Search Results (2,450)

Isomaltulose is produced via sucrose isomerase catalysis by Serratia plymuthica A30. The enzyme was expressed in Bacillus subtilis using surface display, in combination with Bacillus subtilis spore coat CotC. The promoter was further selected and optimized to determine P_amyE as the most suitable promoter, while the spore coat protein assay reveals CotC as optimal. By scanning and analyzing the catalyst motifs with single-point mutation construction, a maximum isomaltulose yield of 27.21 mg/mL was recorded in the F181I-mutant enzyme. Another strain encoding the H363P-mutant reached a maximum yield of 20.84 mg/mL, while the k_cat value also increased from 17.64 to 24.80. Structural analysis showed that the F181I-mutant had higher thermostability, whereas the H363P-mutant had increased k_cat. Both mutants displayed a 5-fold increase in isomaltulose yield with relatively simple construction procedures, making them suitable for high-level isomaltulose production. Full article

Microalgae and probiotics are increasingly considered a promising approach for addressing environmental problems in freshwater aquaculture. However, how this approach affects the resident free-living (FL) and particle-associated (PA) bacterial communities that are essential in biogeochemical processes remains unclear. Here, we established a 60-day mesocosm aquaculture system for Micropterus salmoides to examine the effects of microalgal and probiotic addition on fish growth, water quality, and resident bacterial communities. Four treatments were included: Chlorella vulgaris addition (6.25 × 10⁶ cells/L), Bacillus subtilis addition (7.5 × 10⁷ CFU/L), their combined addition, and a control. Our results indicated that the combined addition significantly improved fish growth performance and reduced total and inorganic nitrogen concentrations than single additions. Moreover, the combined addition enhanced nitrogen- and carbon-related metabolic functions in FL fraction and increased host-associated functions in PA community. Taxa assigned with the classes Bacilli, Bacteroidia, Gammaproteobacteria, and Alphaproteobacteria showed predominant explanations for variations in nitrogen and phosphorus. Furthermore, the combined addition enhanced potential interactions among taxa across lifestyles and strengthened the bacterial community network complexity and stability. These findings provide new insights into the microbial mechanisms underlying microalgae–probiotic synergism and offer a scientific basis for sustainable microbial management in freshwater aquaculture. Full article

Bacterial adaptation to hostile environments depends on the coordinated expression of stress response genes. When adverse conditions persist and nutrients become limiting, sporulating species may initiate sporulation as a last-resort survival strategy. However, sporulation under such conditions may alter the resistance and germination properties of the resulting spores. In this study, we investigated whether stress response regulators that facilitate vegetative cell adaptation to temperature and/or salinity changes during growth can influence the properties of Bacillus subtilis 168 spores. To this end, we examined the resistance and germination of mutant spores lacking key regulators of stress response pathways (SigB, SigW, SigX, Fur, HrcA, CtsR, and CssRS regulon), all produced under optimal sporulation conditions. The constitutive activation of the SigB-mediated general stress response, achieved through the deletion of its negative regulator RsbX, reduced spore heat resistance by 2.2-fold compared to the parental strain, while no effect was observed in vegetative cells. Additionally, ΔrsbX spores displayed both impaired nutrient-induced and CaDPA-induced germination. Collectively, these findings suggest that stress response regulators can influence spore behavior, although their effects may differ from those observed in vegetative cells. Full article

Environmental pollution affects the health of living organisms, provoking the emergence of new diseases and infections. In search of sustainable and effective solutions, this study presents a “green” synthesis of five silver nanocomposites with activated carbon (Ag-NACs) obtained from waste biomass from peach shells. The process is carried out in an aqueous environment and does not use toxic organic solvents. The chemical composition, structural properties and morphology of the synthesized Ag-NACs were characterized using scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FT-IR). Comparative analysis under different conditions, including silver concentration (0.5%, 1.5%, 4.0% and 8.0%) and impregnation time (24 and 72 h), identified the samples with 4.0% and 8.0% Ag as optimally structured, showing the strongest antibacterial activity. The results confirmed the synergistic effect and mechanism of the carbon composites, which effectively attract bacterial cells while the silver ions inhibit the growth of various pathogens. This efficacy was demonstrated against both Gram-positive (Gram⁺), Bacillus subtilis, Bacillus spizizenii, Staphylococcus aureus, Listeria innocua, and Enterococcus faecium, and Gram-negative (Gram⁻) bacterial strains, Escherichia coli, Salmonella typhimurium, Salmonella enteritidis, and Stenotrophomonas maltophilia, which highlights the significant potential of Ag-NACs. Full article

