Green Synthesis of Zinc Oxide Nanoparticles from Althaea officinalis Flower Extract Coated with Chitosan for Potential Healing Effects on Diabetic Wounds by Inhibiting TNF-α and IL-6/IL-1ß Signaling Pathways.

Background: Diabetes Mellitus is a multisystem chronic pandemic, wound inflammation, and healing are still major issues for diabetic patients who may suffer from ulcers, gangrene, and other wounds from uncontrolled chronic hyperglycemia. Marshmallows or Althaea officinalis (A.O.) contain bioactive compounds such as flavonoids and phenolics that support wound healing via antioxidant, anti-inflammatory, and antibacterial properties. Our study aimed to develop a combination of eco-friendly formulations of green synthesis of ZnO-NPs by Althaea officinalis extract and further incorporate them into 2% chitosan (CS) gel. Method and Results: First, develop eco-friendly green Zinc Oxide Nanoparticles (ZnO-NPs) and incorporate them into a 2% chitosan (CS) gel. In-vitro study performed by UV-visible spectrum analysis showed a sharp peak at 390 nm, and Energy-dispersive X-ray (EDX) spectrometry showed a peak of zinc and oxygen. Besides, Fourier transforms infrared (FTIR) was used to qualitatively validate biosynthesized ZnO-NPs, and transmission electron microscope (TEM) showed spherical nanoparticles with mean sizes of 76 nm and Zeta potential +30mV. The antibacterial potential of A.O.-ZnO-NPs-Cs was examined by the diffusion agar method against Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Based on the zone of inhibition and minimal inhibitory indices (MIC). In addition, an in-silico study investigated the binding affinity of A.O. major components to the expected biological targets that may aid wound healing. Althaea Officinalis, A.O-ZnO-NPs group showed reduced downregulation of IL-6, IL-1ß, and TNF-α and increased IL-10 levels compared to the control group signaling pathway expression levels confirming the improved anti-inflammatory effect of the self-assembly method. In-vivo study and histopathological analysis revealed the superiority of the nanoparticles in reducing signs of inflammation and wound incision in rat models. Conclusion: These biocompatible green zinc oxide nanoparticles, by using Althaea Officinalis chitosan gel ensure an excellent new therapeutic approach for quickening diabetic wound healing.

Bacillus subtilis stimulates plant growth and production of bioactive components in saffron.

The effects of B. subtilis on the morphology and physiology of saffron were investigated using two types of soils. Three different bacterial suspensions were applied at 14-day intervals to treat saffron. Morphological attributes were recorded, and the amounts of α-crocin and safranal in the stigma extracts were quantified. The longest stigma, petal, and leaf were observed in the treated groups with 105 and 108 cfu/ml. The highest weight of stigma per corm belonged to the treated groups with 102 cfu/ml in unsterile soil and 105 and 108 cfu/ml in sterile soil. Treatment with 102 and 108 cfu/ml caused a significant increase in safranal production in sterile and unsterile peat/perlite. While treatment with 105 and 108 cfu/ml in sterile peat/perlite and exposure to 102 cfu/ml in unsterile peat/perlite soil resulted in an increase in α-crocin. The data showed that B. subtlis triggers the morphological and physiological processes in saffron.

Combinatorial metabolic engineering of Bacillus subtilis for de novo production of polymyxin B.

