Increasing vineyard sustainability: innovating a targeted chitosan-derived biocontrol solution to induce grapevine resistance against downy and powdery mildews.

The European Green Deal aims to reduce the pesticide use, notably by developing biocontrol products to protect crops from diseases. Indeed, the use of significant amounts of chemicals negatively impact the environment such as soil microbial biodiversity or groundwater quality, and human health. Grapevine (Vitis vinifera) was selected as one of the first targeted crop due to its economic importance and its dependence on fungicides to control the main damaging diseases worldwide: grey mold, downy and powdery mildews. Chitosan, a biopolymer extracted from crustacean exoskeletons, has been used as a biocontrol agent in many plant species, including grapevine, against a variety of cryptogamic diseases such as downy mildew (Plasmopara viticola), powdery mildew (Erysiphe necator) and grey mold (Botrytis cinerea). However, the precise molecular mechanisms underlying its mode of action remain unclear: is it a direct biopesticide effect or an indirect elicitation activity, or both? In this study, we investigated six chitosans with diverse degrees of polymerization (DP) ranging from low to high DP (12, 25, 33, 44, 100, and 470). We scrutinized their biological activities by evaluating both their antifungal properties and their abilities to induce grapevine immune responses. To investigate their elicitor activity, we analyzed their ability to induce MAPKs phosphorylation, the activation of defense genes and metabolite changes in grapevine. Our results indicate that the chitosans with a low DP are more effective in inducing grapevine defenses and possess the strongest biopesticide effect against B. cinerea and P. viticola. We identified chitosan with DP12 as the most efficient resistance inducer. Then, chitosan DP12 has been tested against downy and powdery mildews in the vineyard trials performed during the last three years. Results obtained indicated that a chitosan-based biocontrol product could be sufficiently efficient when the amount of pathogen inoculum is quite low and could be combined with only two fungicide treatments during whole season programs to obtain a good protection efficiency. On the whole, a chitosan-based biocontrol product could become an interesting alternative to meet the chemicals reduction targeted in sustainable viticulture.

Investigation of Bioactive Complexes of Chitosan and Green Coffee Bean or Artichoke Extracts.

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.

Impact of UV Irradiation on the Chitosan Bioactivity for Biopesticide Applications.

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.

Deep Chemical and Physico-Chemical Characterization of Antifungal Industrial Chitosans-Biocontrol Applications.

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.

Functional Chitosan-Calcium Carbonate Coatings for Enhancing Water and Fungal Resistance of Paper Materials.

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.

Polysaccharide Matrices for the Encapsulation of Tetrahydrocurcumin-Potential Application as Biopesticide against Fusarium graminearum.

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%.

Screening of Wood/Forest and Vine By-Products as Sources of New Drugs for Sustainable Strategies to Control Fusarium graminearum and the Production of Mycotoxins.

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.

Silylation of bacterial cellulose to design membranes with intrinsic anti-bacterial properties.

In this work, we report a convenient method of grafting non-leachable bioactive amine functions onto the surface of bacterial cellulose (BC) nanofibrils, via a simple silylation treatment in water. Two different silylation protocols, involving different solvents and post-treatments were envisaged and compared, using 3-aminopropyl-trimethoxysilane (APS) and (2-aminoethyl)-3-aminopropyl-trimethoxysilane (AEAPS) as silylating agents. In aqueous and controlled conditions, water-leaching resistant amino functions could be successfully introduced into BC, via a simple freeze-drying process. The silylated material remained highly porous, hygroscopic and displayed sufficient thermal stability to support the sterilization treatments generally required in medical applications. The impact of the silylation treatment on the intrinsic anti-bacterial properties of BC was investigated against the growth of Escherichia coli and Staphylococcus aureus. The results obtained after the in vitro studies revealed a significant growth reduction of S. aureus within the material.

pH-Triggered release of an antifungal agent from polyglycidol-based nanoparticles against fuel fungus H. resinae.

The goal of this investigation was the development of smart plurifunctional polymeric particles able to deliver a biocide following an acidic trigger due to the presence of microorganisms. Such particles were synthesized by Ring-Opening Metathesis Polymerization of an α-norbornenyl polyglycidol macromonomer functionalized with biocide through a pH-sensitive imine bond. H. resinae was selected as a target strain. In the first part, the pH sensitivity of the functionalized particles was studied. Then, the antifungal activity of both the biocide-functionalized macromonomer and the functionalized particles was evaluated. Incorporation of the particles in a commercial model coating was also tested, to verify that its original barrier properties were maintained.

