Camille Bihanic

Prof. C Kenza Richards

Dr Tomasz K Olszewski

Claude Grison
email: claude.grison@cnrs.fr

Grison

Eco-Mn Ecocatalysts: Toolbox for Sustainable and Green Lewis Acid Catalysis and Oxidation Reactions

Keywords: Green chemistry, Ecocatalysis, Lewis acid catalysis, Sustainable oxidation, Sequential tandem reaction

Phytoextraction is one of the most promising phytotechnologies used to restore natural environments degraded by mining activities. In New Caledonia, a few plant species, which belong to the Grevillea genus, have the ability to extract Mn from soil and accumulate it in abundance (over 1 % of leaves dry weight). This review describes the use of Grevillea Mn-accumulating plant species to produce the first bio-sourced Mn catalysts, called Eco-Mn catalysts. Extensive structural studies of Eco-Mn catalysts have highlighted an original composition characteristic of their vegetal origin. Eco-Mn catalysts have demonstrated competitive catalytic activities compared to conventional Mn catalysts in Lewis acid catalysis, aminoreductions, alcohol oxidations, epoxidation reactions, oxidative cleavage of 1,2-diols and alkenes and "Janus catalysts" for sequential tandem oxidations such as tandem carbonyl-ene cyclisation, synthesis of substituted pyridines and oxidative iodination of ketones.

Introduction

The depletion of the world mineral resources has become a critical issue from economic and environmental points of view. Recycling techniques are still underdeveloped and the rising demand for metallic elements induces a short-term depletion of mineral resources. Access to strategic and primary minerals has become crucial considering this depletion, especially since these minerals are owned by a small number of politically unstable countries. In addition to having values for mining industries, transition metals, such as Mn, are essential for many chemical transformations in the fields of petrochemicals, agrochemicals, polymers, plastics, textiles, dyes, paints, drugs, cosmetics and perfumes. Recently it was reported that the short-term shortage of metal species used in catalytic chemistry is a major issue that chemical industries have now to face. For instance, Mn has been on the list of critical elements in the US since 2017. Referring to the currently known resources, Mn resources could be depleted in 20 to 50 years. [1] The need of metallic elements induces the expansion of mining sites and the use of new extraction techniques, causing significant environmental issues: degradation of natural environments, loss of biodiversity and pollution of ecosystems.

The energy and ecological transition lead to a paradox. While the motives are those of environmental progress through the development of green energies, this transition underlies an increase in mineral resources requirement and ultimately a rising pressure on the demand for metals. The environmental consequences are worrying.