Polymyxin is a lipopeptide antibiotic that is effective against multidrug-resistant Gram-negative bacteria. However, its clinical development is limited due to low titer and the presence of homologs. To address this, the polymyxin gene cluster was integrated into Bacillus subtilis, and sfp from Paenibacillus polymyxa was expressed heterologously, enabling recombinant B. subtilis to synthesize polymyxin B. Regulating NRPS domain inhibited formation of polymyxin B2 and B3. The production of polymyxin B increased to 329.7 mg/L by replacing the native promoters of pmxA, pmxB, and pmxE with PfusA, C2up, and PfusA, respectively. Further enhancement in this production, up to 616.1 mg/L, was achieved by improving the synthesis ability of 6-methyloctanoic acid compared to the original strain expressing polymyxin heterologously. Additionally, incorporating an anikasin-derived domain into the hybrid nonribosomal peptide synthase of polymyxin increased the B1 ratio in polymyxin B from 57.5% to 62.2%. Through optimization of peptone supply in the fermentation medium and fermentation in a 5.0-L bioreactor, the final polymyxin B titer reached 962.1 mg/L, with a yield of 19.24 mg/g maltodextrin and a productivity of 10.02 mg/(L·h). This study demonstrates a successful approach for enhancing polymyxin B production and increasing the B1 ratio through combinatorial metabolic engineering.

Combinatorial metabolic engineering of Bacillus subtilis enables the efficient biosynthesis of isoquercitrin from quercetin.

BACKGROUND: Isoquercitrin (quercetin-3-O-ß-D-glucopyranoside) has exhibited promising therapeutic potentials as cardioprotective, anti-diabetic, anti-cancer, and anti-viral agents. However, its structural complexity and limited natural abundance make both bulk chemical synthesis and extraction from medical plants difficult. Microbial biotransformation through heterologous expression of glycosyltransferases offers a safe and sustainable route for its production. Despite several attempts reported in microbial hosts, the current production levels of isoquercitrin still lag behind industrial standards. RESULTS: Herein, the heterologous expression of glycosyltransferase UGT78D2 gene in Bacillus subtilis 168 and reconstruction of UDP-glucose (UDP-Glc) synthesis pathway led to the synthesis of isoquercitrin from quercetin with titers of 0.37 g/L and 0.42 g/L, respectively. Subsequently, the quercetin catabolism blocked by disruption of a quercetin dioxygenase, three ring-cleavage dioxygenases, and seven oxidoreductases increased the isoquercitrin titer to 1.64 g/L. And the hydrolysis of isoquercitrin was eliminated by three ß-glucosidase genes disruption, thereby affording 3.58 g/L isoquercitrin. Furthermore, UDP-Glc pool boosted by pgi (encoding glucose-6-phosphate isomerase) disruption increased the isoquercitrin titer to 10.6 g/L with the yield on quercetin of 72% and to 35.6 g/L with the yield on quercetin of 77.2% in a 1.3-L fermentor. CONCLUSION: The engineered B. subtilis strain developed here holds great potential for initiating the sustainable and large-scale industrial production of isoquercitrin. The strategies proposed in this study provides a reference to improve the production of other flavonoid glycosides by engineered B. subtilis cell factories.

Prebiotic levan type fructan from Bacillus subtilis PR-C18 as a potent antibiofilm agent: Structural elucidation and in silico analysis.

The global demand for therapeutic prebiotics persuades the quest for novel exopolysaccharides that can retard the growth of pathobionts and healthcare-associated pathogens. In this regard, an exopolysaccharide (3.69 mg/mL) producing strain showing prebiotic and antibiofilm activity was isolated from indigenous pineapple pomace of Tripura and identified as Bacillus subtilis PR-C18. Zymogram analysis revealed EPS PR-C18 was synthesized by levansucrase (∼57 kDa) with a maximal activity of 4.62 U/mg. Chromatography techniques, FTIR, and NMR spectral data revealed the homopolymeric nature of purified EPS with a molecular weight of 3.40 × 104 Da. SEM and rheological study unveiled its microporous structure and shear-thinning effect. Furthermore, EPS PR-C18 showed remarkable emulsification, flocculation, water retention, water solubilization, and antioxidant activity. DSC-TGA data demonstrated its high thermostability and cytotoxicity analysis verified its nontoxic biocompatible nature. In addition, the antibiofilm activity of EPS PR-C18 was validated using molecular docking, molecular simulation, MM-GBSA and PCA studies, which exhibited its strong binding affinity (-20.79 kcal/moL) with PelD, a virulence factor from Pseudomonas aeruginosa. Together, these findings support the future exploitation of EPS PR-C18 as an additive or adjuvant in food and pharmaceutical sectors.

