expressed with each antigen protein. Mice were immunized at a dosage of 10⁹ colony-forming units (CFU) per strain per mouse via intragastric (I.G.), intranasal (I.N.), and intravenous (I.V.) routes. The bacterial mixture was heat-inactivated by boiling for 15 min to destroy viable cells while preserving antigenic structures. I.V. administration caused no hypersensitivity, confirming the method’s safety and effectiveness. Results: Following I.G. administration, rNC8-E120R, rNC8-E183L, rNC8-K78R, and rNC8-A104R induced significant levels of secretory immunoglobulin A (sIgA) in fecal samples, whereas rNC8-H171R and rNC8-D117L failed to induce a comparable response. Meanwhile, rNC8-D117L, rNC8-K78R, and rNC8-A104R also elicited significant levels of sIgA in bronchoalveolar lavage fluid (BALF). Following I.N. immunization, rNC8-E120R, rNC8-K78R, and rNC8-A104R significantly increased sIgA levels in both fecal and BALF immunization. In contrast, I.V. immunization with heat-inactivated rNC8-K78R and rNC8-A104R induced robust serum IgG titers, whereas the remaining antigens elicited minimal or insignificant responses. Flow cytometry analysis revealed expanded CD3⁺CD4⁺ T cells in mice immunized via the I.N. and I.G. and CD3⁺CD4⁺ T cells only in those immunized via the I.N. route. Th1 responses were also significant in the sera of mice immunized via the I.G. and I.N. routes. Conclusions: The rNC8 multiple-antigen cocktail elicited strong systemic and mucosal immune responses, providing a solid foundation for the development of a probiotic-based vaccine against ASF. Full article

Although epidermal growth factor (EGF) has potential wide applications in the cosmetic industry, it still has limitations, such as a costly purification process and low stability in the surrounding environment. To overcome these limitations, we developed genetically modified Pediococcus pentosaceus CBT SL4, which can secrete EGF protein in growth media, thereby producing probiotic-derived PP-EGF culture medium supernatant (PP-EGF-SUP). Even at low EGF concentrations, PP-EGF-SUP exhibited EGF activities, such as cell scratch wound healing, tyrosinase inhibition, and improvements in anti-wrinkle factors, similar to or stronger than those of recombinant human EGF (rhEGF), which was used as a positive control. PP-EGF-SUP exhibited strong additional biological activities, such as antioxidant, anti-inflammatory, and anti-microbial activities, even though rhEGF did not have such properties. PP-EGF-SUP could be easily transformed to PP-EGF-SUP dried powder (PP-EGF-DP) using the freeze-drying method, and it could also be well resolved in water up to 20 mg/mL; furthermore, it still maintained its bioactivity after the manufacturing process. To determine melasma improvement efficacy, a human application test was performed using melasma ampoules containing 1% or 5% PP-EGF-DP formulations for four weeks. When comparing the melasma values before and after treatment, it was found that the light melasma value statistically decreased by 3.38% and 3.79% and that the dark melasma value statistically decreased by 1.74% and 2.93% in the test groups applying the 1% and 5% PP-EGF-DP melasma ampoules, respectively. In addition, the melasma area also decreased by 21.21% and 29.1%, while the control group showed no statistical difference. During the study period, no significant adverse skin reactions were observed due to the application of the PP-EGF-DP melasma ampoule. In conclusion, PP-EGF-DP may offer unique advantages in the cosmetic ingredient market, such as safety (as a probiotic derivative), stability (postbiotics protect EGF activity), and diverse bioactivities (activity potentiation and postbiotic-derived biological activities). Full article

Coffee silverskin (CS), a by-product generated during coffee roasting, contains high levels of xylan hemicellulose and protein, making it a promising substrate for functional ingredient production. This study developed an integrated bioprocess to simultaneously produce bioactive peptides and xylooligosaccharides (CS-XOS) from CS. Conventional alkaline extraction (CAE) under optimized conditions (1.0 M NaOH, 90 °C, 30 min) yielded 80.64 mg of protein per gram of CS and rendered the solid residue suitable for XOS production. Enzymatic hydrolysis of the extracted protein using protease_SE5 generated low-molecular-weight peptides (0.302 ± 0.01 mg/mL), including FLGY, FYDTYY, and FDYGKY. These peptides were non-toxic, exhibited in vitro antioxidant activity (0–50%), and showed ACE-inhibitory activities of 60%, 26%, and 79%, and DPP-IV-inhibitory activities of 19%, 18%, and 0%, respectively. Concurrently, the alkaline-treated CS solid residue (ACSS) was hydrolyzed using recombinant endo-xylanase, yielding 52.5 ± 0.08 mg of CS-XOS per gram of ACSS. The CS-XOS exhibited prebiotic effects by enhancing the growth of probiotic lactic acid bacteria (μ_max 0.100–0.122 h⁻¹), comparable to commercial XOS. This integrated bioprocess eliminates the need for separate processing lines, enhances resource efficiency, and provides a sustainable strategy for valorizing agro-industrial waste. The co-produced peptides and CS-XOS offer significant potential as functional food ingredients and nutraceuticals. Full article