Amid the accelerating spread of antibiotic resistance, medicinal and aromatic plants stand out as powerful natural reservoirs of bioactive compounds, offering innovative prospects for next-generation antimicrobial therapies. To explore its therapeutic potential, this study evaluated the antimicrobial and antioxidant activities of Matricaria pubescens from Southeastern Morocco, supported by a thorough chemical profiling of its essential oils. The oils were obtained by steam distillation and analyzed using gas chromatography–mass spectrometry (GC–MS). The results revealed two distinct chemotypes, with isochrysanthemic ethyl ester (32.7%) as the dominant compound in chemotype EO1 and α-ocimene (19.62%) as the major constituent in chemotype EO2. Antioxidant activities were assessed using DPPH, ABTS, and reducing power assays, while antimicrobial activities were evaluated against bacteria, fungi, and yeasts using both disc diffusion and broth microdilution methods. Both oils exhibited notable antioxidant activities. Significant antimicrobial effects were observed, with Bacillus subtilis, Escherichia coli, and Staphylococcus aureus being the most sensitive strains, whereas Pseudomonas aeruginosa exhibited the highest resistance among all tested microorganisms, with the lowest MIC recorded for B. subtilis (0.612 mg/mL). These findings emphasize that M. pubescens could serve as a valuable source of biologically active compounds, particularly in the development of agents to combat microbial resistance, and further support its potential applications in pharmaceutical, cosmetic, and food industries. Full article

Background/Objectives: Ganoderma lucidum (Curtis) P. Karst. (commonly known as reishi mushroom), a well-characterized medicinal fungus, contains diverse bioactive metabolites. This study aimed to fractionate, characterize and identify the biologically active inhibitors present in G. lucidum and to evaluate their antioxidant, antimicrobial, and antidiabetic activities. Methods: The ethanol extract of G. lucidum was fractionated using column chromatography, yielding ten distinct fractions (designated as A, B, E, F, K, L, M, N, O, and P based on their elution order and visual characteristics). Liquid Chromatography–Mass Spectrometry (LC-MS) analysis identified 46 bioactive compounds, including terpenoids, alkaloids, flavonoids, and polysaccharides. Results: Among the fractions, Fraction L exhibited the strongest antioxidant activity, with an IC₅₀ of 1.59 mg/mL. Fraction O displayed significant antibacterial activity against Escherichia coli ATCC 25922 (24.4 ± 0.238 mm), Klebsiella pneumoniae ATCC 13883 (20.5 ± 0.035 mm), Bacillus subtilis ATCC 6633 (8 ± 0.176 mm), and Staphylococcus warneri ATCC 10209 (20 ± 0.080 mm). Regarding antidiabetic activity, Fraction B demonstrated the strongest inhibition of α-amylase (IC₅₀ 1.69 ± 0.03 mg/mL), while Fraction E showed the strongest α-glucosidase inhibition (IC₅₀ = 1.69 ± 0.02 mg/mL), demonstrating reciprocal selectivity between enzyme targets. Conclusions: These results establish that chromatographic fractionation concentrates specific bioactivities into distinct fractions, supporting its potential for the development of novel therapeutic agents with enhanced specificity and efficacy. Full article

