The current study describes the isolation of exopolysaccharide (EPS) producing lactic acid bacteria (LAB) from marine samples and testing different sugar additives with different proportions for enhanced EPS yield. The isolate MSD8 showed the most potential, yielding 200 mg/L of EPS after being cultivated at 37 °C for 48 h on de Man Rogosa and Sharpe medium (MRS) supplemented with 3% sucrose. The marine isolate MSD8 was identified as Enterococcus faecium with 99.58% probability using 16S rRNA gene sequencing. The obtained sequence was deposited in GenBank and assigned the accession number MW924065. The feature of MSD8-EPS was characterized by estimating the total carbohydrate content by UV-vis to be ~ 71%. The FTIR analysis further indicated the presence of characteristic bands of polysaccharide. The cytotoxicity of the produced MSD8-EPS was assessed using human skin fibroblasts (HSF). The IC50 was determined to be > 100 μg/mL, which signifies that MSD8-EPS is safe for skin application. The produced EPS was used to prepare a novel ointment, which was tested for wound healing ability in male albino rats. The ointment significantly (P ≤ 0.05) shortened the time needed for wound healing, as it successfully healed the wounds by 94.93% on the 7th day and completely (100%) healed the wound by the 12th day. In comparison, the control group was healed by 73.2% and 84.83%, respectively. The data confirm that the prepared ointment can safely be used for pharmaceutical wound care products.

The biosynthetic machinery for cell wall polysaccharide (CWPS) formation in Lactococcus lactis and Lactococcus cremoris is encoded by the cwps locus. The CWPS of lactococci typically consists of a neutral rhamnan component, which is embedded in the peptidoglycan, and to which a surface-exposed side chain oligosaccharide or polysaccharide pellicle (PSP) component is attached. The rhamnan component has been shown for several lactococcal strains to consist of a repeating rhamnose trisaccharide subunit, while the side chain is diverse in glycan content, polymeric status and glycosidic linkage architecture. The observed structural diversity of the CWPS side chain among lactococcal strains is reflected in the genetic diversity within the variable 3\' region of the corresponding cwps loci. To date, four distinct cwps genotypes (A, B, C, D) have been identified, while eight subtypes (C1 through to C8) have been recognized among C-genotype strains. In the present study, we report the identification of three novel subtypes of the lactococcal cwps C genotypes, named C9, C10 and C11. The CWPS of four isolates representing C7, C9, C10 and C11 genotypes were analysed using 2D NMR to reveal their unique CWPS structures. Through this analysis, the structure of one novel rhamnan, three distinct PSPs and three exopolysaccharides were elucidated. Results obtained in this study provide further insights into the complex nature and fascinating diversity of lactococcal CWPSs. This highlights the need for a holistic view of cell wall-associated glycan structures which may contribute to robustness of certain strains against infecting bacteriophages. This has clear implications for the fermented food industry that relies on the consistent application of lactococcal strains in mesophilic production systems.

Exopolysaccharides (EPSs) are carbohydrate polymers that are synthesized and secreted into the extracellular during the growth of microorganisms. Bacillus thuringiensis (Bt) is a type of entomopathogenic bacterium, that produces various insecticidal proteins and EPSs. In our previous study, the EPSs produced by Bt strains were first found to enhance the toxicity of insecticidal crystal proteins against Plutella xylostella. However, the response of the intestinal bacterial communities of P. xylostella under the action of EPSs is still unelucidated. In this study, 16S rRNA amplicon sequencing was used to characterize the intestinal bacterial communities in P. xylostella treated with EPSs alone, Cry1Ac protoxin alone, and both the Cry1Ac protoxin and EPSs. Compared with the control group, alpha diversity indices, the Chao1 and ACE indices were significantly altered after treatment with EPSs alone, and no significant difference was observed between the groups treated with Cry1Ac protoxin alone and Cry1Ac protoxin + EPSs. However, compared with the gut bacterial community feeding on Cry1Ac protoxin alone, the relative abundance of 31 genera was significantly changed in the group treated with Cry1Ac protoxin and EPSs. The intestinal bacteria, through the oral of Cry1Ac protoxin and EPSs, significantly enhanced the toxicity of the Cry1Ac protoxin towards the axenic P. xylostella. In addition, the relative abundance of the 16S rRNA gene in the chloroplasts of Brassica campestris decreased after adding EPSs. Taken together, these results show the vital contribution of the gut microbiota to the Bt strain-killing activity, providing new insights into the mechanism of the synergistic insecticidal activity of Bt proteins and EPSs.