A historical sequence deletion in a commonly used Bacillus subtilis chromosome integration vector generates undetected loss-of-function mutations.

Since the 1980s, chromosome-integration vectors have been used as a core method of engineering Bacillus subtilis. One of the most frequently used vector backbones contains chromosomally derived regions that direct homologous recombination into the amyE locus. Here, we report a gap in the homology region inherited from the original amyE integration vector, leading to erroneous recombination in a subset of transformants and a loss-of-function mutation in the downstream gene. Internal to the homology arm that spans the 3' portion of amyE and the downstream gene ldh, an unintentional 227 bp deletion generates two crossover events. The major event yields the intended genotype, but the minor event, occurring in ~10 % of colonies, results in a truncation of ldh, which encodes lactate dehydrogenase. Although both types of colonies test positive for amyE disruption by starch plating, the potential defect in fermentative metabolism may be left undetected and confound the results of subsequent experiments.

Production of ultra-high-molecular-weight poly-γ-glutamic acid by a newly isolated Bacillus subtilis strain and genomic and transcriptomic analyses.

Poly-γ-glutamic acid (γ-PGA) is a microbial-derived polymer with molecular weight (Mw) from 104 to 107 Da, and the high-Mw (> 7.0 × 105 Da) or ultra-high-Mw (> 5.0 × 106 Da) γ-PGA has important application value as a tissue engineering material, as a flocculant, and as a heavy metal remover. Therefore, how to produce these high-Mw γ-PGAs with low cost and high efficiency has attracted wide attention. In this study, a γ-PGA producer was isolated from the natural environment, and identified and named Bacillus subtilis GXD-20. Then, the ultra-high-Mw (> 6.0 × 106 Da) γ-PGA produced by GXD-20 was characterized. Interestingly, GXD-20 could produce γ-PGA at 42°C, and exhibited a γ-PGA titer of up to 22.29 ± 0.59 g L-1 in a 5-L fermenter after optimization of the fermentation process. Comparative genomic analysis indicated that the specific protein sequence and subcellular localization of PgdS (a γ-PGA-degrading enzyme) were closely related to the ultra-high-Mw of γ-PGA. Transcriptomic analysis revealed that the high γ-PGA titer at 42°C was mainly related to the high expression of genes encoding enzymes for sucrose transportation and utilization, nitrogen transportation, endogenous glutamate synthesis, and γ-PGA synthesis. These results provide new insights into the production of ultra-high-Mw γ-PGA by Bacillus at high temperatures.

Discovery of a Novel Lidocaine Metabolite by Human Liver Microsome and Identification of Microbial Species Which Produces the Same Metabolite.

Preparation of drug metabolites at the milligram scale is essential for determining the structure and toxicity of drug metabolites. However, their preparation using recombinant proteins and human liver microsomes (HLM) is often difficult because of technical and ethical issues. Reproducing human drug metabolism in food-derived microorganisms may be useful for overcoming these challenges. In this study, we identified an unknown metabolite of the anaesthetic drug lidocaine, which is metabolised by HLM. By screening for lidocaine metabolic activity in five types of foods (blue cheese, shiitake mushroom, natto, yoghurt, and dry yeast), we found that bacteria isolated from natto reproduced the lidocaine metabolic reaction that occurs in HLM. A fraction containing the unknown lidocaine metabolite was prepared through mass cultivation of a Bacillus subtilis standard strain, ethyl acetate extraction, open column chromatography, and HPLC purification. We identified the unknown metabolite as 3-(2,6-dimethylphenyl)-1-ethyl-2-methyl-4-imidazolidinone using NMR. Our results showed that food-derived microorganisms can produce large amounts of human drug metabolites via large-scale cultivation. Additionally, food microorganisms that can reproduce drug metabolism in humans can be used to examine drug metabolites at a low cost and without ethical issues.

