Optimization of Lipase-Catalyzed Synthesis of Cetyl Octanoate in Supercritical Carbon Dioxide

Cetyl octanoate, a wax ester of 24 carbons, is widely used in the cosmetic industry as a base oil. The current work focuses on lipase-catalyzed synthesis of cetyl octanoate in supercritical carbon dioxide (SC-CO₂) by esterification of cetyl alcohol and octanoic acid. Three immobilized lipases were screened, and 15 reaction conditions were tested in order to find the combination for maximal yield. The results showed that Novozym^® 435 was the best catalyst for the synthesis, and a reaction time of 20 min was adequate for a maximal yield. Response surface methodology (RSM) with a 3-factor-3-level Box-Behnken design was employed to evaluate the effects of synthesis parameters, including reaction temperature (35–75 °C), pressure (8.27–12.41 MPa), and enzyme amount (5–15% wt of cetyl alcohol). A model for the synthesis was developed and the optimum conditions could be predicted to be reaction pressure of 10.22 MPa, reaction temperature of 63.70 °C, and enzyme amount of 11.20%. An experiment was performed under this optimum condition and a yield of 99.5% was obtained. This experimental yield correlated well with the predicted value of yield (97.6%). We concluded that, in a SC-CO₂ system, nearly 100% molar conversion of cetyl octanoate could be obtained by immobilized Novozym^® 435 in a short reaction time (20 min) under the predicted optimal conditions.     

Enzymatic Interesterification Between Pine Seed Oil and a Hydrogenated Fat to Prepare Semi-Solid Fats Rich in Pinolenic Acid and Other Polyunsaturated Fatty Acids

Enzyme catalyzed interesterification (EIE) of pine seed oil (PSO) and a fully hydrogenated soybean oil (FHSBO) were studied in batch reactors in solvent-free media to prepare different semisolid fats rich in polyunsaturated fatty acids (PUFA). Optimal operation conditions found were: 10 % (w/w) enzyme loading, 75 °C and magnetic agitation at 300 rpm. Quasi-equilibrium conditions were reached after 2, 3 and 6 h, when immobilized lipases from Thermomyces lanuginosus (Lipozyme^® TL IM), Candida antarctica B. (Novozym^® 435) and Rhizomucor miehei (Lipozyme^® RM IM) from Novozymes A/S (Bagsvaerd, Denmark) were employed, respectively. Similar distributions of unsaturated to saturated fatty acid (UFA/SFA) residues along the glycerol backbone of the fat products were obtained with both non-selective and sn-1(3) regioselective lipases due to significant spontaneous acyl migration during the reaction. The products had higher UFA/SFA ratios at the sn-2 position (2.4–2.5, 1.4–1.7, and 0.5–0.8 for the trials involving 20, 40 and 70 % FHSBO, w/w, respectively) than the corresponding physical blends (0.8, 0.4 and 0.5, respectively). Fat products containing 3.1–11.6 % (w/w) pinolenic acid (Pn) and 16.1–35.7 % (w/w) linoleic acid (L) at the sn-2 position were prepared. The free acid contents of EIE products prepared with Lipozyme^® TL IM and Novozym^® 435 were 6.1–6.4 and 2.5–2.6, respectively. Residual activities of Lipozyme^® TL IM and Novozym^® 435 diminish by ca. 20 % after 9 reaction cycles.     

Candida antarctica Lipase B Immobilized onto Chitin Conjugated with POSS? Compounds: Useful Tool for Rapeseed Oil Conversion

A new method is proposed for the production of a novel chitin-polyhedral oligomeric silsesquioxanes (POSS) enzyme support. Analysis by such techniques as X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy confirmed the effective functionalization of the chitin surface. The resulting hybrid carriers were used in the process of immobilization of the lipase type b from Candida antarctica (CALB). Fourier transform infrared spectroscopy (FTIR) confirmed the effective immobilization of the enzyme. The tests of the catalytic activity showed that the resulting support-biocatalyst systems remain hydrolytically active (retention of the hydrolytic activity up to 87% for the chitin + Methacryl POSS^® cage mixture (MPOSS) + CALB after 24 h of the immobilization), as well as represents good thermal and operational stability, and retain over 80% of its activity in a wide range of temperatures (30–60 °C) and pH (6–9). Chitin-POSS-lipase systems were used in the transesterification processes of rapeseed oil at various reaction conditions. Produced systems allowed the total conversion of the oil to fatty acid methyl esters (FAME) and glycerol after 24 h of the process at pH 10 and a temperature 40 °C, while the Methacryl POSS^® cage mixture (MPOSS) was used as a chitin-modifying agent.     