New Caledonia is a representative example of the global context and its current evolution. New Caledonia is an archipelago located in the South Pacific (20°-23°South, 164°-167°East). [2] The ultramafic soils of New Caledonia are naturally enriched in valuable metallic elements, such as nickel, cobalt, manganese, iron, chromium and scandium; [2,3] and nickel, particularly in laterite, represents one of the largest resources of nickel in the world. [4] Access to metal deposits, located at a particular depth, requires the removal of the upper part of soil and hence the destruction of all vegetation cover, leading to serious ecological impacts for the island. [5,6] Indeed, removal of the vegetation cover affects the biodiversity and the landscape environment. In addition, erosion and leaching of soils, denuded by prospecting and ore extraction, cause significant damages to ecosystems. The sediments are dragged away into rivers and then into the New Caledonian lagoon, a World Heritage site by UNESCO, [START_REF]Lagoons of New Caledonia: Reef Diversity and Associated Ecosystems[END_REF] thus threatening the coral reef surrounding the island. [5,[START_REF] Gunkel-Grillon | [END_REF] In this context, our group has developed new rehabilitation programs on six mining sites of New Caledonia. Our approach is based on using the New Caledonian biodiversity and endemic pioneer plants that have naturally evolved to grow on ultramafic soils. Some of them are Ni-or Mn-(hyper)accumulating plants. (Hyper)accumulating plants are able to translocate metals from roots and store them into shoots (Figure 1). [6,[9][10][11][12][13] These plants are accommodated to multiple local constraints: open environment, tropical climate, edaphic and hydric constraints, high altitude, degraded soils depleted of nutrients and micro-organisms, and more interestingly adapt to soils rich in the toxic elements Ni, Co, Mn, Cr, Fe. Thus they are remarkably adapted to the impacts resulting from mining activities and are ideal plants to start the rehabilitation of degraded sites. [14] Among the pioneer Mn-accumulating plants, Grevillea exul ssp exul and ssp rubiginosa, Grevillea meisneri and Grevillea gillivrayi of the genus Grevillea were selected for restoration (Figure 2). [START_REF] Jaffré | Plantes de Nouvelle-Calédonie permettant de revégétaliser des sites miniers[END_REF][START_REF] Huillier | Mines et Environnement En Nouvelle-Caledonie: Les Milieux Sur Substrats Ultramafiques et Leur Restauration[END_REF][START_REF] Udo | Multiplication et Valorisation Horticole de Plantes Indigènes à La Nouvelle-Calédonie: Compte-Rendu Des Essais 2011[END_REF][START_REF] Jaffré | [END_REF] The average foliar manganese concentration in normal plants is 2590 mg/kg (dry weight), which places Grevillea exul among the most Mn-rich species but without being an hyperaccumulator (threshold at 10 000 mg/kg). Individual measurements up to 3900 mg/kg have been reported for the variety exul and up to 6200 mg/kg for the variety rubiginosa. [START_REF] Jaffré | [END_REF]19] These Mn-accumulating plants being particularly abundant in New Caledonia deserve a particular effort of conservation and make them ideal candidates for ecological restoration, in the strict sense of the term.

In 2009, our group showed that it was possible to combine ecological restoration and economic opportunities. Plants used for restoration were transformed through an innovative concept of recycling. Taking advantage of the remarkable adaptive capacity of some plants to accumulate manganese, we directly use the metallic species, originated from this metal-rich biomass, as catalysts in fine organic chemistry. This original approach offered the first perspective of valorization of this unique biomass and initiated a new field of green chemistry: the ecocatalysis (Scheme 1). [START_REF] Grison | Ecocatalysis: A New Integrated Approach to Scientific Ecology[END_REF][START_REF] Grison | [END_REF][22] The concept of ecocatalysis has allowed the preparation of a wide range of ecological catalysts: the ecocatalysts. [23][START_REF] Grison | Use of Certain Metal-Accumulating Plants for the Performance of Organic Chemistry Reactions[END_REF][START_REF] Grison | Use of Certain Manganese-Accumulating Plants for Carrying out Organic Chemistry Reactions[END_REF][START_REF] Grison | Use of Selected Accumulator of PGM Plants for the Implementation of Organic Chemistry Reactions[END_REF][START_REF] Grison | Use of Metal-Accumulating Plants for the Preparation of Catalysts That Can Be Used in Chemical Reactions[END_REF] Ecocatalysts present a flexible reactivity, while respecting the principles of Green chemistry. Ecocatalysts can be used as heterogeneous catalysts in synthetic transformations allowing access to molecules with a high added-value for the production of fine chemicals and in scalable processes. [28,29] Among the different ecocatalysts prepared to date, Eco-Mn catalysts have two major properties: [14,30] a hard and marked Lewis acidity or/ and an oxidative activity allowing the replacement of oxidants prohibited by REACH regulation (Registration, Evaluation, Authorization and Restriction of Chemicals) (European Commission 2006). [START_REF]Candidate List of substances of very high concern for Authorization -ECHA[END_REF] These interesting properties are specific to Eco-Mn catalysts and are the consequence of a particular structure of these materials. The organization of their nanostructures is related to the nature of the plant species they derive from and to the unique and uncommon catalytic species generated during their preparation. [START_REF] Garel | [END_REF] In this review, we illustrate the synthetic potential of these new generations of catalysts, Eco-Mn catalysts, in which the transition metal, Mn, is purely biobased.