Colanic acid (CA) is an exopolysaccharide found in Enterobacteriaceae. Recently, its ability to stimulate physical activity in mice and to prolong the lifespan of invertebrates has been described. In the current work, we use standard MTT assay, fluorescence microscopy, and flow cytometry to describe CA action on several cell lines of different origins. We observed slight antiproliferative activity against colorectal cancer (HCT-116), neuroblastoma (IMR-32), and myoblast (C2C12) cell lines at a concentration of 256 μg/mL, while other cell lines of non-cancerous origin (Vero, HPF) did not show any decrease in the MTT assay. In all cell lines, we observed a rearrangement of mitochondria localization using fluorescence microscopy. CA induces cell differentiation in the myoblast cell line (C2C12) at concentrations of 50-200 μg/mL. Briefly, we observed that the number of apoptotic cells increased and the metabolic activity in the MTT assay decreased, which was accompanied by changes in cell morphology, the quantity of ROS, and the potential of the mitochondrial membrane. Taken together, these results indicate that CA is specific in cytotoxicity to cell lines of different origins and can impact mitochondria and differentiation, consistent with its potential geroprotective function.

In this study 5-((2-((3-methoxy benzylidene)-amino)-phenyl)-diazenyl)-4,6-diphenyl pyrimidine-2(5H)-thione was synthesized. The pharmacological applications of pyrimidine analogs are restricted due to their poor pharmacokinetic properties. As a solution, a microbial exopolysaccharide (curdlan gum) was used to synthesize folic acid-conjugated pyrimidine-2(5H)-thione-encapsulated curdlan gum-PEGamine nanoparticles (FA-Py-CG-PEGamine NPs). The results of physicochemical properties revealed that the fabricated FA-Py-CG-PEGamine NPs were between 100 and 400 nm in size with a majorly spherical shaped, crystalline nature, and the encapsulation efficiency and loading capacity were 79.04 ± 0.79 %, and 8.12 ± 0.39 % respectively. The drug release rate was significantly higher at pH 5.4 (80.14 ± 0.79 %) compared to pH 7.2. The cytotoxic potential of FA-Py-CG-PEGamine NPs against MCF-7 cells potentially reduced the number of cells after 24 h with 42.27 μg × mL-1 as IC50 value. The higher intracellular accumulation of pyrimidine-2(5H)-thione in MCF-7 cells leads to apoptosis, observed by AO/EBr staining and flow cytometry analysis. The highest pyrimidine-2(5H)-thione internalization in MCF-7 cells may be due to folate conjugated on the surface of curdlan gum nanoparticles. Further, internalized pyrimidine-2(5H)-thione increases the intracellular ROS level, leading to apoptosis and inducing the decalin in mitochondrial membrane potential. These outcomes demonstrated that the FA-Py-CG-PEGamine NPs were specificity-targeting folate receptors on the plasma membranes of MCF-7 Cells.

Probiotics are the health beneficial microorganisms and suitable for food industry if found fit for human consumption. In the present study, Lactiplantibacillus plantarum MCC5231, a probiotic bacterium included in vegetable-based beverages, was evaluated for its safety characteristics and gastrointestinal survival using a combined in silico and in vitro approach. The strain was found to be devoid of hemolytic, lecithinase and gelatinase activities. Additionally, it does not consist any transferable antibiotic resistance genes. Further, whole genome sequence analysis revealed the presence of three intact prophages and 14 virulence-associated genes, however, none of them posed a pathogenic threat. Importantly, MCC5231 do not possess any gene associated with toxin production. The strain harbored a CRISPR system, enhancing defense against prophages. Survival assays under simulated gastric and intestinal fluid conditions demonstrated viability rates of 71.4 % and 83.3 %, respectively. Genetic analysis of the mucin binding protein indicated possession of a type II mucin binding domain, suggesting moderate adhesion to intestinal cells. Furthermore, L. plantarum MCC5231 exhibited the ability to produce exopolysaccharides and form biofilms, which may confer additional protection in the gastrointestinal tract. Based on these findings, L. plantarum MCC5231 appears to be a safe probiotic candidate suitable for commercial use in the food industry.

In order to meet growing consumer demands in terms of naturalness, the pharmaceutical, food, and cosmetic industries are looking for active molecules of plant origin. In this context, hairy roots are considered a promising biotechnological system for the sustainable production of compounds of interest. Poplars (genus Populus, family Salicaceae) are trees of ecological interest in temperate alluvial forests and are also cultivated for their industrial timber. Poplar trees also produce specialized metabolites with a wide range of bioactive properties. The present study aimed to assess the hybrid poplar hairy root extracts for antimicrobial and antibiofilm activities against four main life-threatening strains of Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. Ethyl acetate extracts from two hairy root lines (HP15-3 and HP A4-12) showed significant antibacterial properties as confirmed by disc diffusion assay. Antibiofilm activities were found to be dose dependent with significant biofilm inhibition (75-95%) recorded at 1000 µg.mL-1 in all the bacterial strains tested. Dose-dependent enhancement in the release of exopolysaccharides was observed in response to treatment with extracts, possibly because of stress and bacterial cell death. Fluorescence microscopy confirmed loss of cell viability of treated bacterial cells concomitant with increased production of reactive oxygen species compared to the untreated control. Overall, this study demonstrates for the first time a high potential of poplar hairy root extracts as a natural and safe platform to produce antimicrobial agents in pharmaceutical, food, industrial water management, or cosmetic industries.