Bacillus subtilis is aerobic or facultatively anaerobic. After entering the gastrointestinal tract, its spores germinate and colonize the gut, inhibiting the growth of harmful aerobic bacteria (Escherichia coli, Streptococcus, Staphylococcus aureus). However, it remains unclear whether B. subtilis can inhibit Clostridium perfringens type A infection. In this study, B. subtilis PB6 was added to the diets of pregnant sows infected with Clostridium perfringens type A, which significantly improved the reproductive performance and reduced the incidence of bloat in sows and diarrhea in neonatal piglets. The treatment significantly increased the abundance of intestinal probiotics (B. subtilis, Lactobacillus, Limosilactobacillus reuteri, Lactobacillus johnsonii, Muribaculaceae, Lactobacillus amylovorus, and Lactobacillus reuteri) in sows and decreased the relative abundance of Clostridium perfringens type A after feeding B. subtilis administration. These probiotics can repair the intestinal tissue and improve intestinal histomorphology, and enhance the expression of MUC2 and sIgA in sows, thereby further strengthening the mucosal immune function. B. subtilis can also reduce the levels of inflammatory factors (CRP, IL-1β, and IFN-γ) and attenuate the inflammatory response in sows and neonatal piglets. Taken together, our results suggest that dietary supplementation with B. subtilis PB6 could reduce bloat in sows and diarrhea in piglets while improving intestinal barrier function and microbial balance in sows. Full article

This study evaluated activities of crude extracts from different parts of the tree of heaven (Ailanthus altissima (Mill.) Swingle) collected in Slovenia and Hungary, using effect-directed analyses based on hyphenation of high-performance thin-layer chromatography (HPTLC) and nine in vitro assays performed in situ on chromatographic plates after the separation. HPTLC separation combined with a set of four antibacterial assays, two antifungal assays, and three enzyme inhibitor assays to evaluate the extracts of 15 plant parts: young shoots, young leaves, mature leaves, yellow leaves, petioles of leaves, petioles of male inflorescences, petioles of fruits, female inflorescences, young fruits, male inflorescences, mature male inflorescences, bark of 1–2-year branches, bark of 2-year branches, bark of tree trunk, and bark of roots. Antibacterial activities against Gram-positive bacteria (Bacillus subtilis, Rhodococcus fascians) and Gram-negative bacteria (Aliivibrio fischeri, Pseudomonas syringae pv. maculicola (Psm)), as well as inhibition of enzymes α-glucosidase, lipase, and acetylcholinesterase, were observed for all extracts. Extracts differed in their antifungal activities. Extracts of young shoots, mature leaves, petioles of leaves, and bark of roots showed antifungal activity against plant pathogens Fusarium avenaceum and Bipolaris sorokiniana. Extracts of yellow leaves, male inflorescences, bark of 1–2-year branches, and bark of tree trunks were only active against F. avenaceum, whereas extracts of young leaves were only active against B. sorokiniana. This study is the first to report that A. altissima extracts exhibit (1) antifungal activity against F. avenaceum and B. sorokiniana; (2) antibacterial activity against A. fischeri, Psm, R. fascians, and B. subtilis (except leaves, bark of branches and bark of tree trunks); and (3) inhibitory activity toward lipase, α-glucosidase (except bark of tree trunks), and acetylcholinesterase (except bark of tree trunks). Full article

Background: The escalating spread of drug-resistant bacteria is intensifying the antibiotic resistance crisis, necessitating the urgent development of novel antimicrobial agents to address the resulting high global mortality rates and significant socioeconomic burden. Objectives: This study aimed to aminate the C-6 position of β-carboline and investigate the antibacterial activity and mechanism of action of the derivatives. Results: For the first time, 16 derivatives with various nitrogen-containing moieties, including aliphatic- and phenyl-amino, imidazolium, pyridinium, and quinolinium, were synthesized via amination at the C-6 position of β-carboline. These compounds exhibited moderate to good activity against Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and Bacillus subtilis, with minimum inhibitory concentration (MIC) values ranging from 1.56 to 100 μg/mL. The study reveals that elongating an alkyl chain, incorporating a cationic scaffold, and expanding a π-delocalized system can enhance antibacterial activity. The most potent derivative from each series was selected for further mechanistic investigation against MRSA. All studied compounds demonstrated low hemolytic activity and low cytotoxicity. Studies on the antibacterial mechanism indicated that the compounds exert their antibacterial effects by disrupting bacterial cell walls and membranes. Additionally, two of the compounds were found to potentially disrupt the secondary structure of DNA. All tested compounds exhibited antibiofilm activity. Conclusions: Our findings demonstrate that amination modification at the C-6 position of β-carboline can enhance antibacterial activity by disrupting the cell wall membranes and interacting with bacterial DNA. These results provide a basis for further optimization of antibacterial agents based on β-carboline. Full article