The first chapter is dedicated to the Lewis acidity of Eco-Mn catalysts (called Eco-Mn LA ) shown by pyridine sorption capacity and applied to three chemical transformations: the carbonylene cyclisation of citronellal, the enol ether synthesis and the amino-reduction of carbonyl derivatives. In a second chapter, the oxidative capacities of Eco-Mn catalysts (called Eco-Mn Ox ) Claude Grison is CNRS Research Director. She is leading the Laboratory of Bio-inspired Chemistry and Ecological Innovations (Montpellier, France), which is dedicated to the implementation of new ecological solutions and innovative chemical tools for the ambitions of tomorrow's chemistry. C. Grison is the pioneer of the ecocatalysis concept, which represents a major break-through at the interface between ecology and chemistry. Author and co-author of more than 181 scientific articles and patents, she has received 9 Awards for Innovation. are illustrated by major transformations used in the chemical industry: alcohol oxidation, diol oxidative cleavage, ketone iodization, aromatizing dehydrogenation and alkene epoxidation. A final chapter presents the Janus character of the Eco-Mn catalysts (called Eco-Mn LA-Ox ) in tandem sequences of multicomponent-type reactions (synthesis of substituted pyridines) and cyclizations (synthesis of p-cymene).

Preparation and characterization of Eco-Mn catalysts

Preparation of Eco-Mn LA catalysts

A crucial feature of Eco-Mn catalysts is their polymetallic composition resulting from the combination of Mn 2 + at high concentration with physiological elements of the plants (e. g. Fe 3 + , Na + , K + , Ca 2 + , Mg 2 + ).

The preparation of Eco-Mn catalysts requires experimental conditions that allow us to control the composition, structure and degree of oxidation of Mn that modulate the subsequent reactivity. The key steps for the preparation of Eco-Mn are (i) heat treatment and (ii) activation by introduction of new anions (treatment with HCl). The heat treatment burns to ashes the organic material of plants and the acid treatment allows the solubilization of metals as metal chlorides. These experimental conditions generate ecocatalysts with Lewis acidity properties called Eco-Mn LA (Scheme 2).

Eco-Mn LA catalysts are polymetallic catalysts composed of a set of salts, including mixed salts (non-monometallic salts). Eco-Mn LA catalysts are mainly composed of Ca, Mg, Na, K and Mn cations, and contain Fe, Ni and Al cations in smaller amounts. Manganese is exclusively present in the oxidation state II in an octahedral environment; and iron is predominantly present in the oxidation state III in octahedral or tetrahedral environments depending on the catalysts. The anions of Eco-Mn LA catalysts are chlorides due to the chemical treatment, but also of sulphates and phosphates due to the plant origin.

The compositions of Eco-Mn LA catalysts vary according to the plants from which they derive, as illustrated by our study on Grevillea plants that were used in mining restoration. [19] However, the Mn content of Eco-Mn LA catalysts is reproducible, about 4 %, 5.5 %, 6.5 % and 11 % for Grevillea exul ssp exul, gillivrayi, exul ssp. rubiginosa and meisneri respectively. It is directly linked to the accumulation capacities of each species Table 1). [19] We have extensively studied and characterized the crystalline structure of Eco-Mn LA and the chemical environment around active catalytic centers (manganese and iron) by X-Absorption Spectroscopy (EXAFS and XANES) analyses. We found that Eco-Mn LA catalysts are not a juxtaposition of simple metal chlorides, but that their composition is much more complex. Unusual and mixed metal chlorides, such as K 3 NaMnCl 6 and KMnCl 3 , were clearly identified and were further crystallized for an identification by XRD (Figure 3). [START_REF] Garel | [END_REF] To a lesser extent, iron and magnesium are also present as a mixed metal chlorides K 2 FeCl 5 H 2 O, [START_REF] Garel | [END_REF] and KMgCl 3 .6H 2 O. [33] We also demonstrated that Eco-Mn LA catalysts cannot be reproduced, in terms of structure, properties and catalytic activities, by mixtures of commercial metallic salts or by mixing any biomass with a manganese salt. This study highlighted the peculiar vegetal footprint of Eco-Mn catalysts.