Active Packaging Applications for Food.

The traditional role of food packaging is continuing to evolve in response to changing market needs. Current drivers such as consumer's demand for safer, "healthier," and higher-quality foods, ideally with a long shelf-life; the demand for convenient and transparent packaging, and the preference for more sustainable packaging materials, have led to the development of new packaging technologies, such as active packaging (AP). As defined in the European regulation (EC) No 450/2009, AP systems are designed to "deliberately incorporate components that would release or absorb substances into or from the packaged food or the environment surrounding the food." Active packaging materials are thereby "intended to extend the shelf-life or to maintain or improve the condition of packaged food." Although extensive research on AP technologies is being undertaken, many of these technologies have not yet been implemented successfully in commercial food packaging systems. Broad communication of their benefits in food product applications will facilitate the successful development and market introduction. In this review, an overview of AP technologies, such as antimicrobial, antioxidant or carbon dioxide-releasing systems, and systems absorbing oxygen, moisture or ethylene, is provided, and, in particular, scientific publications illustrating the benefits of such technologies for specific food products are reviewed. Furthermore, the challenges in applying such AP technologies to food systems and the anticipated direction of future developments are discussed. This review will provide food and packaging scientists with a thorough understanding of the benefits of AP technologies when applied to specific foods and hence can assist in accelerating commercial adoption.

Preparation and characterization of active emulsified films based on chitosan-carboxymethyl cellulose containing zinc oxide nano particles.

Active nanocomposites based on carboxymethyl cellulose-chitosan-oleic acid (CMC-CH-OL) incorporated with different concentrations (0.5-2wt.%) of zinc oxide nanoparticles (ZnO NPs) were produced by casting method. The effects of ZnO NPs on the morphological, mechanical, thermal, physical and antifungal properties of the films were studied. New interaction between ZnO NPs and polymer matrix were confirmed by Fourier Transform infrared. After addition of ZnO NPs, tensile strength, lightness (L*) and thermal stability decreased however, elongation at break, contact angle, a* (greenness) and b* (yellowness) of the nanocomposite films increased in comparison to the films without nano-filler. UV transmittance at 280nm decreased from 17.3% to 0.2, 0.1 and 0.1 for the nanocomposite films containing 0.5, 1 and 2wt.% ZnO NPs, respectively, suggesting higher UV blocking properties. Disc diffusion test showed considerable antifungal properties of the active nanocomposite films against Aspergillus niger, especially in CMC-CH-OL-ZnO 2wt.% by more than 40% fungal growth inhibition.

Cellulose oligomers production and separation for the synthesis of new fully bio-based amphiphilic compounds.

Cellulose oligomers are water-soluble, on the contrary to cellulose, which greatly increase their application range. In this study, cellulose oligomers were obtained from the acidic hydrolysis of cellulose with phosphoric acid. The global yield in water-soluble oligomers was around 23% with polymerization degree (DP) ranging from 1 to 12. The cellulose oligomers DP distribution was successfully reduced by differential solubilisation in methanol as one of the goals of this work was to avoid the use of a time-consuming full chromatographic separation. The methanol-soluble oligomers were mainly low DP (≤3). The oligomers of higher molar mass, composed of 42% of cellotetraose and 36% of cellopentaose, were then functionalized and coupled with stearic acid through azide-alkyne click chemistry to obtain amphiphilic compounds. The self-assembly of these new bio-based compounds was finally investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM) and their critical micellar concentration (CMC) was found to be in the same range as alkylmaltosides and alkylglucosides.

Active naringin-chitosan films: impact of UV irradiation.

Bioactive citrus extract-chitosan films were prepared through solvent casting-evaporation method. The impact of near UV irradiation was studied to reach a better understanding of the film behavior. The antimicrobial activity of films against Listeria innocua was maintained after UV irradiation. To study the interaction between chitosan and citrus extract components, naringin (main component) was selected as the model compound. UV treatment caused modifications of the flavanone regardless of the solvent used for its dissolution, depending on the concentration of naringin in the film: the greater the concentration the lower the modification. DSC results suggested cross-links due to UV irradiation and interactions between naringin and chitosan. This was confirmed by a decrease in the naringin release from the irradiated samples. Naringin- and citrus extract-chitosan films showed an increased absorbance in the UV region compared to pure chitosan films, showing potentiality for decreasing the lipid oxidation induced by UV light in foodstuffs.