Inhibition of Potato Fusarium Wilt by Bacillus subtilis ZWZ-19 and Trichoderma asperellum PT-29: A Comparative Analysis of Non-Targeted Metabolomics.

Potato Fusarium Wilt is a soil-borne fungal disease that can seriously harm potatoes throughout their growth period and occurs at different degrees in major potato-producing areas in China. To reduce the use of chemical agents and improve the effect of biocontrol agents, the inhibitory effects of the fermentation broth of Bacillus subtilis ZWZ-19 (B) and Trichoderma asperellum PT-29 (T) on Fusarium oxysporum were compared under single-culture and co-culture conditions. Furthermore, metabolomic analysis of the fermentation broths was conducted. The results showed that the inhibitory effect of the co-culture fermentation broth with an inoculation ratio of 1:1 (B1T1) was better than that of the separately cultured fermentation broths and had the best control effect in a potted experiment. Using LC-MS analysis, 134 metabolites were determined and classified into different types of amino acids. Furthermore, 10 metabolic pathways had the most significant variations, and 12 were related to amino acid metabolism in the KEGG analysis. A correlation analysis of the 79 differential metabolites generated through the comprehensive comparison between B, T, and B1T1 was conducted, and the results showed that highly abundant amino acids in B1T1 were correlated with amino acids in B, but not in T.

Strain-Specific Benefits of Bacillus Probiotics in Hybrid Grouper: Growth Enhancement, Metabolic Health, Immune Modulation, and Vibrio harveyi Resistance.

In the realm of modern aquaculture, the utilization of probiotics has gained prominence, primarily due to their ability to enhance growth, boost immunity, and prevent diseases in aquatic species. This study primarily investigates the efficacy of Bacillus subtilis strains, both host-derived and from other sources, in influencing fish growth, immunity, lipid metabolism, and disease resistance. Employing a 42-day feeding trial, we divided hybrid grouper into four distinct groups: a control group on a basal diet and three experimental groups supplemented with 1 × 108 CFU/g of different Bacillus subtilis strains-BS, 6-3-1, and HAINUP40. Remarkably, the study demonstrated that the 6-3-1 and HAINUP40 groups exhibited significant enhancements across key growth parameters: final body weight (FBW), weight gain rate (WGR), feed intake (FI), feed efficiency ratio (FER), and feed conversion ratio (FCR). The investigation into lipid metabolism revealed that the 6-3-1 strain upregulated seven metabolism-related genes, HAINUP40 affected four metabolism-related genes, and the BS strain influenced two metabolism-related genes, indicating diverse metabolic impacts by different strains. Further, a notable reduction in liver enzymes AST and ALT was observed across all supplemented groups, implying improved liver health. Noteworthy was the BS strain's superior antioxidative capabilities, positively affecting all four measured parameters (CAT, GSH-Px, MDA). In the sphere of immune-related gene expression, the BS strain significantly decreased the expression of both inflammation and apoptosis-related genes, whereas the HAINUP40 strain demonstrated an upregulation in these genes. The challenge test results were particularly telling, showcasing improved survival rates against Vibrio harveyi infection in the BS and 6-3-1 groups, unlike the HAINUP40 group. These outcomes highlight the strain-specific nature of probiotics and their varying mechanisms of action within the host. In conclusion, this study reveals that probiotic strains, varying by source, demonstrate unique, strain-specific effects in promoting growth and modulating immunity in hybrid grouper. This research highlights the promise of tailored probiotic applications in improving aquaculture practices. Such advancements contribute to more sustainable and efficient fish farming methods.

Impact of nitrogen fertilizer sustainability on corn crop yield: the role of beneficial microbial inoculation interactions.