This conclusion can be generalized to all Eco-Mn LA derived from plant species of the genus Grevillea used in mining Scheme 1. From the ecological rehabilitation of New Caledonian mining sites to an original valorization: Ecocatalysis. Scheme 2. Experimental conditions for the preparation of Eco-Mn LA catalysts. restoration: Grevillea exul ssp. rubiginosa, Grevillea exul ssp. exul, Grevillea meisneri and Grevillea gillivrayi. [START_REF] Garel | [END_REF]

Preparation of Eco-Mn OX catalysts

Our group has established a general procedure for the preparation of Eco-Mn OX catalysts from Eco-Mn LA catalysts, that are treated with a green co-oxidant (H 2 O 2 or O 2 ) (Scheme 3).

According to the co-oxidant used and the experimental conditions, the catalytic properties differ. We reported three catalytic systems:

-Eco-Mn OX1 : Eco-Mn LA is oxidized by the oxygen from air at atmospheric pressure and at pH 8. Mn(II) is oxidized into Mn 2 O 3 (Mn(III)) and Mn 3 O 4 (Mn(II, III)). Fe(III) also present in Eco-Mn LA catalysts precipitates as Fe 2 O 3 .

Mn 2 O 3 (Mn(III)) and Mn 3 O 4 (Mn(II, III)) are then transformed by disproportionation into soluble Mn 2 + ions (Mn(II)) and MnO 2 (Mn(IV)) in acid conditions at pH = 3. After acidification, Fe 2 O 3 is also converted to soluble hydrated iron chloride ions. The final mixture is composed of MnO 2 , Mn(II) and Fe(III) chlorides, which can all act as Lewis acids (Scheme 4). [34] -Eco-Mn OX2 : Eco-Mn LA is oxidized by hydrogen peroxide in the presence of a large excess of sodium hydroxide. The precipitate formed is composed of a mixture of Mn oxides: Na 0.55 Mn 2 O 4 .1.5H 2 O, MnO(OH), Mn 3 O 4 and CaMn 3 O 8 (Scheme 5). [35] -Eco-Mn OX3 : Eco-Mn LA is supported on montmorillonite K10, then added to the substrate and treated by a mixture of sodium bicarbonate and hydrogen peroxide (Scheme 6). [36] If the experimental conditions allow the preservation of all the metallic elements of Eco-Mn LA , it is not true for Eco-Mn Ox .

Table 2 illustrates the metallic compositions of Eco-Mn LA catalysts and Eco-Mn Ox2 catalysts. [35] The treatment eliminates 2/3 of the Fe present in Eco-Mn LA in favor of a higher rate of Mn. The polymetallic nature of the ecocatalyst is however preserved with an interesting mixture of Mn/Ca/Mg.

Regardless of the preparation of Eco-Mn, its composition is polymetallic and the nature of the species formed is known. The simultaneous presence of a combination of well-defined active sites results from this variety of metallic species. A conventional catalyst would influence only some of the reaction process steps, thus limiting opportunities in organic synthesis.

Here, the richness of the different interactions between the species present in Eco-Mn leads to unusual metal/substrate interactions that is illustrated with functional transformations of high interest in organic synthesis.

Reactivity of Eco-Mn catalysts

Catalysis by Eco-Mn LA catalysts

The Lewis or Brønsted acid character of the eco-catalysts was demonstrated by studying their reactivity towards the cyclic acetal derived from α-bromopropiophenone and the selectivity of the formed products (Scheme 7). This technique is inspired from Corma's works that were on supported palladium and zinc catalysts. [37,38] Eco-Mn LA showed a hard Lewis acid character (43 % yield of the hard Lewis acid products), and a marked Brønsted acidity (18 % yield of the Brønsted acid products) and a negligible soft Lewis acidity (Scheme 7). The hard Lewis acidity is expected and explained by the presence of hard cations; [39,40] and the Brønsted acidity might be due to the presence of iron in Eco-Mn LA (0.7 wt% to 2.8 wt%).

