Enhancing the bioavailability and antioxidant activity of natamycin E235-ferulic acid loaded polyethylene glycol/carboxy methyl cellulose films as anti-microbial packaging for food application.

This study investigated the development of biodegradable films made from a combination of polyethylene glycol (PEG), carboxymethyl cellulose (CMC) and mixtures from natamycin and ferulic acid. The films were characterized for their surface microstructure, antioxidant activity, thermal stability, mechanical properties, permeability and antifungal/bacterial activity. The addition of natamycin and ferulic acid to the film matrix enhanced antioxidant activity, thermal stability, antimicrobial activity, reduced the water vapor permeability (WVP) to 1.083 × 10-10 g × m-1s-1Pa-1, imparted opaque color and increased opacity up to 3.131 A mm-1. The attendance of natamycin and ferulic acid inside films created a clear roughness shape with agglomerates on the surface of films and caused a clear inhibition zone for Aspergillus niger, E. coli and C. botulinum. The utilization of PG/CMC/N-F packaging material on Ras cheese had a noticeable effect, resulting in a slight decrease in moisture content from 34.23 to 29.17 %. Additionally, it helped maintain the titrable acidity within the range of 0.99 % to 1.11 % and the force required for puncture from 0.035 to 0.052 N with non-significant differences. Importantly, these changes did not significantly affect the sensory qualities of Ras cheese during the storage period.

Theoretical Studies of Mutual Effects between 6-m-r Hemiketalization and 26-m-r Lactonization in Pimaricin Thioesterase.

Pimaricin is a small polyene macrolide antibiotic and has been broadly used as an antimycotic and antiprotozoal agent in both humans and foods. As a thioesterase in type-I polyketide synthase, pimTE controls the 26-m-r macrolide main chain release in pimaricin biosynthesis. In this work, we sought to determine whether the 6-m-r hemiketal formation was linked to pimTE-catalyzed 26-m-r lactonization. Compared to non-hemiketal TEs, pimTE is characterized by an aspartic acid residue (D179) accessible to the U-turn motif in the acyl-enzyme intermediate. Both the covalent docking and molecular dynamics simulations demonstrate that the reactive conformations for macrocyclic lactonization are drastically promoted by the 6-m-r hemiketal. Moreover, the small-model quantum mechanistic calculations suggest that protic residues can significantly accelerate the 6-m-r hemiketal cyclization. In addition, the post-hemiketal molecular dynamic simulations demonstrate that hydrogen-bonding networks surrounding the substrate U-turn of the hairpin-shaped conformation changes significantly when the 6-m-r hemiketal is formed. In particular, the R-hemiketal intermediate is not only catalyzed by the D179 residue, but also twists the hairpin structure to the 26-m-r lactonizing pre-reaction state. By contrast, the S-hemiketal formation is unlikely catalyzed by D179, which twists the hairpin in an opposite direction. Our results propose that pimTE could be a bi-functional enzyme, which can synergistically catalyze tandem 6-m-r and 26-m-r formations during the main-chain release of pimaricin biosynthesis.

Fabrication of colloidal stable gliadin-casein nanoparticles for the encapsulation of natamycin: Molecular interactions and antifungal application on cherry tomato.

Natamycin was encapsulated in gliadin-casein nanoparticles (G-C NPs) to control black rot in cherry tomato against Alternaria alternata. The G-C NPs with a mean particle diameter of 211 ± 4 nm were prepared using an anti-solvent method. The composite NPs showed better stability against neutral pH, ion, and storage than gliadin NPs. The quenching of gliadin by natamycin was static with a constant of 5.99 × 10-12 M-1∙S-1, and was spontaneous with a free energy of -23.5 kJ∙M-1 at 298 K. Both hydrophobic stacking and hydrogen bonds between natamycin and gliadin were found as the major driven force in the formation of the complex. The NPs kept the antifungal activity of natamycin with improved photostability. The NPs coatings exhibited better results than natamycin in controlling black rot on cherry tomato. This study shows the potential of the G-C NPs as all-natural delivery systems for natamycin in post-harvest treatments.

Development of Starch-Based Antifungal Coatings by Incorporation of Natamycin/Methyl-ß-Cyclodextrin Inclusion Complex for Postharvest Treatments on Cherry Tomato against Botrytis cinerea.