BACKGROUND: Considering the challenges posed by nitrogen (N) pollution and its impact on food security and sustainability, it is crucial to develop management techniques that optimize N fertilization in croplands. Our research intended to explore the potential benefits of co-inoculation with Azospirillum brasilense and Bacillus subtilis combined with N application rates on corn plants. The study focused on evaluating corn photosynthesis-related parameters, oxidative stress assay, and physiological nutrient use parameters. Focus was placed on the eventual improved capacity of plants to recover N from applied fertilizers (AFR) and enhance N use efficiency (NUE) during photosynthesis. The two-year field trial involved four seed inoculation treatments (control, A. brasilense, B. subtilis, and A. brasilense + B. subtilis) and five N application rates (0 to 240 kg N ha-1, applied as side-dress). RESULTS: Our results suggested that the combined effects of microbial consortia and adequate N-application rates played a crucial role in N-recovery; enhanced NUE; increased N accumulation, leaf chlorophyll index (LCI), and shoot and root growth; consequently improving corn grain yield. The integration of inoculation and adequate N rates upregulated CO2 uptake and assimilation, transpiration, and water use efficiency, while downregulated oxidative stress. CONCLUSIONS: The results indicated that the optimum N application rate could be reduced from 240 to 175 kg N ha-1 while increasing corn yield by 5.2%. Furthermore, our findings suggest that replacing 240 by 175 kg N ha-1 of N fertilizer (-65 kg N ha-1) with microbial consortia would reduce CO2 emission by 682.5 kg CO2 -e ha-1. Excessive N application, mainly with the presence of beneficial bacteria, can disrupt N-balance in the plant, alter soil and bacteria levels, and ultimately affect plant growth and yield. Hence, highlighting the importance of adequate N management to maximize the benefits of inoculation in agriculture and to counteract N loss from agricultural systems intensification.

Genomic characterization and related functional genes of γ- poly glutamic acid producing Bacillus subtilis.

γ- poly glutamic acid (γ-PGA), a high molecular weight polymer, is synthesized by microorganisms and secreted into the extracellular space. Due to its excellent performance, γ-PGA has been widely used in various fields, including food, biomedical and environmental fields. In this study, we screened natto samples for two strains of Bacillus subtilis N3378-2at and N3378-3At that produce γ-PGA. We then identified the γ-PGA synthetase gene cluster (PgsB, PgsC, PgsA, YwtC and PgdS), glutamate racemase RacE, phage-derived γ-PGA hydrolase (PghB and PghC) and exo-γ-glutamyl peptidase (GGT) from the genome of these strains. Based on these γ-PGA-related protein sequences from isolated Bacillus subtilis and 181 B. subtilis obtained from GenBank, we carried out genotyping analysis and classified them into types 1-5. Since we found B. amyloliquefaciens LL3 can produce γ-PGA, we obtained the B. velezensis and B. amyloliquefaciens strains from GenBank and classified them into types 6 and 7 based on LL3. Finally, we constructed evolutionary trees for these protein sequences. This study analyzed the distribution of γ-PGA-related protein sequences in the genomes of B. subtilis, B. velezensis and B. amyloliquefaciens strains, then the evolutionary diversity of these protein sequences was analyzed, which provided novel information for the development and utilization of γ-PGA-producing strains.

Lignocellulose and probiotic supplementation in broiler chicken diet: effect on growth performance, digestive health, litter quality, and genes expression.