These acid properties were explored through the carbonylene cyclisation of citronellal, the synthesis of enol ethers and the reductive amination of carbonyl derivatives. [34] Carbonyl-ene cyclisation of (+)-citronellal into (À )-isopulegol is the key step of the synthesis of (À )-menthol, which is one of the most used flavoring chemicals. [41,[START_REF] Lawrence | Mint: The Genus Mentha[END_REF] Since the skeleton of the (À )-isopulegol has two new stereogenic centers relative to (+)-citronellal, the intramolecular carbonyl-ene reaction can lead to four diastereomers. [START_REF] Feigl | Spot Tests in Inorganic Analysis[END_REF] The (À )-isopulegol is the only isomer of industrial interest.

Carbonyl-ene cyclisation of citronellal

The synthesis of (À )-isopulegol from (+)-citronellal requires a high diastereoselectivity. The carbonyl-ene cyclisation of (+)-citronellal into (À )-isopulegol is favored by Lewis acids, [START_REF] Imachi | [END_REF] while the use of Brønsted acid leads to lower selectivity of (À )isopulegol. [45,46] The selectivity of this cyclisation is directly related to the strength and hardness of Lewis acid metal centers. The Mn 2 + cation is a Lewis acid of ideal hardness, but its use is limited by its low reactivity for this reaction. The polymetallic composition of Eco-Mn LA and the presence of cations such as Fe 3 + and Al 3 + exalts the classical reactivity of Mn-based species. Eco-Mn LA catalyzes the carbonyl-ene reaction, with 95 % yield and 90 % selectivity in the desired (À )isopulegol, with only 2 mol% of Mn in a very short reaction time (0.5 h) (Scheme 8). [34] Alkyl enol ethers are of great synthetic interests. They are very reactive towards electrophilic reagents, in particular carbonyl groups, and are used for the formation of CÀ C bonds in organic synthesis. Compared to their metal enolate counterparts, alkyl enol ethers are more stable and can often be isolated. Alkyl enol ethers are also widely used in cycloaddition reactions, such as the inverse electron demand Diels-Alder reaction. [47,48] Several literature reports about the preparation of alkyl enol ethers and their synthetic applications are available. [START_REF] Chan | Comprehensive Organic Synthesis: Selectivity, Strategy, and Efficiency in Modern Organic Chemistry[END_REF][START_REF] Gassman | [END_REF][51] Notable examples of compounds synthesized with the use of alkyl enol ethers include β-carotene, retinal (vitamin A aldehyde) and its derivatives, [52,53] pyrrolidine alkaloids, [54] iridodial natural products [47] and tetrahydroquinoline derivatives, [55] all having potential biological activity.

Enol ether synthesis

Conventional methods for the preparation of alkyl enol ethers involve the use of hard conditions such as strong bases (for example, n-butyllithium [n-BuLi], lithium diisopropylamide [LDA], sodium hydride) in toxic organic solvents (e. g., tetrahydrofuran, dichloromethane). [START_REF] Gassman | [END_REF]56,57] However, it is also possible to synthesize alkyl enol ethers from aldehydes, by acetalization with an alcohol under acid catalysis, then removal of the alcohol, under acid or base catalysis. [58] The elimination step is generally carried out with the use of aggressive Brønsted acids, such as sulfuric acid or p-toluenesulfonic acid [58] or readily hydrolysable Lewis acids, such as aluminum. [59] This step can also be accomplished by co-catalysis with aluminum chloride and a non-nucleophilic base, such as triethylamine. [59] Noticeably, none of these conditions are in accordance with the principles of green chemistry.