Synthetic dyes discharged from the textile and dyeing industry present a significant environmental and health hazard due to their inherent toxicity, environmental persistence, and potential carcinogenicity. Microbial degradation has garnered significant interest as a cost-effective and eco-friendly strategy for dye wastewater treatment in recent years. The study systematically evaluated the decolorization performance, degradation pathways, and detoxification effects of three bacterial strains, including Rhodopseudomonas palustris gh32, Bacillus cereus HL7, and Bacillus safensis X64, on the dye indigo carmine (IC) and three azo dyes: reactive black 5 (RB5), direct black G (DBG), and direct blue 15 (DB15). The degradation mechanisms were elucidated through UV-Vis spectroscopy, UPLC-Orbitrap-HRMS analysis, and enzyme activity assays. Molecular docking simulations were employed to investigate the interactions between key redox enzymes (such as laccase, tyrosinase, and azoreductase) and the dye molecules. The results demonstrated that the strain-specific enzymatic systems effectively disrupted the dye structures. Significant detoxification effects were further confirmed through a series of bio toxicity assays involving Escherichia coli, Bacillus subtilis, plant seeds, and erythrocytes. The addition of Fe³⁺, sodium citrate, or yeast extract significantly enhanced both the decolorization efficiency and enzyme activity. This study provides an in-depth understanding of the bacterial dye degradation process at the mechanistic level, highlighting the potential of customized bacterial systems for eco-friendly dye wastewater treatment. It offers theoretical support for elucidating the mechanisms of bacterial dye degradation and advancing bioremediation technologies. Full article

The development of sustainable plant protection strategies requires stable and environmentally compatible delivery systems for beneficial microorganisms. In this study, Bacillus subtilis was encapsulated within chitosan-based cast films to evaluate bacterial viability, sustained biological activity, and antifungal efficacy. Films prepared from chitooligosaccharide (COS) and chitosans of low, medium, and high molecular weight (CS-LMW, CS-MMW, CS-HMW) were characterized in terms of morphology, mechanical performance, and pH-dependent swelling behavior. The viscosity of the chitosan solutions increased markedly with molecular weight from 73 cP (COS) to 614 cP (CS-HMW), while film thickness ranged from 34 ± 1.5 to 57 ± 2.3 µm. Mechanical performance improved significantly with increasing molecular weight, with maximum tensile stress exceeding 200 MPa for CS-HMW films, while swelling studies confirmed pronounced pH-dependent behavior consistent with the polyelectrolyte nature of chitosan. Encapsulation effectively preserved bacterial viability and metabolic activity over time. The intrinsic antifungal activity of chitosan synergized with the biocontrol activity of B. subtilis against Fusarium avenaceum and Alternaria solani. The highest antifungal performance was observed for CS-HMW films, which produced inhibition zones up to 84.6 ± 5.0 mm against A. solani. These findings demonstrate that chitosan-based cast films serve as effective carriers for beneficial microorganisms, providing environmental protection and regulated biological activity. The combination of a bioactive polymer matrix with a potent biocontrol agent represents a promising eco-friendly approach to sustainable plant protection. Full article