Three hundred one-day-old Avian 48 broiler chicks were used to investigate the effect of lignocellulose (LC) and probiotic supplementation in broiler chicken diet on growth performance, digestive health, litter quality, and some gene expression. Experimental treatments consisted of 3 × 2 factorial arrangements with 3 levels of LC without or with probiotics to formulate 6 experimental groups. Groups 1, 2, and 3 were fed on the basal diet with dietary LC inclusion at 0, 0.5, and 1.0%, respectively, while groups 4, 5, and 6 were fed on the previously mentioned design with Bacillus subtilis at 100 gm/ton. The results revealed that Dietary LC inclusion nonsignificantly (P ≥ 0.05) reduced body weight (BW), body weight gain (BWG), and feed intake. Meanwhile, B. subtilis supplementation improved BW and BWG and enhanced the effect of LC on the broilers' weight. The group fed a 0.5% LC and B. subtilis-supplemented diet recorded the best (P ≥ 0.05) BW, BWG, FCR, PER, EEU, and PI. LC and or B. subtilis supplementation improved carcass traits of broiler (higher dressing% with lower abdominal fat% compared with a control group), intestinal health, and absorptive capacity. LC potentiates the effect of B. subtilis supplementation in broilers' diet in modulating intestinal microflora (lowered (P ≥ 0.05) cecal Coliform and increased Lactobacillus counts), the highest Coliform counts were recorded in group fed 0.5 or 1.0% LC plus B. subtilis. LC at 0.5 or 1.0% and or B. subtilis supplementation reduced (P ≥ 0.05) litter moisture% at the 2nd, 4th, and 6th wk compared to the control group. Dietary inclusion of LC and or B. subtilis supplementation significantly (P < 0.001) up-regulated hepatic growth-related genes (growth hormone receptor (GHR) and insulin growth factor1 (IGF-1)) and antioxidant-related genes (superoxide dismutase 1 (SOD1), glutathione peroxidase (GPX1) and uncoupling protein (UCP) and down-regulated (P < 0.001) splenic toll-like receptor 4 (TLRP) gene expression while had no significant effect on splenic interleukin 8 (IL8) and tumor necrosis factor (TNF) with the best-obtained results with 1.0% followed by 0.5% LC with B. subtilis supplementation. We concluded that dietary LC and/or B. subtilis supplementation positively affected the growth performance, feed efficiency, carcass quality, intestinal absorptive capacity and health, litter quality and growth, and antioxidant and immune-related gene expression.

Purification, characterization and application of collagenolytic protease from Bacillus subtilis strain MPK.

A new extracellular protease from Bacillus subtilis strain MPK with collagenolytic activity was isolated and purified. Fish skin which otherwise would be treated as waste is used as substrate for the production of protease. Using various techniques such as ammonium sulphate precipitation and ion exchange chromatography, protease was purified and characterized subsequently. Protease of approximately 61 kDa molecular weight was purified by 135.7-fold with 18.42% enzyme recovery. The protease showed effective properties like pH and temperature stability over a broad range with optimum pH 7.5 and temperature 60 °C. Km and Vmax were found to be 1.92 mg ml-1 and 1.02 × 10-4 mol L-1 min-1, respectively. The protease exhibited stability in various ions, surfactants, inhibitors and organic solvents. Subsequently, the protease was successfully utilized for collagen hydrolysis to generate collagen peptides; thus, the produced protease would be a potential candidate for multifaceted applications in food and pharmaceutical industries due to its significant characteristics and collagenolytic properties.

From Growth Inhibition to Ultrastructural Changes: Toxicological Assessment of Lambda Cyhalothrin and Fosetyl Aluminium against Bacillus subtilis and Pseudomonas aeruginosa.