The application of natamycin as a natural fungicide in edible coatings is challenging because of its low aqueous solubility. In this study, the natamycin/methyl-ß-cyclodextrin (N/ME-ß-CD) inclusion complex was fabricated and incorporated into waxy corn starch-based coatings for postharvest treatments. The phase solubility of natamycin in the presence of ME-ß-CD at 293.2 K, 303.2 K, and 313.2 K is determined and used to calculate the process thermodynamic parameters. The N/ME-ß-CD inclusion complex was confirmed and characterized by FTIR and 1H NMR spectroscopy. The results indicated that the inclusion complex was formed and the hydrophobic part (C16-C26) of natamycin might be partially inserted into the cavity of ME-ß-CD form the wide rim. The effects of N/ME-ß-CD incorporated starch-based coatings (N/ME-ß-CD S coatings) on postharvest treatments of cherry tomatoes were evaluated in vivo. The N/ME-ß-CD S coatings could reduce weight loss, delay fruit ripening, and inhibit fruit decay caused by Botrytis cinerea in tomato fruit during storage.

Evaluation and characterization of some protective culture metabolites in free and nano-chitosan-loaded forms against common contaminants of Egyptian cheese.

This research attempted to inspect the contribution of lactic acid bacteria (LAB) with nanoparticle application in antimicrobial enhancement. Seven lactic acid cultures-free supernatants (CFSs) in both free and nanoparticles-loaded states were examined against seven foodborne microorganisms. Lactobacillus helveticus followed by Lactobacillus Plantarum possessed considerable antimicrobial activity. Headspace GC-MS characterization of Lactobacillus helveticus CFS identified a mixture of antimicrobial and health-promoting compounds. Minimal inhibitory concentration (MIC) values for tested Gram-positive bacteria represented 50% of that for Gram-negative bacteria, 20% and 7.35% of those for fungus and yeast respectively. Nanoparticles were prepared through chitosan-tripolyphosphate nanoparticle formation giving nanospheres from in the range from 5 to 10 nm, and narrow size distribution. CFS-loaded chitosan nanoparticles (CS-NPs) significantly enhanced the overall inhibition zone diameter, as well as, the decline in MIC values for Salmonella enterica (50%) and Penicillium chrysogenum (12.5%) was observed. Lactobacillus helveticus CFS, however, displayed lower antimicrobial activity vs. nisin and natamycin, it has both antibacterial and antifungal promising activities.

Effect of Chemically Transformed Macrocyclic Polyene Antibiotics on Tumor Cells.

Comparative analysis of the effects of chemically transformed polyene antibiotics pimaricin, nystatin, lucensomycin, amphotericin B, and levorin on biological objects in vivo and in vitro revealed the greatest biological activity of original amphotericin B and levorin with its derivatives. The study also examined the effects of alkyl derivatives of amphotericin B and levorin modified in certain parts of the lactone ring on the lipid and biological membranes. It is established that methylated levorin possesses larger biological activity than the original antibiotic. Examination of the effects of alkyl derivatives of levorin and amphotericin B on cell cultures C6 (rat glioma) and HeLa (human cervical carcinoma) in vitro revealed the antitumor action of methylated levorin and original amphotericin B.

Systematic optimization of cationic surface engineered mucoadhesive vesicles employing Design of Experiment (DoE): A preclinical investigation.

Fungal keratitis (FK) is treated by topical natamycin (Nat) which is an effective antifungal agent. However, it has numerous therapeutic limitations i.e. toxicity, tolerance, need of frequent dosing and patient incompliance. The aim of the present study was to develop Nat loaded trimethyl chitosan (TMC) coated mucoadhesive cationic niosomes (Muc-Cat-Nios) for prolonged and effective delivery to eyes. Niosomes were prepared using thin film hydration method and optimized using a Box-Behnken design (BBD) with the help of Design-Expert® Software. Three independent variables were considered: amount of Span 60 (X1), amount of Cholesterol [Chol(X2)] and TMC concentration (X3). The encapsulation efficiency (R1: EE%), vesicle size (R2: VS) and Zeta potential (R3: ZP) were selected as dependent variables or responses. The optimized Nios displayed spherical shape, 1034.14 nm vesicle size and 81.76% EE. Nat loaded niosomes were incubated with TMC to get mucoadhesive cationic vesicular system. Uncoated and TMC coated niosomes were characterized for mucoadhesive properties, in vitro drug release, rheological behaviour, and ex vivo permeation studies. Cationic Nios showed greater mucoadhesive potential that provided drug release for a long period of time. The promising outcomes suggest that natamycin delivery using cationic mucoadhesive niosomes could be employed for the effective treatment of fungal keratitis.