Lactic acid bacteria (LAB) are central to food, feed, and health biotechnology, yet their genomes have long resisted rapid, precise manipulation. This review charts the evolution of LAB genome-editing strategies from labor-intensive RecA-dependent double-crossovers to state-of-the-art CRISPR and CRISPR-associated transposase systems. Native homologous recombination, transposon mutagenesis, and phage-derived recombineering opened the door to targeted gene disruption, but low efficiencies and marker footprints limited throughput. Recent phage RecT/RecE-mediated recombineering and CRISPR/Cas counter-selection now enable scar-less point edits, seamless deletions, and multi-kilobase insertions at efficiencies approaching model organisms. Endogenous Cas9 systems, dCas-based CRISPR interference, and CRISPR-guided transposases further extend the toolbox, allowing multiplex knockouts, precise single-base mutations, conditional knockdowns, and payloads up to 10 kb. The remaining hurdles include strain-specific barriers, reliance on selection markers for large edits, and the limited host-range of recombinases. Nevertheless, convergence of phage enzymes, CRISPR counter-selection and high-throughput oligo recombineering is rapidly transforming LAB into versatile chassis for cell-factory and therapeutic applications. Full article

Background/Objectives: The ongoing evolution of SARS-CoV-2 has highlighted the limitations of parenteral vaccines in preventing viral transmission, largely due to their failure to elicit robust mucosal immunity. Methods: Here, we evaluated an intranasal (IN) vaccine formulation consisting of recombinant receptor-binding domain (RBD) adsorbed onto human probiotic Bacillus subtilis DG101 spores. Results: In BALB/c mice, IN spore-RBD immunization induced strong systemic and mucosal humoral responses, including elevated specific IgG, IgM, and IgA levels in serum, bronchoalveolar lavage fluid (BALF), nasal-associated lymphoid tissue (NALT), and saliva. It further promoted mucosal B cell and T cell memory, along with a Th1/Tc1-skewed T cell response, characterized by increased IFN-γ-expressing CD4⁺ and CD8⁺ T cells in the lungs. Conclusions: All in all, these findings highlight the potential of intranasal vaccines adjuvanted with probiotic B. subtilis spores in inducing sterilizing immunity and limiting SARS-CoV-2 transmission. Full article

CRISPR technology, which is derived from the bacterial adaptive immune system, has transformed traditional genetic engineering techniques, made strain engineering significantly easier, and become a very versatile genome editing system that allows for precise, programmable modifications to a wide range of microbial genomes. The economies of fermentation-based manufacturing are changing because of its quick acceptance in both academic and industry labs. CRISPR processes have been used to modify industrially significant bacteria, including the lactic acid producers, Clostridium spp., Escherichia coli, and Corynebacterium glutamicum, in order to increase the yields of bioethanol, butanol, succinic acid, acetone, and polyhydroxyalkanoate precursors. CRISPR-mediated promoter engineering and single-step multiplex editing have improved inhibitor tolerance, raised ethanol titers, and allowed for the de novo synthesis of terpenoids, flavonoids, and recombinant vaccines in yeasts, especially Saccharomyces cerevisiae and emerging non-conventional species. While enzyme and biopharmaceutical manufacturing use CRISPR for quick strain optimization and glyco-engineering, food and beverage fermentations benefit from starter-culture customization for aroma, texture, and probiotic functionality. Off-target effects, cytotoxicity linked to Cas9, inefficient delivery in specific microorganisms, and regulatory ambiguities in commercial fermentation settings are some of the main challenges. This review provides an industry-specific summary of CRISPR–Cas9 applications in microbial fermentation and highlights technical developments, persisting challenges, and industrial advancements. Full article