Background/Objectives: The gut–brain axis is increasingly recognized as a critical modulator of cognitive function. This study investigated the neurotoxic effects of combined exposure to bacterial lipopolysaccharide (LPS) and the antiretroviral drug zidovudine (ZDV) in a mouse model, and evaluated the protective potential of two probiotic interventions: Bacillus subtilis and a mixture of lactobacilli. Methods: Cognitive function was assessed using the Morris water maze (MWM). Gut microbiota composition was analyzed by 16S rRNA sequencing, and intestinal morphology was examined histologically. Gene expression of neuroinflammatory markers and mitophagy-related genes in brain tissue was quantified by RT-PCR. Plasma levels of cell-free mitochondrial DNA (cf-mtDNA) were measured as a marker of mitochondrial damage. Results: Combined LPS + ZDV exposure induced systemic inflammation, impaired spatial memory, damaged the intestinal mucosa, and caused dysbiosis characterized by an increase in pro-inflammatory Muribaculaceae. In the brain, LPS + ZDV significantly upregulated Tnfa expression, confirming neuroinflammation. Bacillus subtilis administration prevented cognitive deficits, maintained Tnfa at control levels, and significantly reduced Il1b and Il6 expression compared to the LPS + ZDV group. This was accompanied by activation of the PINK1/PTEN-dependent mitophagy pathway, prevention of cf-mtDNA release, and restoration of gut microbial diversity. In contrast, the Lactobacilli mixture not only failed to improve outcomes but was associated with exacerbated intestinal damage, more pronounced cognitive dysfunction, and no reduction in neuroinflammatory markers. Conclusions: Combined exposure to LPS and ZDV induces gut–brain axis dysfunction characterized by neuroinflammation, cognitive impairment, intestinal damage, and dysbiosis. Bacillus subtilis effectively preserves cognitive function through activation of PINK1/PTEN-dependent mitophagy and suppression of neuroinflammation, highlighting its potential as a therapeutic candidate for cognitive impairments associated with gut–brain axis dysfunction. The contrasting effects of the lactobacilli mixture underscore the critical importance of strain-specificity in probiotic interventions. Full article

The enzymatic production of low-molecular-weight chitosan and chitooligosaccharides (COS), with broad application potential in agriculture, food, medicine, and cosmetics, has emerged as an attractive alternative to chemical chitosan depolymerization owing to its substrate specificity and environmentally benign catalytic action. However, the functional properties of available chitosanases need to be enhanced to meet the demands of industrial COS manufacturing under high temperature and substrate concentrations. In this work, we performed directed evolution on a recombinant Bacillus subtilis chitosanase to increase chitosan hydrolysis performance and thermal resistance. Three rounds of directed evolution screening (~9000 clones) yielded variants MT1, MT2, and MT3 with higher specific activity, achieved through Vmax improvement and increased T_1/2 at 60 °C. HPLC, DLS, and MALDI-TOF results indicate differences in the hydrolysis kinetics and size distribution of COS products over reaction time, suggesting a narrower distribution and a lower average molecular weight. Molecular dynamics simulations and docking studies revealed potential modulation of chitosanase activity via changes in the opening and closing dynamics of the active-site cleft. These results suggest that future efforts targeting the cleft interface could significantly advance both the catalytic performance and the mechanistic understanding of GH46 family chitosanases. Full article

