Finding a Needle in a Haystack: Producing Antimicrobial Cutin-Derived Oligomers from Tomato Pomace.

Agro-industrial residues comprise a rich diversity of plant polymers and bioactive compounds, constituting promising sources for the development of materials, including bioplastics, and food supplements, among other applications. In particular, the polyester cutin is abundant in fruit peel, a plentiful constituent of pomace agro-industrial residues. The potential of diverse fruit pomaces as a source for the development of cutin-derived materials/products has been extensively sought out. This study expands the established knowledge: it sets proof of concept for the production of antimicrobial oligomers from cutin-rich materials isolated in a single step from tomato pomaces generated by two remote agro-industries. Specifically, it first analyzed how the chemical signature (nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS)) of a pomace (and of its major constituents) mirrors that of the corresponding cutin-rich material isolated using an ionic liquid extractant. The cutin-rich materials were then deconstructed (using mild hydrolyses), and the resultant mixtures were chemically characterized and screened for bactericidal activity against Escherichia coli and Staphylococcus aureus. The presence of esterified structures, linear and/or branched, likely comprising dioic acids as a major building block (but not exclusively) is a prerequisite for activity against E. coli but not against S. aureus that was susceptible to monomers as well. Further studies are required to optimize the production of broad bactericidal oligomers from any cutin-rich pomace source, moving ahead toward their circular usage.

Quantification of Structure-Property Relationships for Plant Polyesters Reveals Suberin and Cutin Idiosyncrasies.

Polyesters, as they exist in planta, are promising materials with which to begin the development of "green" replacements. Cutin and suberin, polyesters found ubiquitously in plants, are prime candidates. Samples enriched for plant polyesters, and in which their native backbones were largely preserved, were studied to identify "natural" structural features; features that influence critical physical properties. Quantitative nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and X-ray scattering methods were used to quantify structure-property relationships in these polymeric materials. The degree of esterification, namely, the presence of acylglycerol linkages in suberin and of secondary esters in cutin, and the existence of mid-chain epoxide groups defining the packing of the aliphatic chains were observed. This packing determines polymer crystallinity, the resulting crystal structure, and the melting temperature. To evaluate the strength of this rule, tomato cutin from the same genotype, studying wild-type plants and two well-characterized mutants, was analyzed. The results show that cutin's material properties are influenced by the amount of unbound aliphatic hydroxyl groups and by the length of the aliphatic chain. Collectively, the acquired data can be used as a tool to guide the selection of plant polyesters with precise structural features, and hence physicochemical properties.

An Ionic Liquid Extraction That Preserves the Molecular Structure of Cutin Shown by Nuclear Magnetic Resonance.

The biopolyester cutin is ubiquitous in land plants, building the polymeric matrix of the plant's outermost defensive barrier, the cuticle. Cutin influences many biological processes in planta; however, due to its complexity and highly branched nature, the native structure remains partially unresolved. Our aim was to define an original workflow for the purification and systematic characterization of the molecular structure of cutin. To purify cutin we tested the ionic liquids cholinium hexanoate and 1-butyl-3-methyl-imidazolium acetate. The ensuing polymeric materials are highly esterified, amorphous, and have a typical monomeric composition as demonstrated by solid-state NMR, complemented by spectroscopic, thermal, and x-ray scattering analyses. We performed a systematic study by solution-state NMR of cryogenically milled cutins extracted from tomatoes (Solanum lycopersicum 'Micro-Tom'; the wild type and the GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE [GPAT6] and CUTIN SYNTHASE [CUS1] mutants). We resolved their molecular structures, relative distribution of ester aliphatics, free acid end-groups and free hydroxyl groups, differentiating between those derived from primary and secondary esters. Our data demonstrate the existence of free hydroxyl groups in cutin and provide insight into how the mutations affect the esterification arrangement of cutin. The usage of ionic liquids for studying plant polyesters has advantages over conventional approaches, since simple modifications can be applied to recover a biopolymer carrying distinct types/degrees of modifications (e.g. preservation of esters or cuticular polysaccharides), which in combination with the solution NMR methodologies developed here, constitutes essential tools to fingerprint the multifunctionality and the structure of cutin in planta.

Antimicrobial Contact-Active Oligo(2-oxazoline)s-Grafted Surfaces for Fast Water Disinfection at the Point-of-Use.