Although rare, the ability to produce surface S-layer proteins is beneficially associated with particular Lactobacillus strains being investigated as probiotics. Therefore, this work aimed to study specific probiotic functionalities of selected Levilactobacillus brevis strains MB1, MB2, MB13 and MB20, isolated from human milk microbiota, and to assess the contribution of S-proteins. Firstly, Rapid Annotation using Subsystem Technology revealed that cell wall-related genes were abundant in analysed L. brevis genomes. Furthermore, the results demonstrated that S-proteins mediate aggregation capacity and competitive exclusion of selected pathogens by L. brevis strains. The improvement of Caco-2 epithelial monolayer barrier function was demonstrated by the increase in JAM-A and occludin expressions when L. brevis strains or S-proteins were added, with the effect being most pronounced after treatment with MB2 and S-proteins of MB1. L. brevis strains, especially MB20, exerted the potential to adhere to recombinant human ZG16. Strain MB2 and MB20-S-proteins improved the barrier function of HT29 epithelial monolayer, as evidenced by increased ZG16 expression. Analysed L. brevis strains and S-proteins differentially affected the protein expression of IL-1β, IL-6 and IL-8, and IL-10 cytokines. The most prominent effect was observed by S-proteins of MB20, since IL-1β production was decreased while IL-10 production was significantly increased. Full article

Saccharomyces boulardii, the only commercially available probiotic yeast, has gained attention as a recombinant live biotherapeutic product (rLBP) empowered with the expression of heterologous therapeutic proteins for treating gastrointestinal diseases. However, the genetic modification of S. boulardii intended for clinical use is hindered by regulatory and technical challenges. In this study, we developed a dihydrofolate reductase (DHFR)-based selection system as an innovative alternative to traditional auxotrophic selection strategies for engineering S. boulardii. The DHFR selection system overcame inherent resistance of the yeast to methotrexate (MTX) by incorporating sulfanilamide, a dihydrofolate synthesis inhibitor, to enhance selection efficiency. The system demonstrated robust functionality, enabling the efficient screening of high-expression clones and tunable expression of therapeutic proteins, such as cytokines and antibodies, by modulating MTX concentrations. Furthermore, the yeast’s endogenous DHFR homolog, DFR1, was shown to be a viable selection marker, providing greater host compatibility while maintaining functionality compared to DHFR. This selection system avoids reliance on foreign antibiotic selection markers and the construction of auxotrophic strains, thus simplifying engineering and allowing for a tunable protein expression. These advancements establish the DHFR/DFR1 selection system as a robust and versatile platform for developing S. boulardii-based live biotherapeutics. Full article

Probiotics show beneficial effects on diabetes mellitus (DM). If probiotics can secrete the recombinant insulins that may help suppress DM development, then it would likely have very few adverse side effects. To produce insulin analogs in bacteria, recombinant insulin (insulin-CBT1) should be the single-chain insulin (SCI) similar to proinsulin. However, insulin-CBT1 should allow the protein to activate insulin receptors directly without the need for proteolytic cleavage. In this study, we evaluated the effect of the flexible linker peptide on the physical and structural characteristics of insulin-CBT1 compared with commercial insulin (c-insulin). In the results, the linker peptide had marked effects on polarity and structure by increasing the α-helix content (19.3%→25.6%). Furthermore, insulin-CBT1 induced MIN6 proliferation 1.75-fold more than c-insulin, whereas differentiation and glucose uptake rates by 3T3-L1 were 39% and 15% lower, respectively. The biological anti-diabetes properties of insulin-CBT1 were well evaluated compared with c-insulin. Furthermore, we first suggest a special method for oral administration of insulin-CBT 1 without damage to the digestive tract. We developed an insulin-CBT1 delivery system using Pediococcus pentosaceus (PP), which has been reported as a potential bacteria in DM. First, insulin-CBT1 was harbored in pCBT2-24, which verified the expression and secretion vector system of PP. We finally confirmed that PP-insulin-CBT1 successfully secreted insulin-CBT1 proteins to culture media. These results presented herein open up new avenues to developing therapeutic options for DM. Full article

