Enhanced antimicrobial activity of lactic acid bacteria through genome shuffling and genetic variability among shuffled strains.

In this investigation, lactic acid bacteria (LAB) isolated from milk were tested for their antibacterial properties and improved the antimicrobial activity of these isolates using genome shuffling. A total of sixty-one isolates were found in eleven samples, which were then tested using the agar diffusion method for their antibacterial activity against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa. Thirty-one strains exhibited antibacterial activity against at least one of the tested pathogens, with an inhibitory zone's diameter varying between 15.0 and 24.0 mm. Two isolates that showed the highest antimicrobial activity were identified as Lactobacillus plantarum CIP 103151 and Lactobacillus plantarum JCM 1149 according to 16S rRNA analysis. In the present study, applying genome shuffling approach significantly enhanced the antibacterial activity of L. plantarum. The initial populations were obtained via ultraviolet irradiation and were treated using the protoplast fusion method. The ideal condition for the production of protoplasts was 15 mg/ml of lysozyme and 10 µg/ml of mutanolysin. After two rounds of fusion, ten recombinants exhibited a significant increase in the inhibition zones versus S. aureus, S. typhimurium, P. aeruginosa, and E. coli, reaching up to 1.34, 1.31, 1.37, and 1.37-fold increase in inhibitory zone respectively. Random Amplified Polymorphic DNA results showed clear differences in DNA banding patterns among the wild strain of L. plantarum CIP 103151 and the three selected shuffled strains using primers 1283 & OPA09. On the other hand, no change was obtained using primers OPD03 neither among the wild strain and the three recombinant strains nor among the three shuffled strains.

Stable, efficient, and cost-effective system for the biosynthesis of recombinant bacterial cellulose in Escherichia coli DH5α platform.

BACKGROUND: Owing to its remarkable mechanical properties that surpass the plant-based cellulose, bacterial cellulose production has been targeted for commercialization during the last few years. However, the large-scale production of cellulose is generally limited by the slow growth of producing strains and low productivity which ultimately makes the commercial production of cellulose using the conventional strains non cost-effective. In this study, we developed a novel plasmid-based expression system for the biosynthesis of cellulose in E. coli DH5α and assessed the cellulose productivity relative to the typically used E. coli BL21 (DE) expression strain. RESULTS: No production was detected in BL21 (DE3) cultures upon expression induction; however, cellulose was detected in E. coli DH5α as early as 1 h post-induction. The total yield in induced DH5α cultures was estimated as 200 ± 5.42 mg/L (dry weight) after 18 h induction, which surpassed the yield reported in previous studies and even the wild-type Gluconacetobacter xylinum BRC5 under the same conditions. As confirmed with electron microscope micrograph, E. coli DH5α produced dense cellulose fibers with ~ 10 µm diameter and 1000-3000 µm length, which were remarkably larger and more crystalline than that typically produced by G. hansenii. CONCLUSIONS: This is the first report on the successful cellulose production in E. coli DH5α which is typically used for plasmid multiplication rather than protein expression, without the need to co-express cmcax and ccpAx regulator genes present in the wild-type genome upstream the bcs-operon, and reportedly essential for the biosynthesis.

Alginate/κ-carrageenan oral microcapsules loaded with Agaricus bisporus polysaccharides MH751906 for natural killer cells mediated colon cancer immunotherapy.

The current study explores the effect of the extracted novel Mushroom polysaccharides and its formulation onto Alginate (Alg.)/kappa carrageenan microcapsules to exert immunotherapeutic effect upon activating gut resident natural killer cells (NK) against colon cancer. The extracted polysaccharides of Agaricus bisporus MH751906 was microcapsulated in Alg/κ-carrageenan microcapsules as an oral delivery system for colon cancer. The microcapsule is characterized by SEM, FTIR, Raman and TGA; and showed a superior acidic stability, controlled release, and thermal stability at high temperature with higher hydrogel swelling rate in colon-mimicking pH. Upon activation of human NK cells with microcapsules (ANK cells), a significant increase in CD16+CD56+ NK cell populations were recorded. These activated NK cells showed 74.09% cytotoxic effects against human colon cancer Caco-2 cells where majority of cancer cell populations arrested at G0/G1 phase leading to apoptosis. The apoptotic molecular mechanism induced by ANK cells on Caco-2 treated cells is through down regulations of both BCL2 and TGF surviving genes and up regulation in IkappaB-α gene expression. Therefore, this novel polysaccharides-alginate/κ-carrageenan microcapsules can be used as an oral targeted delivery system for colon cancer immunotherapy.

Antimicrobial synergism and antibiofilm activity of amoxicillin loaded citric acid-magnesium ferrite nanocomposite: Effect of UV-illumination, and membrane leakage reaction mechanism.

Magnesium ferrite nanoparticles (Mg Fe2O4 NPs) was synthesized by a chemical co-precipitation method and characterized via structural and optical properties. The surface of Mg Fe2O4 NPs was stabilized with citric acid (CA) by a direct addition method (CA-Mg Fe2O4 NPs), then Amoxicillin (AX) was loaded with CA-Mg Fe2O4 nanocomposites. Furthermore, their antimicrobial, and antibiofilm activities, growth curve, and effect of UV-illumination methods were examined against different pathogenic microbes. Based on XRD, HRTEM and SEM analyses, it is found that Mg Fe2O4 NPs are located at the core, while the CA and AX are coated this core. In-vitro zone of inhibition (ZOI) and minimum inhibitory concentration (MIC) results verified that AX-loaded CA-Mg Fe2O4 nanocomposites exhibited its encouraged antimicrobial activity against S. aureus, E. coli, and C. albicans (32.2, 22.0, and 19.0 mm ZOI, respectively) & (0.312, 0.625, and 1.25 µg/ml MIC, respectively). AX-CA-Mg Fe2O4 nanocomposites are showed antibiofilm percentage against S. aureus (95.34%), E. coli (93.93%), and C. albicans (76.23%). AX-CA-MgFe2O4 nanocomposites are an excellent disinfectant agents once they are excited by UV light. Membrane leakage assay explains the formation of holes on the surface of bacteria, and confirms SEM reaction mechanism. AX-loaded CA-Mg Fe2O4 NPs are promising for potential applications in biomedical uses.