Optimization of Enzymatic Synthesis of Cetyl 2-Ethylhexanoate by Novozym<Superscript>®</Superscript> 435

Waxes are esters obtained from long-chain fatty acids and long-chain alcohols which are biodegradable, biocompatible and nontoxic. Seafowl feather oil is a natural wax ester that exists on seafowl feathers. Cetyl 2-ethylhexanoate is the major ingredient of seafowl feather oil. Cetyl 2-ethylhexanoate is widely used in cosmetics as a base oil because of its lubricity, moisture retention and non-toxic properties. An optimal production of cetyl 2-ethylhexanoate by direct esterification of cetyl alcohol with 2-ethylhexanoic acid was developed using an immobilized lipase (Novozym^® 435) as a catalyst in n-hexane. Response surface methodology (RSM) and 5-level-4-factor central composite rotatable design (CCRD) were employed to evaluate the effects of reaction time, reaction temperature, substrate molar ratio, and enzyme amount on the yield of cetyl 2-ethylhexanoate. The results show that reaction time, reaction temperature, substrate molar ratio, and enzyme amount have significant effects on the yield of the esterification reaction. On the basis of ridge-max analysis, the optimum conditions were as follows: a reaction time of 2.65 days, a reaction temperature of 56.18 °C, a substrate molar ratio of 2.55:1, and an enzyme amount of 251.39%. The predicted and experimental values of molar conversion were 91.95 and 89.75 ± 1.06%, respectively.     

Enzymatic Synthesis of High sn-2 DHA and ARA Modified Oils for the Formulation of Infant Formula Fat Analogues

High sn‐2 docosahexaenoic and arachidonic acid oils (DHAO_m and ARAO_m, respectively) were produced independently via enzymatic interesterification of DHA‐rich and ARA‐rich single cell oils (DHASCO and ARASCO, respectively) using a mix of immobilized lipases, Lipozyme^® TL IM and Novozym^® 435 (weight ratio 1:1) as the biocatalyst system. Response surface methodology (RSM) was employed to model and optimize the reactions conditions. Three independent variables, namely reaction time, reaction temperature, and enzyme load, were investigated in DHAO_m and ARAO_m models. The prediction power of the model was further confirmed by solvent‐free scale‐up reactions of 100 g per batch. Final results showed that DHAO_m contained 46.53 mol% of total DHA (49.70 % at the sn‐2 position), while ARAO_m contained 47.25 mol% of total ARA (36.08 % at the sn‐2 position). This represents a significant increment in the amount of DHA and ARA at the sn‐2 position when compared to DHASCO (47.8 mol%; 30.30 % at the sn‐2) and ARASCO (47.79 mol%; 28.50 % at the sn‐2), respectively. These products have potential as additions to infant formulas where DHA and ARA supplementation is required.     

Optimization of β-Glucosidase, β-Xylosidase and Xylanase Production by Colletotrichum graminicola under Solid-State Fermentation and Application in Raw Sugarcane Trash Saccharification

Efficient, low-cost enzymatic hydrolysis of lignocellulosic residues is essential for cost-effective production of bioethanol. The production of β-glucosidase, β-xylosidase and xylanase by Colletotrichum graminicola was optimized using Response Surface Methodology (RSM). Maximal production occurred in wheat bran. Sugarcane trash, peanut hulls and corncob enhanced β-glucosidase, β-xylosidase and xylanase production, respectively. Maximal levels after optimization reached 159.3 ± 12.7 U g⁻¹, 128.1 ± 6.4 U g⁻¹ and 378.1 ± 23.3 U g⁻¹, respectively, but the enzymes were produced simultaneously at good levels under culture conditions optimized for each one of them. Optima of pH and temperature were 5.0 and 65 °C for the three enzymes, which maintained full activity for 72 h at 50 °C and for 120 min at 60 °C (β-glucosidase) or 65 °C (β-xylosidase and xylanase). Mixed with Trichoderma reesei cellulases, C. graminicola crude extract hydrolyzed raw sugarcane trash with glucose yield of 33.1% after 48 h, demonstrating good potential to compose efficient cocktails for lignocellulosic materials hydrolysis.     