In modern agricultural practices, agrochemicals and pesticides play an important role in protecting the crops from pests and elevating agricultural productivity. This strategic utilization is essential to meet global food demand due to the relentless growth of the world's population. However, the indiscriminate application of these substances may result in environmental hazards and directly affect the soil microorganisms and crop production. Considering this, an in vitro study was carried out to evaluate the pesticides' effects i.e. lambda cyhalothrin (insecticide) and fosetyl aluminum (fungicide) at lower, recommended, and higher doses on growth behavior, enzymatic profile, total soluble protein production, and lipid peroxidation of bacterial specimens (Pseudomonas aeruginosa and Bacillus subtilis). The experimental findings demonstrated a concentration-dependent decrease in growth of both tested bacteria, when exposed to fosetyl aluminum concentrations exceeding the recommended dose. This decline was statistically significant (p <.000). However, lambda cyhalothrin at three times of recommended dose induces 10 % increase in growth of Pseudomonas aeruginosa (P. aeruginosa) and 76.8 % decrease in growth of Bacillus subtilis (B. subtilis) respectively as compared to control. These results showed the stimulatory effect of lambda cyhalothrin on P. aeruginosa and inhibitory effect on B. subtilis. Pesticides induced notable alterations in biomarker enzymatic assays and other parameters related to oxidative stress among bacterial strains, resulting in increased oxidative stress and membrane permeability. Generally, the maximum toxicity of both (P. aeruginosa and B. subtilis) was shown by fosetyl aluminum, at three times of recommended dose. Fosetyl alumimium induced morphological changes like cellular cracking, reduced viability, aberrant margins and more damage in both bacterial strains as compared to lambda cyhalothrin when observed under scanning electron microscope (SEM). Conclusively the, present study provide an insights into a mechanistic approach of pyrethroid insecticide and phosphonite fungicide induced cellular toxicity towards bacteria.

Bacillus subtilis Edible Films for Strawberry Preservation: Antifungal Efficacy and Quality at Varied Temperatures.

Fungal infestations, particularly from Rhizopus stolonifer, pose significant post-harvest challenges for strawberries, compromising their shelf life and quality. Traditional preservation methods, including refrigeration, offer limited protection against such pathogens. This study introduces an innovative approach, utilizing edible films infused with Bacillus subtilis strains GOS 01 B-67748 and HFC 103, known for their antifungal properties. We demonstrate that these bioactive films not only inhibit fungal growth effectively but also enhance the preservation of strawberries at varying temperatures. The inclusion of Bacillus subtilis in edible films represents a significant advancement in extending the viability of strawberries, surpassing the efficacy of conventional methods. Our findings suggest a promising avenue for natural, safe food preservation techniques, aligning with current consumer preferences for additive-free products. This research contributes to the broader understanding of microbial-based food preservation strategies, offering potential applications across a range of perishable commodities.

Effects of adding Bacillus subtilis natto NTU-18 in paste feed on growth, intestinal morphology, gastrointestinal microbiota diversity, immunity, and disease resistance of Anguilla japonica glass eels.

Japanese eel, Anguilla japonica, holds significant importance in Taiwanese aquaculture. With the intensification of eel farming, the impact of Edwardsiella tarda has become increasingly severe. Consequently, the abusive use of antibiotics has risen. Bacillus subtilis natto NTU-18, a strain of Bacillus with a high survival rate in feed processing, plays a crucial role in promoting intestinal health through competitive rejection, enhancing immune responses against bacterial pathogens, and improving intestinal health by modulating gastrointestinal microbiota to produce beneficial metabolites of mice and grass carp, Ctenopharyngodon idella. This study investigated the effects of different proportions (control, 0.25 %, 0.5 %, 1 %, and 2 %) of B. subtilis natto NTU-18 added to paste feed on the growth performance, intestinal morphology, and microbiota, expression of immune-related genes, and resistance to E. tarda in Japanese glass eel. The results indicated that the growth performance of all groups with B. subtilis natto NTU-18 added was significantly higher than that of the control group and did not impact the villi morphology. The expression of immune-related genes in the kidney, specifically HSP70 and SOD, was significantly higher from 0.5 % and above than the control; however, no significant differences were observed in CAT, POD, and HSP90. In the liver, significant differences were found in HSP70 and IgM above 0.25 % compared to the control group, with no significant differences in SOD, CAT, POD, and HSP90 among all groups. Additionally, intestinal microbiota analysis revealed that the 2 % additional group had significantly lower diversity than other groups, with Cetobacterium as the dominant species. The challenge test observed that the survival rates of the 0.5 % and 1 % groups were significantly higher. This research suggests that adding 0.5 % and 1 % of B. subtilis natto NTU-18 to the diet is beneficial for Japanese glass eel's immunity, growth performance, and disease resistance.