Aeromonas hydrophila presents a significant threat to global aquaculture due to its ability to infect freshwater and marine fish species, leading to substantial economic losses. Effective mitigation methods are essential to address these challenges. Vaccination has emerged as a promising strategy to reduce A. hydrophila infections; however, it faces several obstacles, including variability in immune responses, pathogen diversity, and environmental factors affecting vaccine efficacy. To enhance vaccine performance, researchers focus on adjuvants to boost immune responses and develop multivalent vaccines targeting multiple A. hydrophila strains. Tailoring vaccines to specific environmental conditions and optimizing vaccination schedules can further address the challenges posed by pathogen diversity and variable immune responses. This review provides an in-depth analysis of the immunological hurdles associated with A. hydrophila vaccine development. Current vaccine types—live attenuated, inactivated, subunit, recombinant, and DNA—exhibit diverse mechanisms for stimulating innate and adaptive immunity, with varying levels of success. Key focus areas include the potential of advanced adjuvants and nanoparticle delivery systems to overcome existing barriers. The review also highlights the importance of understanding host–pathogen interactions in guiding the development of more targeted and effective immune responses in fish. Complementary approaches, such as immunostimulants, probiotics, and plant-based extracts, are explored as adjuncts to vaccination in aquaculture health management. Despite notable progress, challenges remain in translating laboratory innovations into scalable, cost-effective solutions for aquaculture. Future directions emphasize the integration of advanced genomic and proteomic tools to identify novel antigen candidates and the need for industry-wide collaborations to standardize vaccine production and delivery. Addressing these challenges can unlock the potential of innovative vaccine technologies to safeguard fish health and promote sustainable aquaculture practices globally. Full article

Oligosaccharides containing galactosyl moieties belong to two main groups: raffinose family oligosaccharides (RFO, α-GOS) and lactose-type β-galactooligosaccharides (β-GOS), both well-known for their prebiotic effect. The present review investigates the vast amounts of recent research on the structures of GOS and their beneficial impact. It focuses on the molecular interactions between GOS and probiotics in vitro and in vivo, the enzymology of the processes, and the genetic prerequisites for the synthesis and degradation of GOS by probiotic bacteria. The preferences of probiotic strains belonging to the Bifidobacterium and Lactobacillus genera are elucidated to form and degrade GOS of a certain length, structure, and linkages between monomers. A brief overview of the industrial production of β-GOS by natural and recombinant strains included the methods and production efficiency evaluation. Full article

Bile salt hydrolase (BSH), a probiotic-related enzyme with cholesterol-assimilating and anti-hypercholesterolemic abilities, has been isolated from intestinal bacteria; however, BSH activity of bacteria in bile-salt-free (non-intestinal) environments is largely unknown. Here, we aimed to identify BSH from non-intestinal Enterococcus faecium and characterize its enzymatic function. We successfully isolated a plasmid-encoded bsh (efpBSH) from E. faecium, and the recombinant EfpBSH showed BSH activity that preferentially hydrolyzed taurine-conjugated bile salts, unlike the activity of known BSHs. EfpBSH functioned optimally at pH 4.0 and 50 °C. EfpBSH exhibited very low amino acid sequence similarity (48.46%) to EfBSH from E. faecalis T2 isolated from human urine, although 241 sequences with 100% identity to EfpBSH were found in both plasmids and chromosomes of E. faecium strains inhabiting intestinal and non-intestinal environments. Phylogenetically, EfpBSH was not affiliated with any known BSH phylogroup and was clearly distinguished from previously identified BSHs from intestinal lactic acid bacteria. Our genome database analysis demonstrated that horizontal gene transfer causes global efpBSH distribution among E. faecium strains in various environments (soil, water, and intestinal samples) and geographical regions (Asia, Africa, Europe, North America, South America, and Australia/Oceania). Overall, our findings are the first to indicate that BSH is not an intestine-specific enzyme and that hitherto-overlooked probiotic candidates with BSH activity can exist in diverse environments. Full article

Bile salt hydrolase (BSH; EC 3.5.1.24) is the microbial enzyme that catalyzes the conversion of primary bile acids (BAs) into secondary ones, promoting microbial adaptation and modulating several host’s biological functions. Probiotics with BSH activity are supposed to survive harsh intestinal conditions and exert a cholesterol-lowering effect. Here, Lacticaseibacillus rhamnosus strains (VB4 and VB1), isolated from the vaginal ecosystem, were submitted to a genomic survey, in vitro BSH activity, and BAs tolerance assay to unravel their probiotic potential as BAs modulators. The draft genomes of Lcb. rhamnosus VB4 and VB1 strains comprised 2769 and 2704 CDSs, respectively. Gene annotation revealed numerous strain-specific genes involved in metabolism and transport, as well as in DNA recombination. Each strain harbors a single bsh gene, encoding a C-N amide hydrolase, which conserved the essential residues required in the BSH core site. According to the results, compared to VB1, the VB4 strain tolerated better BAs stress and was more active in deconjugating BAs. However, BAs stress increased the bsh gene transcription in the VB1 strain but not in the VB4 strain, suggesting a partially nonlinear relationship between BSH activity and gene expression. In conclusion, despite the complexity of the BSH transcriptional system, the results support the VB4 strain as a promising BAs-deconjugating probiotic candidate. Full article