Structural assessment of the bioplastic (poly-3-hydroxybutyrate) produced by Bacillus flexus Azu-A2 through cheese whey valorization.

The demand for the production of biodegradable plastics has significantly increased. Bioplastics have become an essential alternative to the threats of the daily consumable plastics, sourced from fossil fuels, to the environment. Polyhydroxyalkonates (PHAs) are a ubiquitous group of bioderived and biodegradable plastics, however their production is limited by the costs associated mainly with the carbon sources. Herein, this study aims to reduce the PHAs production cost by using a by-product from the dairy industry, i.e., cheese whey (CW), as a sole carbon source. The developed process recruits an aquatic isolate, Bacillus flexus Azu-A2, and is optimized via studying various parameters using the shaking flasks technique. The results showed that the maximum PHA production (0.95 g L-1) and PHA content (20.96%, w/w), were obtained after incubation period 72 h at 45 °C, 100 rpm agitation rate, 50% CWS concentration, pH 8.5, and 1.0 g L-1 ammonium chloride. Physiochemically, Fourier transform infrared spectroscopy (FTIR), gas chromatography-mass spectroscopy (GC-MS), nuclear magnetic resonance (NMR), and energy-dispersive X-ray (EDX) techniques, emphasized the type of the extracted PHA as polyhydroxybutyrate (PHB). The thermal properties of PHB were measured using differential scanning calorimetry (DSC), recording melting transition temperature (Tm) at 170.96 °C. Furthermore, a scanning electron microscope (SEM) visualized a homogenous microporous structure for the thin PHB biofilm. In essence, this study highlights the ability of Bacillus flexus Azu-A2 to produce a good yield of highly purified PHB at reduced production cost from dairy CW. Consequently, the current study paves the way for an improved whey management strategy.

Statistical optimization and characterization of a biocellulose produced by local Egyptian isolate Komagataeibacter hansenii AS.5.

Optimization of the culture parameters used for biocellulose (BC) production by a previously isolated bacterial strain (Komagataeibacter hansenii AS.5) was carried out. The effect of nine culture parameters on BC production was evaluated by implementing the Plackett-Burman design, and the results revealed that, the most significant variables affecting BC production were MgSO4, ethanol, pH and yeast extract. A three-level and four-factor Box-Behnken design was applied to determine the optimum level of each significant variable. According to the results of the Plackett-Burman (PBD) and Box-Behnken designs (BBD), the following medium composition and parameters were calculated to be optimum (g/l): glucose 25, yeast extract 13, MgSO4 0.15, KH2PO4 2, ethanol 7.18 ml/l, pH 5.5, inoclume size 7%, cultivation temperature 20 °C and incubation time 9 days. Characterization of purified BC was performed to determine the network morphology by scanning electron microscopy, crystallinity by X-ray diffraction, chemical structure and functional groups by Fourier-transform infrared spectroscopy, thermal stability by thermogravimetric analysis and mechanical properties such as Young's modulus, tensile strength and elongation at beak % of BC.

Biodiesel Production by Aspergillus niger Lipase Immobilized on Barium Ferrite Magnetic Nanoparticles.

In this study, Aspergillus niger ADM110 fungi was gamma irradiated to produce lipase enzyme and then immobilized onto magnetic barium ferrite nanoparticles (BFN) for biodiesel production. BFN were prepared by the citrate sol-gel auto-combustion method and characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) and scanning electron microscopy with energy dispersive analysis of X-ray (SEM/EDAX) analysis. The activities of free and immobilized lipase were measured at various pH and temperature values. The results indicate that BFN-Lipase (5%) can be reused in biodiesel production without any treatment with 17% loss of activity after five cycles and 66% loss in activity in the sixth cycle. The optimum reaction conditions for biodiesel production from waste cooking oil (WCO) using lipase immobilized onto BFN as a catalyst were 45 °C, 4 h and 400 rpm. Acid values of WCO and fatty acid methyl esters (FAMEs) were 1.90 and 0.182 (mg KOH/g oil), respectively. The measured flash point, calorific value and cetane number were 188 °C, 43.1 MJ/Kg and 59.5, respectively. The cloud point (-3 °C), pour point (-9 °C), water content (0.091%) and sulfur content (0.050%), were estimated as well.

Induced resistance in tomato plants by IAA against Fusarium oxysporum lycopersici.

The phytohormone IAA (indol-3-acetic acid) was tested in vitro on growth of tomato wilt pathogen Fusarium oxysporum lycopersici. The hormone reduced spore germination, mycelial dry weight and protein content. Such reduction was matched with the elevation in the hormone concentration. The in vivo application of IAA to soil of the uninoculated plants (controls) improved growth and yielded longer shoot and root, particularly at low concentrations. Moreover, the hormone could prevent completely any chance for disease incidence by soil pathogens. Presence of IAA in soil of inoculated plants not only reduced the infection rate but also increased plant growth, causing that they appeared healthy and normal. Disease suppression in tomato plants, exerted by application of IAA, was achieved through either increasing plant growth, exerting a direct harmful effect on the target pathogen and/or inducing resistance in host tissue. The induced resistance was correlated with induction of certain secondary metabolites which may have a role in increasing tolerance in tomato plants to the pathogen.