Synthesis of Wax Esters from Crude Fish Fat by Lipase of Burkholderia sp. EQ3 and Commercial Lipases

The lipase from Burkholderia sp. EQ3 was used to synthesize wax esters in comparison with commercial lipases. The supernatant of Burkholderia sp. EQ3 was collected from a liquid basal medium with 1 % fish oil after 12 h cultivation (1.90 U/ml of lipase activity). The crude lipase was prepared by acetone precipitation of the culture supernatant (4.70 U/mg and 9.40 purification folds). The crude fish fat obtained by hexane extraction of waste fat from the wastewater pond of a canned tuna factory and cetyl alcohol were used for wax esters synthesis. Five commercial lipases were screened in comparison with crude lipase from Burkholderia sp. EQ3 in wax esters synthesis. The optimum conditions for wax esters synthesis from crude fish fat using Novozyme 435 were enzyme 1 U, substrate molar ratio of crude fish fat to cetyl alcohol 1:2 (115.30 mg of crude fish fat and 66.67 mg of cetyl alcohol) in hexane at 37 °C and 200 rpm with 90.81 % (TLC–FID peak area) after one h of reaction. The optimum conditions for the synthesis by crude lipase from Burkholderia sp. EQ3 were crude lipase 40 U, substrate molar ratio of crude fish fat and cetyl alcohol 1:2 in isooctane at 30 °C and 200 rpm with 95.07 % (TLC–FID peak area) after 6 h of reaction. The synthesized wax esters were mainly composed of cetyl palmitate and cetyl oleate.     

In Vitro Activity of Copper(II) Complexes,Loaded or Unloaded into a Nanostructured Lipid System,against Mycobacterium tuberculosis

Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis (Mtb), presenting 9.5 million new cases and 1.5 million deaths in 2014. The aim of this study was to evaluate a nanostructured lipid system (NLS) composed of 10% phase oil (cholesterol), 10% surfactant (soy phosphatidylcholine, sodium oleate), and Eumulgin^® HRE 40 ([castor oil polyoxyl-40-hydrogenated] in a proportion of 3:6:8), and an 80% aqueous phase (phosphate buffer pH = 7.4) as a tactic to enhance the in vitro anti-Mtb activity of the copper(II) complexes [CuCl₂(INH)₂]·H₂O (1), [Cu(NCS)₂(INH)₂]·5H₂O (2) and [Cu(NCO)₂(INH)₂]·4H₂O (3). The Cu(II) complex-loaded NLS displayed sizes ranging from 169.5 ± 0.7095 to 211.1 ± 0.8963 nm, polydispersity index (PDI) varying from 0.135 ± 0.0130 to 0.236 ± 0.00100, and zeta potential ranging from −0.00690 ± 0.0896 to −8.43 ± 1.63 mV. Rheological analysis showed that the formulations behave as non-Newtonian fluids of the pseudoplastic and viscoelastic type. Antimycobacterial activities of the free complexes and NLS-loaded complexes against Mtb H₃₇Rv ATCC 27294 were evaluated by the REMA methodology, and the selectivity index (SI) was calculated using the cytotoxicity index (IC₅₀) against Vero (ATCC^® CCL-81), J774A.1 (ATCC^® TIB-67), and MRC-5 (ATCC^® CCL-171) cell lines. The data suggest that the incorporation of the complexes into NLS improved the inhibitory action against Mtb by 52-, 27-, and 4.7-fold and the SI values by 173-, 43-, and 7-fold for the compounds 1, 2 and 3, respectively. The incorporation of the complexes 1, 2 and 3 into the NLS also resulted in a significant decrease of toxicity towards an alternative model (Artemia salina L.). These findings suggest that the NLS may be considered as a platform for incorporation of metallic complexes aimed at the treatment of TB.     