Bacterial and fungal infections, together with oxidative stress-mediated damage, remain major challenges in human health and in the protection of materials, highlighting the need for new multifunctional molecules that combine antioxidant and antimicrobial properties. In this context, a new chloropyridine-based derivative, N4,N4-bis((6-chloropyridin-3-yl)methyl)-N1,N1-diethylpentane-1,4-diamine (AMZ), was synthesized via a simple, catalyst-free N-alkylation of N1,N1-diethylpentane-1,4-diamine with 2-chloro-4-(chloromethyl)pyridine in acetonitrile at 55 °C, affording a 62% yield. The structure of AMZ was confirmed by melting point determination, ¹H and ¹³C NMR spectroscopy, and EI–MS analysis. Its antioxidant activity was evaluated using DPPH and FRAP assays with BHT as a reference standard, while antibacterial and antifungal activities were assessed via disk diffusion and microdilution methods to determine inhibition zones and MIC/MBC values. In silico investigations included drug-likeness and ADMET predictions, as well as molecular docking on catalase (PDB: 2CAG) and fungal CYP51 (PDB: 1EA1). AMZ exhibited dose-dependent radical scavenging in the DPPH assay, reaching 76.88 ± 3.20% inhibition at 1000 µg/mL, with an EC₅₀ of 26.03 ± 0.21 µg/mL, close to that of BHT (23.65 ± 0.22 µg/mL). In the FRAP assay, AMZ showed a higher reducing power than BHT at a low concentration (OD₅₀ µg/mL 0.177 ± 0.023 vs. 0.134 ± 0.017), although its FRAP EC₅₀ was higher (700.48 ± 22.54 vs. 400.16 ± 8.67 µg/mL). AMZ displayed broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria and fungi, with particularly strong effects on Bacillus subtilis (44.5 ± 0.5 mm; MIC/MBC 0.008 mg/mL) and Aspergillus niger (30 mm; MIC/MBC 0.030 mg/mL), in some cases comparable or superior to streptomycin and fluconazole. In silico analysis indicated that AMZ fulfilled major drug-likeness rules, showed high predicted intestinal absorption (91.14%), and was classified as non-AMES toxic, while docking predicted favorable binding to catalase and CYP51, in agreement with the experimental antioxidant and antifungal activities. These findings highlight the potential of AMZ as a multi-target pyridine-based lead compound that warrants further structural optimization and in vivo evaluation for applications in oxidative-stress-related and infectious conditions. Full article

Background: Nigella sativa (black cumin) seeds are renowned for their ethnomedicinal significance and are rich in bioactive phytochemicals, which contribute to food preservation and the prevention of various diseases through their antimicrobial and antioxidant properties. Accordingly, this study aimed to characterize the phytochemical composition of N. sativa seed extracts, isolate thymoquinone, and assess their antibacterial, antibiofilm, antioxidant, anti-inflammatory and antidiabetic activities. Methods: Nigella sativa seed extracts were prepared using solvents of increasing polarity and analyzed for phytochemical content. Metabolite profiling was performed using UHPLC/QTOF-MS. Thymoquinone, the major constituent, was isolated via thin-layer chromatography (TLC), further purified using semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC), and evaluated in vitro for antibacterial, antibiofilm, antioxidant, anti-inflammatory, and antidiabetic activities. Results: Extraction yields ranged from 5.5% to 8.4% (w/w), with methanol yielding the highest phenol (6.34 ± 0.31 mg GAE/mL) and flavonoid (5.12 ± 0.26 mg QE/mL) contents. UHPLC/QTOF-MS revealed a chemically diverse profile dominated by thymoquinone (58% relative abundance), alongside p-cymene, carvacrol, longifolene, and nigellidine. Thymoquinone (Rf = 0.56) was initially isolated from the methanolic extract with a yield of 270 mg/g and further purified from preparative TLC fractions using semi-preparative RP-HPLC, affording 82 mg of >95% pure compound with a 68.3% recovery, suitable for subsequent biological assays. It inhibited Gram-positive and Gram-negative bacteria, with MICs of 62.5 µg/mL against Staphylococcus aureus, Bacillus subtilis, and Listeria monocytogenes; 125–250 µg/mL against Escherichia coli and Salmonella typhimurium; and 500 µg/mL against Pseudomonas aeruginosa. Thymoquinone reduced biofilm formation (>80% at 25–50 µg/mL; MBIC₅₀ ≈ 5.4–11.6 µg/mL), exhibited antioxidant activity (DPPH IC₅₀ = 52.3 ± 2. 1 µg/mL; ABTS IC₅₀ = 41.6 ± 1.9 µg/mL), stabilized erythrocyte membranes (IC₅₀ ≈ 14.8 µg/mL), and inhibited carbohydrate-hydrolyzing enzymes, with stronger inhibition of α-glucosidase (~92%) than α-amylase (~84%) at 128 µg/mL. Conclusions: Thymoquinone is a major bioactive constituent of N. sativa seeds, exhibiting consistent multi-target in vitro activity. These findings highlight its functional relevance and in vivo investigations to establish therapeutic potential. Full article