Short communication: Mycosporine-like amino acids protect natamycin against photodegradation in milk exposed to fluorescent or light-emitting diode light.

Natamycin has been used as a natural antimicrobial in dairy products, but the poor light stability of natamycin can limit usefulness in food products. Mycosporine-like amino acids are strong UV absorbers and might be useful as an additive to decrease light-induced degradations. Therefore, the objective of this study was to evaluate the photoprotective effect of mycosporine-like amino acids (MAA) in cow milk stored under 2 lighting conditions in retail commercial display cabinets with fluorescent and high intensity light-emitting diode illumination. When milk was exposed to both fluorescent and light-emitting diode light, natamycin degradation was very fast, and only 17.1 ± 0.9% of its original concentration was observed after 8 d at 4.0°C. On the other hand, by adding MAA into milk, natamycin was retained at 82.2 ± 0.9% and 92.2 ± 0.9% when low and high MAA levels were used, respectively. However, high MAA levels resulted in color changes. Therefore, MAA at low levels had a photoprotective effect for natamycin stability in cow milk exposed to light in refrigerated glass containers.

Modelling growth/no growth interface of Zygosaccharomyces bailii in simulated acid sauces as a function of natamycin, xanthan gum and sodium chloride concentrations.

Probabilistic microbial modelling using logistic regression was used to predict the growth/no growth (G/NG) interfaces of Zygosaccharomyces bailii in simulated acid sauces as a function of natamycin, xanthan gum (XG) and sodium chloride concentrations. The growth was assessed colorimetrically by using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride and 2-methoxy-1,4-naphthoquinone as detection reagents. The logistic regression model successfully predicted G/NG probability. The detection reagents used allowed the evaluation of G/NG interfaces in opaque systems with an excellent agreement with the plate count method. Natamycin concentration of 12 mg/L was needed to inhibit Z. bailii growth independently of the presence of XG and/or NaCl. Addition of 3.00 and 6.00% of NaCl exerted an antagonistic effect on natamycin action. Furthermore, addition of 0.25 and 0.50% XG decreased natamycin and/or NaCl action. However, an increased in XG concentration to 1.00% decreased yeast growth. Mentioned results highlighted the importance of the correct selection of stress factors applied to inhibit Z. bailii growth.

Stability study of a nisin/natamycin blend by LC-MS.

Nisin and natamycin are natural preservatives used in a broad range of food products. In the present study, the stability of an aqueous nisin/natamycin blend (pH 4.0) for the beverage market was tested at room temperature (22 °C) under dark conditions by means of liquid chromatography. Both an HPLC-UV and a UPLC-MS method were developed to monitor the concentration of natamycin and nisin, respectively. After 6 months at room temperature (22 °C) under dark conditions, the nisin/natamycin blend (pH 4.0) still contained 85% of the original amount of nisin and 95% of natamycin. Even after 1 year, 50% of nisin and 90% of natamycin remained in the formulation. The resulting degradation products were studied by means of high resolution mass spectrometry. For both nisin and natamycin, degradation products originating from hydration (natamycin + H2O; nisin + H2O) and oxidation reactions (natamycin + O; nisin + O; nisin + 2O) were observed.

Ion-sensitive in situ hydrogels of natamycin bilosomes for enhanced and prolonged ocular pharmacotherapy: in vitro permeability, cytotoxicity and in vivo evaluation.