Enzymatic production of alkyl esters through alcoholysis: A critical evaluation of lipases and alcohols

This paper focuses on a detailed evaluation of commercially available immobilized lipases and simple monohydric alcohols for the production of alkyl esters from sunflower oil by enzymatic alcoholysis. Six lipases were tested with seven alcohols, including straight and branched-chain primary and secondary alcohols. The reactions were conducted in a batch stirred reaction vessel using stoichiometric amounts of substrates under solvent-free conditions. Dramatic differences in alcoholysis performance were observed among the different lipases. For most of the alcohols, Novozym 435 produced the highest yield of FA alkyl esters, with yields well over 90% for methanol, absolute ethanol, and 1-propanol. Overall, 96% ethanol was the preferred alcohol for all lipases except Novozym 435, and ethanolysis reactions reached the maximal conversion efficiency. Increasing the water content in the system resulted in an increased degree of conversion for all lipases except Novozym 435. The secondary alcohol 2-propanol significantly reduced the alcoholysis reaction with all lipases; however, the branch-chain isobutanol was more advantageous than linear 1-butanol for Novozym 435, Lipozyme RMIM, and Lipase PS-C. Many commercial immobilized lipases are highly efficient and promising for the production of alkyl esters, offering high reaction yields and a simple operation process.     

One-Dimensional Hydrogen-Bonded Infinite Chain from Nickel(II) Tetraaza Macrocyclic Complex and 1,2-Cyclopentanedicarboxylate Ligand

The reaction of [Ni(L)]Cl₂·2H₂O (L = 3,14-dimethyl-2,6,13,17-tetraazatricyclo [14,4,0^1.18,0^7.12]docosane) with trans-1,2-cyclopentanedicarboxylic acid (H₂-cpdc) yields a 1D hydrogen-bonded infinite chain with formula [Ni(L)(H-cpdc⁻)₂] (1). This complex has been characterized by X-ray crystallography, spectroscopy and cyclic voltammetry. The crystal structure of 1 exhibits a distorted octahedral geometry about Ni atom with four nitrogen atoms of the macrocycle and two oxygen atoms of the H-cpdc⁻ ligand at the axial position. Compound 1 crystallizes in the monoclinic system P2₁/c with a = 8.7429(17), b = 10.488(2), c = 18.929(4) Å, β = 91.82(2), V = 1734.8(6) ų, Z = 2. Electronic spectrum of 1 reveals a high-spin octahedral environment. Cyclic voltammetry of 1 undergoes two waves of a one-electron transfer corresponding to Ni^II/Ni^III and Ni^II/Ni^I processes.     

Mitochondrial Protection and Anti-aging Activity of Astragalus Polysaccharides and Their Potential Mechanism

The current study was performed to investigate mitochondrial protection and anti-aging activity of Astragalus polysaccharides (APS) and the potential underlying mechanism. Lipid peroxidation of liver and brain mitochondria was induced by Fe²⁺–Vit C in vitro. Thiobarbituric acid (TBA) colorimetry was used to measure the content of thiobarbituric acid reactive substances (TBARS). Mouse liver mitochondrial permeability transition (PT) was induced by calcium overload in vitro and spectrophotometry was used to measure it. The scavenging activities of APS on superoxide anion (O₂^•−) and hydroxyl radical (•OH), which were produced by reduced nicotinamide adenine dinucleotide (NADH)—N-Methylphenazonium methyl sulfate (PMS) and hydrogen peroxide (H₂O₂)–Fe²⁺ system respectively, were measured by 4-nitrobluetetrazolium chloride (NBT) reduction and Fenton reaction colorimetry respectively. The Na₂S₂O₃ titration method was used to measure the scavenging activities of APS on H₂O₂. APS could inhibit TBARS production, protect mitochondria from PT, and scavenge O₂^•−, •OH and H₂O₂ significantly in a concentration-dependent manner respectively. The back of the neck of mice was injected subcutaneously with D-galactose to induce aging at a dose of 100 mg/kg/d for seven weeks. Moreover, the activities of catalase (CAT), surperoxide dismutase (SOD) and glutathione peroxidase (GPx) and anti-hydroxyl radical which were assayed by using commercial monitoring kits were increased significantly in vivo by APS. According to this research, APS protects mitochondria by scavenging reactive oxygen species (ROS), inhibiting mitochondrial PT and increasing the activities of antioxidases. Therefore, APS has the effect of promoting health.     