Pilot-scale production of Bacillus subtilis MSCL 897 spore biomass and antifungal secondary metabolites in a low-cost medium.

OBJECTIVES: Bacillus subtilis is a plant growth promoting bacterium (PGPB) that acts as a microbial fertilizer and biocontrol agent, providing benefits such as boosting crop productivity and improving nutrient content. It is able to produce secondary metabolites and endospores simultaneously, enhancing its ability to survive in unfavorable conditions and eliminate competing microorganisms. Optimizing cultivation methods to produce B. subtilis MSCL 897 spores on an industrial scale, requires a suitable medium, typically made from food industry by-products, and optimal temperature and pH levels to achieve high vegetative cell and spore densities with maximum productivity. RESULTS: This research demonstrates successful pilot-scale (100 L bioreactor) production of a biocontrol agent B. subtilis with good spore yields (1.5 × 109 spores mL-1) and a high degree of sporulation (>80%) using a low-cost cultivation medium. Culture samples showed excellent antifungal activity (1.6-2.3 cm) against several phytopathogenic fungi. An improved methodology for inoculum preparation was investigated to ensure an optimal seed culture state prior to inoculation, promoting process batch-to-batch repeatability. Increasing the molasses concentration in the medium and operating the process in fed-batch mode with additional molasses feed, did not improve the overall spore yield, hence, process operation in batch mode with 10 g molasses L-1 is preferred. Results also showed that the product quality was not significantly impacted for up to 12 months of storage at room temperature. CONCLUSION: An economically-feasible process for B. subtilis-based biocontrol agent production was successfully developed at the pilot scale.

Single-cell analysis reveals microbial spore responses to sodium hypochlorite.

Pollution from toxic spores has caused us a lot of problems because spores are extremely resistant and can survive most disinfectants. Therefore, the detection of spore response to disinfectant is of great significance for the development of effective decontamination strategies. In this work, we investigated the effect of 0.5% sodium hypochlorite on the molecular and morphological properties of single spores of Bacillus subtilis using single-cell techniques. Laser tweezers Raman spectroscopy showed that sodium hypochlorite resulted in Ca2+-dipicolinic acid release and nucleic acid denaturation. Atomic force microscopy showed that the surface of treated spores changed from rough to smooth, protein shells were degraded at 10 min, and the permeability barrier was destroyed at 15 min. The spore volume decreased gradually over time. Live-cell imaging showed that the germination and growth rates decreased with increasing treatment time. These results provide new insight into the response of spores to sodium hypochlorite.

Structural and functional analyses of nematode-derived antimicrobial peptides support the occurrence of direct mechanisms of worm-microbiota interactions.

The complex relationships between gastrointestinal (GI) nematodes and the host gut microbiota have been implicated in key aspects of helminth disease and infection outcomes. Nevertheless, the direct and indirect mechanisms governing these interactions are, thus far, largely unknown. In this proof-of-concept study, we demonstrate that the excretory-secretory products (ESPs) and extracellular vesicles (EVs) of key GI nematodes contain peptides that, when recombinantly expressed, exert antimicrobial activity in vitro against Bacillus subtilis. In particular, using time-lapse microfluidics microscopy, we demonstrate that exposure of B. subtilis to a recombinant saposin-domain containing peptide from the 'brown stomach worm', Teladorsagia circumcincta, and a metridin-like ShK toxin from the 'barber's pole worm', Haemonchus contortus, results in cell lysis and significantly reduced growth rates. Data from this study support the hypothesis that GI nematodes may modulate the composition of the vertebrate gut microbiota directly via the secretion of antimicrobial peptides, and pave the way for future investigations aimed at deciphering the impact of such changes on the pathophysiology of GI helminth infection and disease.