In situ rheometry of concentrated cellulose fibre suspensions and relationships with enzymatic hydrolysis.

This work combines physical and biochemical analyses to scrutinize liquefaction and saccharification of complex lignocellulose materials. A multilevel analysis (macroscopic: rheology, microscopic: particle size and morphology and molecular: sugar product) was conducted at the lab-scale with three matrices: microcrystalline cellulose (MCC), Whatman paper (WP) and extruded paper-pulp (PP). A methodology to determine on-line viscosity is proposed and validated using the concept of Metzner and Otto (1957) and Rieger and Novak's (1973). The substrate suspensions exhibited a shear-thinning behaviour with respect to the power law. A structured rheological model was established to account for the suspension viscosity as a function of shear rate and substrate concentration. The critical volume fractions indicate the transition between diluted, semi-diluted and concentrated regimes. The enzymatic hydrolysis was performed with various solid contents: MCC 273.6 gdm/L, WP 56.0 gdm/L, PP 35.1 gdm/L. During hydrolysis, the suspension viscosity decreased rapidly. The fibre diameter decreased two fold within 2 h of starting hydrolysis whereas limited bioconversion was obtained (10-15%).

Influence of the variation of the alkyl chain length of N-alkyl-ß-D-glycosylamine derivatives on antifungal properties.

Twelve new glucosidic and galactosidic derivatives of N-alkylaminosugars with different alkylamines from 6 to 18 carbons were synthesized and characterized by (1)H and (13)C NMR. Their antifungal activity against the food fungal pathogen Aspergillus niger was evaluated using the radial growth assay. The influence of the variation of the alkyl chain length of N-alkylaminosugars on the mycelium growth was then discussed. Inhibition by the different alkylamines is shown as a biostatic effect rather than a biocidal effect. It was observed that alkylamines keep their antifungal properties after a thermal treatment compatible with food packaging and processing.

Synthesis of D-glucosamine quaternary ammonium derivatives and evaluation of their antifungal activity together with aminodeoxyglucose derivatives against two wood fungi Coriolus versicolor and Poria placenta: structure-activity relationships.

BACKGROUND: Structure-activity relationships are often reported in scientific studies. These may be employed in searching for new acceptable biocides to use against harmful microorganisms, because the biocides used hitherto encounter various problems, including lack of efficiency, high toxicity and persistence. Nowadays, scientists are trying to find new, environmentally acceptable biocides to replace these earlier biocides. Different compounds from renewable materials have been studied and have shown pronounced antifungal activity against wood fungi. These include aminopolysaccharide derivatives and different quaternary ammonium polymers. A biological study carried out with these products indicated a possible relationship between amino groups and differences in biological activity observed. RESULTS: In this study, an amino group was successively fixed to different carbon atoms of glucose, and glucosamine was also modified by both N-alkylation and quaternisation. The impact of the amino group position on antifungal activity against two wood decay fungi was investigated. The amino group at the anomeric position showed the highest antifungal activity against both Coriolus versicolor Quel. and Poria placenta (Fr.) Cooke. Furthermore, the positive impact of both N-alkylation and quaternisation on the growth of both strains was demonstrated. CONCLUSION: The anomeric position of the amino group and the N-alkylation and quaternisation of amino sugars considerably increase the antifungal activity of these compounds.

In vitro inhibitory effect of tetrahydrocurcuminoids on Fusarium proliferatum growth and fumonisin B1 biosynthesis.

