Véronique Coma

Chitosan’s effectiveness as an antimicrobial coating for biocontrol depends on its resistance to rain. Unfortunately, to the best of our knowledge, there is currently no satisfactory method for assessing this resistance, which means that field tests have to be carried out to evaluate it in situ, which are difficult to implement and therefore unsuitable for optimizing formulations. This article explores the use of genipin to detect residual chitosan on surfaces after simulated rain, using fluorescence microscopy. A first study on real vine leaves using MacroFluo microscopy was carried out but showed limitations for the intended application, notably due to the requirement for high chitosan concentrations to achieve detectable signals. A semi-quantitative method based on confocal laser scanning microscopy was then developed on model leaves, as real leaves were unsuitable due to their autofluorescence. Among the tested models, Parafilm^® proved to be the most effective, showing sufficient fluorescence after reaction with genipin, even at low chitosan concentrations. For the first time, a method that does not require chromophore grafting onto chitosan has been proposed, allowing for the comparison of chitosan solution rainfastness under laboratory conditions. As an application, the effect of the counter ion on chitosan’s rain resistance was evaluated.

Active, fully biobased film-forming dispersions (FFDs) with highly promising results for sliced soft bread preservation were successfully elaborated from carboxymethyl cellulose (CMC) and chitosan (CH) using a simple method based on pH adjustments. They consisted of the association of polysaccharides and oleic acid (OL) added with cinnamon (CEO) or ginger (GEO) essential oils. The chemical compositions of the commercial essential oils were first determined via GC/MS, with less than 3% of compounds unidentified. The films obtained from FFDs were characterized by SEM, FTIR and DSC, indicating specific microstructures and some interactions between essential oils and the polymer matrix. CEO-based films exhibited higher antioxidant properties and a lower minimal inhibitory concentration in terms of antifungal properties. From experiments on sliced soft bread, the ginger-based films could increase the shelf life up to 20 days longer than that of the control. Even more promising, cinnamon-based films led to complete fungal inhibition in bread slices that was maintained beyond 60 days. Enumeration of the yeasts and molds for the FFD-coated breads revealed complete inhibition even after 15 days of storage with the FFDs containing the highest concentration of CEO.

The formation of water-insoluble complexes between chitosan (ChS) and caffeoylquinic acid (CQ) derivatives present in artichoke (AE) and green coffee bean (GCBE) extracts was investigated by the equilibrium adsorption method. The UPLC/HPLC analysis revealed that the phenolic compounds accounted for 8.1% and 74.6% of AE and GCBE respectively, and CQ derivatives were the predominant compounds. According to the applied Langmuir adsorption model, anionic compounds present in natural extracts were adsorbed onto the active centers of ChS, i.e., primary amino groups. The driving forces of adsorption were electrostatic interactions between cationic groups of ChS and anionic compounds of natural extracts. Chromatographic analysis revealed that not only CQ derivatives, but also other phenolic compounds of natural extracts were attached to ChS. The release of adsorbed compounds into different media as well as the bioactive properties of complexes were also studied. With the immobilization of bioactives onto ChS, increased and prolonged ABTS^•+ radical scavenging activity and decreased antifungal activity against Fusarium graminearum and Botrytis cinerea were observed compared to those of ChS. The findings of the current study highlight that the adsorption approach could be used to successfully prepare water-insoluble complexes of ChS and components of natural extracts with prolonged antioxidant activity.

Chitosan is known for its antimicrobial and antifungal properties that make it a promising candidate for plant protection. However, when sprayed in open fields, the bioactivity of chitosan significantly diminishes, suggesting a possible influence of sunlight on chitosan structure. This study aimed to investigate the effects of UV radiation, by using artificial UV sources simulating sunlight, on the stability of chitosan. A powdered chitosan with a low polymerization degree was selected and analyzed using various physicochemical methods, both before and after irradiation. Some minor differences appeared. UV spectra analysis revealed the disappearance of initially present chromophores and the emergence of a new band around 340 nm, potentially indicating the formation of carbonyl compounds. However, elemental analysis, MALDI-TOF spectra, polymerization degree, and infrared spectra did not exhibit any clear structural modifications of chitosan. Interestingly, irradiated powdered chitosan samples maintained their bioactivity, including their eliciting and antifungal properties. In the case of grapevine, irradiated chitosan demonstrated effectiveness in controlling grapevine diseases such as downy mildew, contradicting the assumption that sunlight is responsible for the decreased effectiveness of chitosan in open field conditions.