Variants of Oxidized Regenerated Cellulose and Their Distinct Effects on Neuronal Tissue

Based on oxidized regenerated cellulose (ORC), several hemostyptic materials, such as Tabotamp^®, Equicel^® and Equitamp^®, have been developed to approach challenging hemostasis in neurosurgery. The present study compares ORC that differ in terms of compositions and properties, regarding their structure, solubility, pH values and effects on neuronal tissue. Cytotoxicity was detected via DNA-binding fluorescence dye in Schwann cells, astrocytes, and neuronal cells. Additionally, organotypic hippocampal slice cultures (OHSC) were analyzed, using propidium iodide, hematoxylin-eosin, and isolectin B₄ staining to investigate the cellular damage, cytoarchitecture, and microglia activation. Whereas Equicel^® led to a neutral pH, Tabotamp^® (pH 2.8) and Equitamp^® (pH 4.8) caused a significant reduction of pH (p < 0.001). Equicel^® and Tabotamp^® increased cytotoxicity significantly in several cell lines (p < 0.01). On OHSC, Tabotamp^® and Equicel^® led to a stronger and deeper damage to the neuronal tissue than Equitamp^® or gauze (p < 0.01). Equicel^® increased strongly the number of microglia cells after 24 h (p < 0.001). Microglia cells were not detectable after Tabotamp^® treatment, presumably due to an artifact caused by strong pH reduction. In summary, our data imply the use of Equicel^®, Tabotamp^® or Equitamp^® for specific applications in distinct clinical settings depending on their localization or tissue properties.     

Purification and Properties of an Insecticidal Metalloprotease Produced by Photorhabdus luminescens Strain 0805-P5G,the Entomopathogenic Nematode Symbiont

A total of 13 Photorhabdus luminescens strains were screened for proteolytic activity. The P. luminescens strain 0805-P5G had the highest activity on both skim milk and gelatin plates. The protease was purified to electrophoretical homogeneity by using a two-step column chromatographic procedure. It had a molecular weight of 51.8 kDa, as determined by MALDI-TOF mass spectrometry. The optimum pH, temperature, as well as pH and thermal stabilities were 8, 60 °C, 5–10, and 14–60 °C, respectively. It was completely inhibited by EDTA and 1,10-phenanthroline. Bioassay of the purified protease against Galleria mellonella by injection showed high insecticidal activity. The protease also showed high oral toxicity to the diamondback moth (Plutella xylostella) of a Taiwan field-collected strain, but low toxicity to an American strain. To our knowledge, this is the first report to demonstrate that the purified protease of P. luminescens has direct toxicity to P. xylostella and biopesticide potentiality.     

Biochemical Properties and Structure Analysis of a DAG-Like Lipase from Malassezia globosa

Diacylglycerol (DAG)-like lipases are found to play an important role in the life sciences and industrial fields. A putative DAG-like lipase (MgMDL2) from Malassezia globosa was cloned and expressed in recombinant Pichia pastoris. The recombinant MgMDL2 was expressed as a glycosylated protein and purified into homogeneity by anion exchange chromatography. The activity of recombinant MgMDL2 was optimal at 15 °C and pH 6.0, and it keeps over 50% of relative activity at 5 °C, suggesting that MgMDL2 was a cold active lipase. MgMDL2 retained over 80% of initial activity after incubation at 30 and 40 °C for 2.5 h, but it was not stable at 50 °C. Incubation of methanol and ethanol at a concentration of 30% for 2 h did not affect the recombinant enzyme activity, while metal ions, including Ca²⁺, Mn²⁺ and Ni²⁺, sharply inhibited the MgMDL2 activity at 5 mM by 42%, 35% and 36%, respectively. MgMDL2 exhibited a preference for medium chain-length esters with highest activity toward p-nitrophenyl caprylate, while it was active on mono- and diacylglycerol but not on triacylglycerol, indicating that it was a typical DAG-like lipase. By homology modeling, Phe278 was predicted to be involved in the preference of MgMDL2 for monoacyl- and diacyl-glyceride substrates, but not triglycerides.     