Given the double acid nature of Eco-Mn LA1 as hard Lewis and Brønsted acids, the formation of ethyl enol ether from carbonyl derivatives is possible (Scheme 9). Aliphatic aldehydes were treated with 10 mol% of Mn in anhydrous ethanol at 80 °C and lead to aliphatic acetals (path A) in good yields (77 %-96 %). The alcohol elimination of the acetals was performed by distillation and led to the corresponding ethyl enol ethers in good yields (82 %-89 %). When citronellal was used as substrate (path B), the corresponding enol ether was directly obtained and with a good yield (96 %); no trace of acetal was detected. The conversion of the intermediate acetal was facilitated by the double Lewis and Brønsted acidity of Eco-Mn LA1 catalyst.

Eco-Mn LA1 catalyst has proved to be a green alternative to conventional acid catalysts used for the synthesis of enol ethers, avoiding the use of strong acid catalysts or sensitive reagents. [60] Amines are very important industrial organic compounds that have found widespread applications in the pharmaceutical, agrochemical, material and chemical industries.

Reductive amination of ketones

Reductive amination is one of the most common methods to prepare amines from carbonyl compounds in the presence of a reducing agent. Stoichiometric boron hydrides and heterogeneous hydrogenation have been mainly used to reduce reaction intermediates. However, the use of borohydrides generates abundant and hazardous wastes and heterogeneous hydrogenation is limited by its relatively poor chemoselectivity. [61] An alternative method is based on the use of 1,4-dihydropyridines (DHP) and conducts to a competitive regio-and enantioselectivity compared to classical methods. [START_REF] Kellog | Comprehensive Organic Synthesis. Selectivity, Strategy and Efficiency in Modern Organic Chemistry[END_REF] DHP are hydridedonating agents in this reaction [START_REF] He | [END_REF] as they behave as biomimetic reducing agents of nicotinamide adenine dinucleotide (NADH). Commonly available 1,4-dihydropyridines are the Hantzsch esters (HEH). The reductive amination with HE requires an acid promotor to activate the imine or ketiminium intermediates. Dry Mg(ClO 4 ) 2 and zinc ions are particularly effective.

The reductive amination is usually applied to aldehydes by using hazardous reductive agents. Using Eco-Mn LA1 catalysts, the reductive amination was applied to less reactive ketones in milder conditions respecting the principles of Green chemistry. The reaction was conducted under solvent-free conditions and with an easy to prepare, soft and non-toxic HEH. [64] The catalytic efficiency of the Eco-Mn LA1 catalyst was compared to conventional Mn chloride salts, which constitute the majority of the anions in Eco-Mn LA1 structure (Table 3). To highlight the differences between catalysts, the reaction was carried out with the poorly reactive 3-methyl-2-butanone. The use of Eco-Mn LA1 catalyst led to a higher conversion than commercial Mn chlorides, illustrating the original properties of the polymetallic complexes present in the plant-derived catalyst. This was confirmed by using a "reconstituted Eco-Mn LA1 " catalyst, prepared by mixing together commercial chlorides of K, Mg, Ca, Al, Mn, Fe and Ni, in the same proportions as the ones in Eco-Mn LA catalyst. The reconstituted Eco-Mn LA1 catalyst behaved similarly to the original Eco-Mn LA1 catalyst. The catalytic performance of Eco-Mn LA1 catalyst is therefore specific to its particular polymetallic composition induced by its plant origin.

The reductive amination by Eco-Mn LA was extended to various ketones using aliphatic and cyclic amines (Table 4). Eco-Mn LA catalyst showed a high catalytic efficiency, since only 0.25 mol% of catalyst allowed 96 % yield for the model reaction with cyclohexanone and aniline. Interestingly, the aliphatic and Scheme 9. Acetalization/elimination of aldehydes catalyzed by Eco-Mn LA1 . a Reaction conditions: aldehyde (1.0 mmol), Eco-Mn LA (82 mg, amount corresponding to 10 mol% of Mn), anhydrous ethanol (3 mL), 80 °C, 5 h. b Yields were determined by GC-MS after calibration, using biphenyl as internal standard.