Five different chitosan samples (CHI-1 to CHI-5) from crustacean shells with high deacetylation degrees (>93%) have been deeply characterized from a chemical and physicochemical point of view in order to better understand the impact of some parameters on the bioactivity against two pathogens frequently encountered in vineyards, Plasmopara viticola and Botrytis cinerea. All the samples were analyzed by SEC-MALS, ¹H-NMR, elemental analysis, XPS, FTIR, mass spectrometry, pyrolysis, and TGA and their antioxidant activities were measured (DPPH method). Molecular weights were in the order: CHI-4 and CHI-5 (MW >50 kDa) > CHI-3 > CHI-2 and CHI-1 (MW < 20 kDa). CHI-1, CHI-2 and CHI-3 are under their hydrochloride form, CHI-4 and CHI-5 are under their NH₂ form, and CHI-3 contains a high amount of a chitosan calcium complex. CHI-2 and CHI-3 showed higher scavenging activity than others. The bioactivity against B. cinerea was molecular weight dependent with an IC50 for CHI-1 = CHI-2 (13 mg/L) ≤ CHI-3 (17 mg/L) < CHI-4 (75 mg/L) < CHI-5 (152 mg/L). The bioactivity on P. viticola zoospores was important, even at a very low concentration for all chitosans (no moving spores between 1 and 0.01 g/L). These results show that even at low concentrations and under hydrochloride form, chitosan could be a good alternative to pesticides.

The objective of this study was to increase the water resistance of paper while providing fungal resistance using a bio-based coating made from chitosan. The water resistance was improved through the surface control of roughness using modified calcium carbonate particles. The higher the quantity of particles in the film-forming solution, the higher the surface hydrophobicity of the paper. The addition of particles was found to counterbalance the chitosan hydrophilicity through the control of the coatings’ penetration in the paper bulk. As a consequence, the wetting time and liquid water resistance were enhanced. The antifungal activity of the film-forming solutions and coated paper was also investigated against the growth of Chaetomium globosum, which was selected as a model strain able to contaminate paper materials. The results reveal that the antifungal activity of chitosan was improved by a possible synergic effect with the bicarbonate ions from the mineral particles.

Cereals are subject to contamination by pathogenic fungi, which damage grains and threaten public health with their mycotoxins. Fusarium graminearum and its mycotoxins, trichothecenes B (TCTBs), are especially targeted in this study. Recently, the increased public and political awareness concerning environmental issues tends to limit the use of traditional fungicides against these pathogens in favor of eco-friendlier alternatives. This study focuses on the development of biofungicides based on the encapsulation of a curcumin derivative, tetrahydrocurcumin (THC), in polysaccharide matrices. Starch octenylsuccinate (OSA-starch) and chitosan have been chosen since they are generally recognized as safe. THC has been successfully trapped into particles obtained through a spray-drying or freeze-drying processes. The particles present different properties, as revealed by visual observations and scanning electron microscopy. They are also different in terms of the amount and the release of encapsulated THC. Although freeze-dried OSA-starch has better trapped THC, it seems less able to protect the phenolic compound than spray-dried particles. Chitosan particles, both spray-dried and lyophilized, have shown promising antifungal properties. The IC₅₀ of THC-loaded spray-dried chitosan particles is as low as 0.6 ± 0.3 g/L. These particles have also significantly decreased the accumulation of TCTBs by 39%.

Fusarium graminearum is a fungal pathogen that can colonize small-grain cereals and maize and secrete type B trichothecene (TCTB) mycotoxins. The development of environmental-friendly strategies guaranteeing the safety of food and feed is a key challenge facing agriculture today. One of these strategies lies on the promising capacity of products issued from natural sources to counteract crop pests. In this work, the in vitro efficiency of sixteen extracts obtained from eight natural sources using subcritical water extraction at two temperatures was assessed against fungal growth and TCTB production by F. graminearum. Maritime pine sawdust extract was shown to be extremely efficient, leading to a significant inhibition of up to 89% of the fungal growth and up to 65% reduction of the mycotoxin production by F. graminearum. Liquid chromatography/mass spectrometry analysis of this active extract revealed the presence of three families of phenolics with a predominance of methylated compounds and suggested that the abundance of methylated structures, and therefore of hydrophobic compounds, could be a primary factor underpinning the activity of the maritime pine sawdust extract. Altogether, our data support that wood/forest by-products could be promising sources of bioactive compounds for controlling F. graminearum and its production of mycotoxins.