A Newly Isolated Thermostable Lipase from Bacillus sp

A thermophilic lipolytic bacterium identified as Bacillus sp. L2 via 16S rDNA was previously isolated from a hot spring in Perak, Malaysia. Bacillus sp. L2 was confirmed to be in Group 5 of bacterial classification, a phylogenically and phenotypically coherent group of thermophilic bacilli displaying very high similarity among their 16S rRNA sequences (98.5–99.2%). Polymerase chain reaction (PCR) cloning of L2 lipase gene was conducted by using five different primers. Sequence analysis of the L2 lipase gene revealed an open reading frame (ORF) of 1251 bp that codes for 417 amino acids. The signal peptides consist of 28 amino acids. The mature protein is made of 388 amino acid residues. Recombinant lipase was successfully overexpressed with a 178-fold increase in activity compared to crude native L2 lipase. The recombinant L2 lipase (43.2 kDa) was purified to homogeneity in a single chromatography step. The purified lipase was found to be reactive at a temperature range of 55–80 °C and at a pH of 6–10. The L2 lipase had a melting temperature (Tm) of 59.04 °C when analyzed by circular dichroism (CD) spectroscopy studies. The optimum activity was found to be at 70 °C and pH 9. Lipase L2 was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) (100%), whereas phenylmethylsulfonyl fluoride (PMSF), pepstatin-A, 2-mercaptoethanol and dithiothreitol (DTT) inhibited the enzyme by over 40%. The CD spectra of secondary structure analysis showed that the L2 lipase structure contained 38.6% α-helices, 2.2% ß-strands, 23.6% turns and 35.6% random conformations.     

Coumarins from the Herb Cnidium monnieri and Chemically Modified Derivatives as Antifoulants against Balanus albicostatus and Bugula neritina Larvae

In the search for new environmental friendly antifouling (AF) agents, four coumarins were isolated from the herbal plant Cnidium monnieri, known as osthole (1), imperatorin (2), isopimpinellin (3) and auraptenol (4). Furthermore, five coumarin derivatives, namely 8-epoxypentylcoumarin (5), meranzin hydrate (6), 2′-deoxymetranzin hydrate (7), 8-methylbutenalcoumarin (8), and micromarin-F (9) were synthesized from osthole. Compounds 1, 2, 4, 7 showed high inhibitory activities against larval settlement of Balanus albicostatus with EC₅₀ values of 4.64, 3.39, 3.38, 4.67 μg mL⁻¹. Compound 8 could significantly inhibit larval settlement of Bugula neritina with an EC₅₀ value of 3.87 μg mL⁻¹. The impact of functional groups on anti-larval settlement activities suggested that the groups on C-5′ and C-2′/C-3′ of isoamylene chian could affect the AF activities.     

Lower Temperature Cultures Enlarge the Effects of Vitreoscilla Hemoglobin Expression on Recombinant Pichia pastoris

An heterologous expression of Vitreoscilla hemoglobin (VHb) for improving cell growth and recombinant protein production has been successfully demonstrated in various hosts, including Pichia pastoris. Lower temperature cultures can enhance target protein production in some studies of P. pastoris. In this study, the strategy of combining heterologous VHb expression and lower temperature cultures in P. pastoris showed that final cell density and viability of VHb⁺ strain at 23 °C were higher than that at 30 °C. In addition, the effects of VHb expression on recombinant β-galactosidase production and oxygen uptake rate were also higher at 23 °C than at 30 °C. Consequently, lower temperature cultures can enlarge VHb effectiveness on cell performance of P. pastoris. This is because VHb activity obtained at 23 °C cultures was twofold higher than that at 30 °C cultures, due to a different heme production. This strategy makes P. pastoris an excellent expression host particularly suitable for increasing the yields of the low-stability and aggregation-prone recombinant proteins.     