Many plant pathogens produce toxic metabolites when growing on food and feed. Some antioxidative components seem to prevent fungal growth and mycotoxin formation. Recently, we synthesized a new class of powerful antioxidative compounds, i.e. tetrahydrocurcuminoids, and its structure/antioxidant activity relationships have been established. The South West of France produces large amounts of corn, which can be infected by Fusarium species, particularly F. proliferatum. In this context, the efficiency of tetrahydrocurcuminoids, which can be obtained from natural curcuminoids, was investigated to control in vitro the growth of F. proliferatum and the production of its associated mycotoxin, fumonisin B1. The relation between structure and antifungal activity was studied. Tetrahydrocurcumin (THC1), with two guaiacyl phenolic subunits, showed the highest inhibitory activity (measured as radial growth on agar medium) against the F. proliferatum development (67% inhibition at a concentration of 13.6 µmol ml⁻¹). The efficiencies of THC2 (36% at a concentration of 11.5 µmol ml⁻¹), which contains syringyl phenolic units, and THC3 (30% at a concentration of 13.6 µmol ml⁻¹), which does not have any substituent on the aromatic rings, were relatively close. These results indicate that the simultaneous presence of guaiacyl phenols and the enolic function of the ß-diketone moiety play an important role in the inhibition mechanisms. The importance of this combination was confirmed using n-propylguaiacol and acetylacetone as molecular models. Under the same conditions, ferulic acid and eugenol, other natural phenolic antioxidants, were less efficient in inhibiting fungal growth. THC1 also reduced fumonisin B1 production in liquid medium by approximately 35, 50 and 75% at concentrations of 0.8, 1.3, and 1.9 µmol ml⁻¹, respectively. These very low inhibitory concentrations show that tetrahydrocurcuminoids could be one of the most promising biobased molecules for the control of mycotoxinogen fungal strains.

Hydrophobization and antimicrobial activity of chitosan and paper-based packaging material.

This study reports the elaboration of water-resistant, antimicrobial, chitosan and paper-based materials as environmentally friendly food packaging materials. Two types of papers were coated with chitosan-palmitic acid emulsions or with a blend of chitosan and O,O'-dipalmitoylchitosan (DPCT). Micromorphology studies showed that inclusion of hydrophobic compounds into the chitosan matrix was enhanced by grafting them onto chitosan and that this led to their penetration of the paper's core. Compared to chitosan-coated papers, the coating of chitosan-palmitic emulsion kept vapor-barrier properties unchanged (239 and 170 g.m(-2).d(-1) versus 241 and 161 g.m(-2).d(-1)), while the coating of chitosan-DPCT emulsion dramatically deteriorated them (441 and 442 g.m(-2).d(-1)). However, contact angle measurements (110-120 degrees after 1 min) and penetration dynamics analysis showed that both strategies improved liquid-water resistance of the materials. Kit-test showed that all hydrophobized chitosan-coated papers kept good grease barrier properties (degree of resistance 6-8/12). Finally, all chitosan-coated materials exhibited over 98% inhibition on Salmonella Typhimurium and Listeria monocytogenes .

Synthesis and antibacterial activity of aminodeoxyglucose derivatives against Listeria innocua and Salmonella typhimurium.

In this study aminodeoxyglucose derivatives were synthesized and evaluated for their antibacterial activity against two food bacteria, Listeria innocua and Salmonella typhimurium . 6-Amino-6-deoxy-alpha-D-methylglucopyranose (GSA-6), 3-amino-3-deoxy-D-glucopyranoside (GSA-3), and beta-D-glucopyranosylamine (GSA-1) were synthesized and concurrently tested with commercially available D-glucosamine (GSA-2) for antibacterial activity. Results obtained from this study showed a pronounced antagonist effect due to the position of amino groups of aminoglucose derivatives on the antibacterial activity. GSA-3 was the most active compound. At a concentration of 2 x 10(-4) mol mL(-1), it delayed the growth of both bacteria with percentages of inhibition of 29 and 15% for L. innocua and S. typhimurium, respectively. At the same concentration the percentages of inhibition for other aminodeoxyglucoses varied between 5 and 18% and between 2 and 11% for L. innocua and S. typhimurium, respectively. All compounds were characterized by FTIR, (1)H NMR, and (13)C NMR spectroscopy.

Advances on selective C-6 oxidation of chitosan by TEMPO.

The specific C-6 oxidation by TEMPO of chitosan and chitosan derivatives were studied to obtain tailored bioactive biopolymers. The modifications on chitosan presented many difficulties and showed the adverse effect of the amine moieties of chitosan on this reaction. Thus, protections of the amino groups by N-acetylation or N-phthaloylation were studied and followed by the C-6 specific oxidations of the resulting polymers. The desired 6-carboxychitosan could not be obtained after deprotection; the reactions with TEMPO led to degradation of the polymers. The specific oxidation of a potentially bioactive derivative of chitosan was then achieved by the oxidation of a quaternized chitosan: N, N, N-trimethylchitosan. N, N, N-Trimethyl-6-carboxychitosan was characterized by FTIR spectroscopy, 1H, and 13C NMR spectroscopy.