Delivery of therapeutic molecules into the deeper ocular compartments is mainly hampered by short precorneal residence and limited transmembrane permeability of topically administered drugs. Hence, the current study was undertaken to fabricate the ion-sensitive in situ gels of natamycin (NT) bilosomes (NB) for efficient ocular delivery. The effect of cholesterol and sodium taurocholate proportion on the properties of the bilosomes were studied and the formulation with better physicochemical properties was optimized and utilized to derive the in situ gelling system (IG). The impact of type/composition of gelling agent on the formation and characteristics of the hydrogel was investigated. The hydrogel formed from IG with 0.3% w/v gellan gum showed optimal viscoelastic and adhesive characteristics. The ocular safety and cytocompatibility of NB and its IG was confirmed by corneal histology and in vitro cytotoxicity evaluation. A 6- to 9-fold enhancement in the transcorneal flux of NB demonstrated efficient ocular penetration of bilosomes. Moreover, the superior mean dose normalized NT levels in the ocular tissues of rabbits treated with optimized NB and IG illustrated the effectiveness of bilosomes loaded ion-sensitive in situ hydrogels as a potential platform for the improved and prolonged ocular pharmacotherapy.

Directed accumulation of less toxic pimaricin derivatives by improving the efficiency of a polyketide synthase dehydratase domain.

Pimaricin is an important polyene antifungal antibiotic that binds ergosterol and extracts it from fungal membranes. In previous work, two pimaricin derivatives (1 and 2) with improved pharmacological activities and another derivative (3) that showed no antifungal activity were produced by the mutant strain of Streptomyces chattanoogensis, in which the P450 monooxygenase gene scnG has been inactivated. Furthermore, inactivation of the DH12 dehydratase domain of the pimaricin polyketide synthases (PKSs) resulted in specific accumulation of the undesired metabolite 3, suggesting that improvement of the corresponding dehydratase activity may reduce or eliminate the accumulation of 3. Accordingly, the DH12-KR12 didomain within the pimaricin PKS was swapped with the fully active DH11-KR11 didomain. As predicted, the mutant was not able to produce 3 but accumulated 1 and 2 in high yields. Moreover, the effect of the flanking linker regions on domain swapping was evaluated. It was found that retention of the DH12-KR12 linker regions was more critical for the processivity of hybrid PKSs. Subsequently, high-yield production of 1 or 2 was obtained by overexpressing the scnD gene and its partner scnF and by disrupting the scnD gene, respectively. To our knowledge, this is the first report on the elimination of a polyketide undesired metabolite along with overproduction of desired product by improving the catalytic efficiency of a DH domain using a domain swapping technology.

Active edible coating and γ-irradiation as cold combined treatments to assure the safety of broccoli florets (Brassica oleracea L.).

The antimicrobial activity of essential oils (EOs), organic acid (OA) salts and natamycin, a natural antifungal produced during fermentation by the bacterium Streptomyces natalensis, was assessed against four pathogens (Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium and Aspergillus niger). The Minimum Inhibitory Concentration (MIC) of each antimicrobial (AM) was assessed to determine their efficiency on tested microbial species in order to select the most efficient. Then, the interactions between different antimicrobial compounds showing the lowest MIC were determined by the checkerboard method. The most effective antimicrobial formulation showing synergistic or additive effects was then encapsulated in an alginate matrix to protect the antimicrobial efficiency during storage. The effectiveness of the formulation was then evaluated in situ using broccoli as a food model. A combined treatment of active coating and γ-irradiation (0.4 and 0.8kGy) was also done in order to evaluate the possible synergistic effect between treatments. The results of this study allowed the selection of 4 EOs, one OA salt and the natamycin as an antifungal agent exhibiting lower MIC values. The interactive antimicrobial effects between them showed that an antimicrobial formulation composed of 300ppm of lemongrass EO, 5000ppm of sodium diacetate and 80ppm of natamycin resulted in an additive effect against A. niger, E. coli and S. Typhimurium and showing synergistic effect against L. monocytogenes. Finally, in situ analyses showed a synergistic antimicrobial activity between active coating and γ-irradiation and allowed the extension of the shelf-life of ready-to-eat (RTE) broccoli during storage at 4°C.

Innovative Approaches Using Lichen Enriched Media to Improve Isolation and Culturability of Lichen Associated Bacteria.