Development of an Efficient Electroporation Method for Iturin A-Producing Bacillus subtilis ZK

In order to efficiently introduce DNA into B. subtilis ZK, which produces iturin A at a high level, we optimized seven electroporation conditions and explored an efficient electroporation method. Using the optimal conditions, the electroporation efficiency was improved to 1.03 × 10⁷ transformants/μg of DNA, an approximately 10,000-fold increase in electroporation efficiency. This efficiency is the highest electroporation efficiency for B. subtilis and enables the construction of a directed evolution library or the knockout of a gene in B. subtilis ZK for molecular genetics studies. In the optimization process, the combined effects of three types of wall-weakening agents were evaluated using a response surface methodology (RSM) design, which led to a two orders of magnitude increase in electroporation efficiency. To the best of our limited knowledge, this study provides the first demonstration of using an RSM design for optimization of the electroporation conditions for B. subtilis. To validate the electroporation efficiency, a case study was performed and a gene (rapC) was inactivated in B. subtilis ZK using a suicide plasmid pMUTIN4. Moreover, we found that the rapC mutants exhibited a marked decrease in iturin A production, suggesting that the rapC gene was closely related to the iturin A production.     

In vitro evaluation of the antimicrobial effectiveness and moisture binding properties of wound dressings

A variety of silver-coated dressings and some impregnated with other chemicals are now available in the market; however, there have been few studies analyzing their comparative efficacies as antimicrobial agents. Moreover, their properties for retaining an appropriate level of moisture that is critical for effective wound healing have never been reported. Five commercially available silver-containing and chlorhexidine dressings, Urgotul SSD^®, Bactigras^®, Acticoat^®, Askina Calgitrol Ag^® and Aquacel Ag^®, were tested to determine their comparative antimicrobial effectiveness in vitro against five common wound pathogens, namely methicillin-sensitive and -resistant Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. Mepitel^®, a flexible polyamide net coated with soft silicone, was used as a control. The zones of inhibition and both the rapidity and the extent of killing of these pathogens were evaluated. All five antimicrobial dressings investigated exerted some bactericidal activity, particularly against E. coli. The spectrum and rapidity of action ranged widely for the different dressings. Acticoat^® had a broad spectrum of action against both Gram-positive and -negative bacteria. Other dressings demonstrated a narrower range of bactericidal activities. Regarding the absorption and release of moisture, Askina Calgitrol Ag^® absorbed and released the most moisture from the environment. Aquacel Ag^® also exhibited good moisture absorption and moisture release, but to a lower degree. The other tested dressings absorbed or released very little moisture. Askina Calgitrol Ag^® and Aquacel Ag^® are good alternative dressings for treating wounds with high exudates and pus. An understanding of the characteristics of these dressings will be useful for utilizing them for specific requirements under specified conditions.     

Anti-Adhesive Activity of Cranberry Phenolic Compounds and Their Microbial-Derived Metabolites against Uropathogenic Escherichia coli in Bladder Epithelial Cell Cultures

Cranberry consumption has shown prophylactic effects against urinary tract infections (UTI), although the mechanisms involved are not completely understood. In this paper, cranberry phenolic compounds and their potential microbial-derived metabolites (such as simple phenols and benzoic, phenylacetic and phenylpropionic acids) were tested for their capacity to inhibit the adherence of uropathogenic Escherichia coli (UPEC) ATCC^®53503™ to T24 epithelial bladder cells. Catechol, benzoic acid, vanillic acid, phenylacetic acid and 3,4-dihydroxyphenylacetic acid showed anti-adhesive activity against UPEC in a concentration-dependent manner from 100–500 µM, whereas procyanidin A2, widely reported as an inhibitor of UPEC adherence on uroepithelium, was only statistically significant (p < 0.05) at 500 µM (51.3% inhibition). The results proved for the first time the anti-adhesive activity of some cranberry-derived phenolic metabolites against UPEC in vitro, suggesting that their presence in the urine could reduce bacterial colonization and progression of UTI.