Table 3. Reductive amination of ketones -comparison of Eco-Mn LA1 with classical Mn chlorides. [a] Entry Catalyst Catalyst loading cyclic amines can also react with ketones under similar conditions and the desired products are formed in very good yields (88-96 %, Table 4, entries 3). Using a cyclic ketone, the amino partner can be aniline substituted with electron withdrawing or donating groups (95-99 %). The methodology was applied to the preparation of amines derived from 2-tetralone and 2-indanone as precursors of pharmaceutically compounds (Table 4, entries 5 & 6). The Eco-Mn LA1 catalyst has been successfully used in the reductive amination of various ketones in combination with a non-toxic, affordable, and safe reducing agent, under a solventfree methodology and in short reaction times (1-4 h). This illustrates well the capacity of Eco-Mn LA1 for the synthesis of substituted amines, precursors of pharmaceutically compounds. [34] The oxidation of benzylic alcohols to aldehydes is an important industrial chemical reaction. Many oxidizing agents are available to perform this key reaction. Although several stoichiometric reagents based on activated dimethylsulfoxide (Swern oxidation), [START_REF] Lee | Comprehensive Organic Synthesis. Selectivity, Strategy and Efficiency in Modern Organic Chemistry[END_REF] hypervalent iodine compounds (Drawing-Martin periodinane) [START_REF] Dess | [END_REF] and reactive oxo-metallic compounds (MnO 2 and pyridinium chlorochromate) [START_REF] Ley | Comprehensive Organic Synthesis. Selectivity, Strategy and Efficiency in Modern Organic Chemistry[END_REF] have been developed. The search for new catalytic procedures for selective oxidation of benzyl or allylic alcohols remains an area of intense interest. Indeed, despite their efficiency, the disadvantage of the abovementioned oxidants in the perspective of their use in multi-step synthesis is their lack of selectivity. For example, the overoxidation of aldehydes to carboxylic acids and the degradation of unsaturated substrates are often unavoidable side reactions.

[mol %] [b] Yield [%] [c,d]

Catalysis by Eco-Mn Ox catalysts

Eco-Mn Ox1 catalyst, a versatile oxidative agent of alcohols

The oxidative potential of Eco-Mn Ox1 was first tested using benzyl alcohol (Table 5). Using less than 4 molar equivalents of Mn, Eco-Mn Ox1 catalyzed quantitatively the formation of benzaldehyde, while MnO 2 could not (Table 5, entry 1 & 2). The amounts of Mn species were quite small compared to conventional conditions (typically 10 equivalents). [START_REF] Cahiez | Encyclopedia of Reagents for Organic Synthesis[END_REF] The polymetallic composition of Eco-Mn Ox1 , in particular the presence of Fe(III) species, increases the oxidizing properties of Mn(IV) while driving the reaction to the expected aldehyde. Indeed, only 20 % of benzaldehyde was obtained using a mixture of MnO 2 and FeCl 3 (Table 5, entry 4). The combination of Mn(IV) and Fe (III) cannot explain the quantitative yield when using Eco-Mn Ox1 . We assumed a synergetic effect between the metallic species within Eco-Mn Ox1 or a specific structure of the catalyst due to its vegetal origin. [START_REF] Garel | [END_REF] The absence of benzoic acid among the reaction products is an important advantage of the presented procedure. It was applied to the oxidation of 4-hydroxy-3-methoxybenzyl alcohol (vanillyl alcohol) to vanillin, which was quantitatively catalyzed by Eco-Mn Ox1 (Table 6, entry 1). It is noteworthy for an industrial development that this synthesis is entirely bio-based: the alcohol is a natural and abundant substance present in Table 4. Reductive amination of ketones catalyzed by Eco-Mn LA /HEH system -substrate scope. [a] Entry Ketone Amine Product Yield [%] Table 5. Oxidation of alcohols -comparison of Eco-Mn OX1 with classical Mn oxides. [a] Entry Catalyst Catalyst loading [eq.] Yield [%] [b] 1 Eco-Mn Ox1 3.9 100 2 MnO 2 3.9. traces 3 [START_REF] Cahiez | Encyclopedia of Reagents for Organic Synthesis[END_REF] MnO 2 10 89 4