Water is one of the most valuable resources today and its purity is crucial to health and society well-being. The access to safe drinking water is decreasing in the world, which can have a huge socio-economic impact especially in developing countries, more prone to water-associated diseases. The goal of this work was to develop an innovative, fast, and cost-effective 3D material capable of decontaminating water. We have used an eco-friendly strategy, combining plasma surface activation and supercritical fluid technology to produce, for the first time, a 2-oxazoline-grafted 3D surface with broad-spectrum contact-active antimicrobial properties. Oligo(2-methyl-2-oxazoline) quaternized with N,N-dimethyldodecylamine and grafted to a chitosan (CHT) scaffold (CHT-OMetOx-DDA) efficiently and quickly (<3 min) killed >99.999% of Staphylococcus aureus and Escherichia coli cells upon direct contact and avoided bacterial adhesion to the materials surface, which is important for the prevention of biofilm formation. As a proof of concept, CHT-OMetOx-DDA scaffold was demonstrated to be suitable for water purification efficiently killing the microorganisms present in different water samples within minutes of contact and without leaching to the water. Additionally, we report for the first time a new method to clearly distinguish two mechanisms of action of bioactive surfaces: contact-active and releasing systems.

Supercritical carbon dioxide design strategies: from drug carriers to soft killers.

The integrated use of supercritical carbon dioxide (scCO(2)) and micro- and nanotechnologies has enabled new sustainable strategies for the manufacturing of new medications. 'Green' scCO(2)-based methodologies are well suited to improve either the synthesis or materials processing leading to the assembly of three-dimensional multifunctional constructs. By using scCO(2) either as C1 feedstock or as solvent, simple, economic, efficient and clean routes can be designed to synthesize materials with unique properties such as polyurea dendrimers and oxazoline-based polymers/oligomers. These new biocompatible, biodegradable and water-soluble polymeric materials can be engineered into multifunctional constructs with antimicrobial activity, targeting moieties, labelling units and/or efficiently loaded with therapeutics. This mini-review highlights the particular features exhibited by these materials resulting directly from the followed supercritical routes.

Antimicrobial activity of new green-functionalized oxazoline-based oligomers against clinical isolates.

BACKGROUND: The search for new antimicrobial compounds able to overcome the global issue of microbial resistance to antibiotics is a priority worldwide. Moreover, several commensal microorganisms have been increasingly associated to opportunistic microbial infections. Having previously disclosed the green synthesis and preliminary characterization of the oligomers [linear oligo(ethylenimine) hydrochloride and oligo(2-methyl-2-oxazoline) quaternized with N,N-dimethyldodecylamine] we herein report on the screening of these oligomers against a battery of 69 clinical isolates of Aerococcus spp., Candida spp., Staphylococcus spp. and Streptococcus spp. FINDINGS: The isolates' susceptibility to both oligomers was evaluated by determining their minimal inhibitory concentration (MIC) and the biocidal effectiveness of each compound was further confirmed through spectrophotometric measurements and fluorescence microscopy. The MIC values of the 69 isolates were highly variable, yet favourably comparable with those of other antimicrobial polymers. The viability assays resulted in 100% of microbial killing rate after only 5 min, highlighting the promising antimicrobial action of these oligomers. CONCLUSIONS: Though further studies are required, evidence suggests that a strong effort should be done in order to confirm these compounds as valid alternatives for several clinical applications. This is reinforced by their well described biocompatibility with human tissues and by their proposed mechanism of action which difficult the development of microbial resistance to these compounds.

Anti-biofouling 3D porous systems: the blend effect of oxazoline-based oligomers on chitosan scaffolds.

The production, characterization and anti-biofouling activity of 3D porous scaffolds combining different blends of chitosan and oxazoline-based antimicrobial oligomers is reported. The incorporation of ammonium quaternized oligo(2-oxazoline)s into the composition of the scaffold enhances the stability of the chitosan scaffold under physiological conditions as well as its ability to repel protein adsorption. The blended scaffolds showed mean pore sizes in the range of 18-32 µm, a good pore interconnectivity and high porosity, as well as a large surface area, ultimate key features for anti-biofouling applications. Bovine serum albumin (BSA) adhesion profiles showed that the composition of the scaffolds plays a critical role in the chitosan-oligooxazoline system. Oligobisoxazoline-enriched scaffolds (20% w/w, CB8020) decreased protein adsorption (BSA) by up to 70%. Moreover, 1 mg of CB8020 was able to kill 99.9% of Escherichia coli cells upon contact, demonstrating its potential as promising material for production of tailored non-fouling 3D structures to be used in the construction of novel devices with applications in the biomedical field and water treatment processes.

Oxazoline-based antimicrobial oligomers: synthesis by CROP using supercritical CO2.

A method using supercritical CO(2) to obtain biocompatible 2-oxazoline-based oligomers quaternized with different amines is described. The synthesized oligo(2-oxazoline)s display partial carbamic-acid insertion at one end. The syntheses of quaternary oligo(2-bisoxazoline)s and linear oligoethylenimine hydrochlorides are reported. Oligo(2-methyl-2-oxazoline) and oligo(2-bisoxazoline) quaternized with N,N-dimethyldodecylamine are the most efficient biocidal agents showing fast killing rates against Staphylococcus aureus and Escherichia coli. Linear oligoethylenimine hydrochloride shows the lowest MIC values but higher killing times against both bacteria. Based on the antimicrobial activity studies, a cooperative action of carbamic acid with the ammonium end group is proposed.