Lichens, self-supporting mutualistic associations between a fungal partner and one or more photosynthetic partners, also harbor non-photosynthetic bacteria. The diversity and contribution of these bacteria to the functioning of lichen symbiosis have recently begun to be studied, often by culture-independent techniques due to difficulties in their isolation and culture. However, culturing as yet unculturable lichenic bacteria is critical to unravel their potential functional roles in lichen symbiogenesis, to explore and exploit their biotechnological potential and for the description of new taxa. Our objective was to improve the recovery of lichen associated bacteria by developing novel isolation and culture approaches, initially using the lichen Pseudevernia furfuracea. We evaluated the effect of newly developed media enriched with novel lichen extracts, as well as the influence of thalli washing time and different disinfection and processing protocols of thalli. The developed methodology included: i) the use of lichen enriched media to mimic lichen nutrients, supplemented with the fungicide natamycin; ii) an extended washing of thalli to increase the recovery of ectolichenic bacteria, thus allowing the disinfection of thalli to be discarded, hence enhancing endolichenic bacteria recovery; and iii) the use of an antioxidant buffer to prevent or reduce oxidative stress during thalli disruption. The optimized methodology allowed significant increases in the number and diversity of culturable bacteria associated with P. furfuracea, and it was also successfully applied to the lichens Ramalina farinacea and Parmotrema pseudotinctorum. Furthermore, we provide, for the first time, data on the abundance of culturable ecto- and endolichenic bacteria that naturally colonize P. furfuracea, R. farinacea and P. pseudotinctorum, some of which were only able to grow on lichen enriched media. This innovative methodology is also applicable to other microorganisms inhabiting these and other lichen species.

Electrophilic, Activation-Free Fluorogenic Reagent for Labeling Bioactive Amines.

Herein we report the preparation of BODIPY mesoionic acid fluorides through a short sequence involving an isocyanide multicomponent reaction as the key synthetic step. These novel BODIPY acid fluorides are water-stable electrophilic reagents that can be used for the fluorescent derivatization of amine-containing biomolecules using mild and activation-free reaction conditions. As a proof of principle, we have labeled the antifungal natamycin and generated a novel fluorogenic probe for imaging a variety of human and plant fungal pathogens, with excellent selectivity over bacterial cells.

Casein/natamycin edible films efficiency for controlling mould growth and on microbiological, chemical and sensory properties during the ripening of Kashar cheese.

BACKGROUND: The objective of the present study was to investigate the effects of the dipping application of coating materials such as casein (Cas), casein/natamycin (Cas/N) and natamycin (N) solutions on chemical (e.g. pH, dry matter, fat, acidity, salt, protein, water-soluble nitrogen, ripening index, 12% trichloroacetic acid-soluble nitrogen and pH 4.6 soluble nitrogen), microbial (e.g. total number of aerobic mesophilic bacteria, lactic acid bacteria in MRS, lactic acid bacteria in M17, coliforms and mould) and organoleptic properties of Kashar cheese samples during ripening (3, 30, 60 and 90 days). RESULTS: The difference in microbiological and chemical changes between samples were found to be significant (P < 0.05) during the ripening period. The highest and lowest mould counts were determined for the control (2.87 log cfu g(-1) ) and the Cas/N (<1 log cfu g(-1) ) samples, respectively, at 90 days of ripening. Compared to other cheeses, control and Cas/N-coated cheeses had higher levels of water-soluble nitrogen and ripening index at in the end of storage. The Cas/N-coated cheese samples were preferred more by the panellists, while N-coated cheese samples received the lowest scores. CONCLUSION: This study suggests that coating could be used to improve the quality of Kashar cheese during ripening.

Formulation and antifungal performance of natamycin-loaded liposomal suspensions: the benefits of sterol-enrichment.