Introduction: B. animalis subsp. lactis HN019 is a commercially available well-characterized probiotic with documented effects on human health, such as the ability to enhance the immune function and to balance the intestinal microbiome. Therefore, optimizing the manufacturing process to improve sustainability, increasing biomass yields and viability, and avoiding animal -derived nutrients in the medium to meet vegan consumer\'s needs, is currently of interest. Besides the established use of live probiotic cells, alternative supplements indicated as postbiotics, like non-viable cells and/or probiotics derived bioactive molecules might be considered as potential next generation biotherapeutics. In fact, advantages of postbiotics include fewer technological limitations, such as easier production processes and scale-up, and even higher specificity. Methods: In this work, medium design together with different fermentation strategies such as batch, fed-batch and in situ product removal on lab-scale bioreactors were combined. Medium pretreatment by ultrafiltration and protease digestion was performed to reduce polysaccharidic contaminants and facilitate the purification of secreted exopolysaccharides (EPS). The latter were isolated from the fermentation broth and characterized through NMR, GC-MS and SEC-TDA analyses. The expression of TLR-4, NF-kb and IL-6 in LPS challenged differentiated CaCo-2 cells treated with EPS, live and heat-killed B. lactis cells/broth, was evaluated in vitro by western blotting and ELISA. Zonulin was also assessed by immunofluorescence assays. Results and Discussion: The titer of viable B. lactis HN019 was increased up to 2.9 ± 0.1 x 1010 on an animal-free semidefined medium by applying an ISPR fermentation strategy. Medium pre-treatment and a simple downstream procedure enriched the representativity of the EPS recovered (87%), the composition of which revealed the presence of mannuronic acid among other sugars typically present in polysaccharides produced by bifidobacteria. The isolated EPS, live cells and whole heat inactivated broth were compared for the first up to date for their immunomodulatory and anti-inflammatory properties and for their ability to promote intestinal barrier integrity. Interestingly, EPS and live cells samples demonstrated immune-stimulating properties by downregulating the expression of TLR-4 and NF-kb, and the ability to promote restoring the integrity of the intestinal barrier by up-regulating the expression of zonulin, one of the tight junctions forming proteins. Postbiotics in the form of heat killed broth only reduced NF-kb expression, whereas they did not seem effective in the other tested conditions.

The present study investigates the low temperature tolerance strategies of thermophilic bacterium Anoxybacillus rupiensis TPH1, which grows optimally at 55 °C , by subjecting it to a temperature down-shift of 10 °C (45 °C) for 4 and 6 h followed by studying its growth, morphophysiological, molecular and proteomic responses. Results suggested that although TPH1 experienced increased growth inhibition, ROS production, protein oxidation and membrane disruption after 4 h of incubation at 45 °C yet maintained its DNA integrity and cellular structure through the increased expression of DNA damage repair and cell envelop synthesizing proteins and also progressively alleviated growth inhibition by 20% within two hours i.e., 6 h, by inducing the expression of antioxidative enzymes, production of unsaturated fatty acids, capsular and released exopolysaccharides and forming biofilm along with chemotaxis proteins. Conclusively, the adaptation of Anoxybacillus rupiensis TPH1 to lower temperature is mainly mediated by the synthesis of large numbers of defense proteins and exopolysaccharide rich biofilm formation.

Drought stress is a significant environmental challenge affecting global agriculture, leading to substantial reductions in crop yields and overall plant productivity. It induces a cascade of physiological and biochemical changes in plants, including reduced water uptake, stomatal closure, and alterations in hormonal balance, all of which contribute to impaired growth and development. Drought stress diminishes crop production by impacting crucial plant metabolic pathways. Plants possess the ability to activate or deactivate specific sets of genes, leading to changes in their physiological and morphological characteristics. This adaptive response enables plants to evade, endure, or prevent the effects of drought stress. Drought stress triggers the activation of various genes, transcription factors, and signal transduction pathways in plants. In this context, imposing plant growth-promoting rhizobacteria (PGPR) emerges as a promising strategy. PGPR, employing diverse mechanisms such as osmotic adjustments, antioxidant activity, and phytohormone production, not only ensures the plant\'s survival during drought conditions but also enhances its overall growth. This comprehensive review delves into the various mechanisms through which PGPR enhances drought stress resistance, offering a thorough exploration of recent molecular and omics-based approaches to unravel the role of drought-responsive genes. The manuscript encompasses a detailed mechanistic analysis, along with the development of PGPR-based drought stress management in plants.