The aim of this study is to develop and evaluate food-grade liposomal delivery systems for the antifungal compound natamycin. Liposomes made of various soybean lecithins are prepared by solvent injection, leading to small unilamellar vesicles (<130 nm) with controlled polydispersity, able to encapsulate natamycin without significant modification of their size characteristics. Presence of charged phospholipids and reduced content of phosphatidylcholine in the lecithin mixture are found to be beneficial for natamycin encapsulation, indicating electrostatic interactions of the preservative with the polar head of the phospholipids. The chemical instability of natamycin upon storage in these formulations is however significant and proves that uncontrolled leakage out of the liposomes occurs. Efficient prevention of natamycin degradation is obtained by incorporation of sterols (cholesterol, ergosterol) in the lipid mixture and is linked to higher entrapment levels and reduced permeability of the phospholipid membrane provided by the ordering effect of sterols. Comparable action of ergosterol is observed at concentrations 2.5-fold lower than cholesterol and attributed to a preferential interaction of natamycin-ergosterol as well as a higher control of membrane permeability. Fine-tuning of sterol concentration allows preparation of liposomal suspensions presenting modulated in vitro release kinetics rates and enhanced antifungal activity against the model yeast Saccharomyces cerevisiae.

Development of novel magnetic solid phase extraction materials based on Fe3O4/SiO2/poly(acrylamide-N,N'-methylene bisacrylamide)-Pluronic L64 composite microspheres and their application to the enrichment of natamycin.

Novel magnetic adsorbents based on Fe3O4/SiO2/poly(acrylamide-N,N'-methylene bisacrylamide) magnetic microspheres modified with non-ionic triblock copolymer surfactant were successfully prepared as a magnetic solid phase extraction adsorbent for the determination of trace natamycin in jam samples. The adsorbent was characterized by scanning electron microscopy, transmission electron microscopy, Fourier transformed infrared spectroscopy, vibrating sample magnetometer, and X-ray diffractometer analysis, confirming that Pluronic L64 was effectively functionalized on the magnetic materials. Various experimental parameters affecting the extraction capacity were investigated including adsorbent amount, extraction time, desorption time, sample pH, and ionic strength. For recovery evaluations, the jam samples were spiked at two concentration levels of 100 and 200µgkg(-1) of natamycin and the recovery values were in the range of 78.8-93.4%. The relative standard deviations (RSD) for the recoveries were less than 3.5%. The novel magnetic solid phase extraction method provided several advantages, such as simplicity, low environmental impact, convenient extraction procedure, and short analysis time when used for natamycin analysis.

Engineered biosynthesis of pimaricin derivatives with improved antifungal activity and reduced cytotoxicity.

Pimaricin is an important antifungal antibiotic for antifungal therapy and prevention of mould contamination in the food industry. In this study, three new pimaricin derivatives, 12-decarboxy-12-methyl pimaricin (1), 4,5-desepoxy-12-decarboxy-12-methyl pimaricin (2), and 2-hydro-3-hydroxy-4,5-desepoxy-12-decarboxy-12-methyl pimaricin (3), were generated through the inactivation of P450 monooxygenase gene scnG in Streptomyces chattanoogensis L10. Compared with pimaricin, 1 displayed a twofold increase in antifungal activity against Candida albicans ATCC 14053 and a 4.5-fold decrease in cytotoxicity with erythrocytes, and 2 had comparable antifungal activity and reduced cytotoxicity, whereas 3 showed nearly no antifungal and hemolytic activities. Genetic and biochemical analyses proved that 1 is converted from 2 by P450 monooxygenase ScnD. Therefore, the overexpression of scnD in scnG-null strain eliminated the accumulation of 2 and improved the yield of 1 by 20 %. Conversely, scnG/scnD double mutation abolished the production of 1 and improved the yield of 2 to 2.3-fold. These results indicate that the pimaricin derivatives with improved pharmacological properties obtained by genetic engineering can be further developed into antifungal agents for potential clinical application.

Complexation of synthetic CDM-AM copolymer with natamycin and carbendazim to improve solubility and fungicidal activity.

The ß-cyclodextrin-acrylamide (CDM-AM) copolymer was prepared from acrylamide and ß-CD maleate (CDM) using K2S2O8 as initiator. The effects of the CDM-AM copolymer on the solubility and fungicidal activity of natamycin (NM) and carbendazim (MBC) were investigated. The stability constant of NM·CDM-AM and MBC·CDM-AM complexes at 303 K were of 10,725.45 M(-1) and 3000.89 M(-1), respectively. The complexes were characterized using phase solubility diagrams, NMR spectra and FT-IR spectra. The analysis of the biological activities of these two complexes indicated that they possessed enhancing fungicidal activities compared to NM and MBC alone.