MnO 2 + FeCl 3

[c]

3.9 20

[a] Reaction conditions: alcohol (10 mmol), catalyst (38.9 mmol Mn), EtOAc (20 mL), 75 °C, 3 h. [b] Yields were determined by GC-MS after calibration, using biphenyl as internal standard.

[c] 5.30 mmol of FeCl 3 were used, in order to reproduce the ratio Mn/Fe of Eco-Mn Ox1 .

numerous plant species such as Picea sitchensis (Pinaceae); [START_REF] Buckingham | Dictionary of Natural Products[END_REF] the oxidant is completely natural, since it derives from Grevillea exul rubiginosa. This synthesized vanillin can be described as a natural aroma of vanillin. [START_REF] Stanzione | [END_REF] The oxidation procedure was easily transposable to the controlled oxidation of allyl alcohols under similar conditions; for example, geraniol led to geranial (trans-citral) (Table 6, entry 2). This molecule faces a strong demand for its lemon scent in the food industry or as an important intermediate for the production of perfumes and pharmaceuticals. [71] The particular case of the polyhydroxyalkyl furan allowed to evaluate the chemoselectivity of the oxidation process catalyzed by Eco-Mn Ox1 (Table 6, entry 3). The furan aldehyde was obtained directly and in a quantitative This result reflects the ability of Eco-Mn Ox1 to promote the oxidative cleavage of a polyol in the benzylic position. The reaction showed no concurrent side reaction, which confirmed the excellent regioselectivity of the cleavage. The yield of the reaction is remarkably higher with Eco-Mn Ox1 than with Ce(IV) reagents, which require a large excess of oxidant (11 equivalents, 48 % yield). [72] This oxidative cleavage of polyhydroxyalkyl furan to furan aldehyde could be an interesting way to produce functionalized furfural compounds, with new functionalities compared to the widely studied furfural compounds. [73,74] This method can easily replace toxic or unstable oxidants such as NaIO 4 , Pb(OAc) 4 , NaBiO 3 , sodium and calcium hypochlorite, Ce(IV), Cr(VI), Co(II) or V(IV) reagents, Niodosuccinimide. [START_REF] Shing | Comprehensive Organic Synthesis. Selectivity, Strategy and Efficiency in Modern Organic Chemistry[END_REF] In addition, it can also be advantageously compared to recent methods using O 2 as an oxidant with ruthenium catalysis, [START_REF] Takezawa | [END_REF] palladium [77] or cobalt. [78] Although these methods represent valuable ecological improvements in the oxidative cleavage of glycols, the catalysts involved rely on rare and expensive metals, which considerably limit their use. [35] The catalytic properties of EcoMn Ox2 were also evaluated in the aerobic oxidative cleavage of 1,2-activated diols under atmospheric pressure of O 2 and with 10 mol% of Mn. A study of the solvent effect was carried out on meso-hydrobenzoin as a model substrate (Table 7). Almost complete conversion was obtained in 1 h in 1-butanol at 100 °C (98 %, Table 7, entry 2). Complete selectivity for benzaldehyde was observed, with no traces of benzoic acid or other oxidation by-products. This result clearly contrasts with previous work on MnO 2 , which requires 40 equivalents of Mn in dichloromethane. [79] Eco-Mn OX2 was recovered by simple filtration after reaction and reused up to 6 times without any loss of activity. The Efactor of this reaction using EcoMn Ox2 shows that 4 to 17 times Table 6. Oxidation of alcohols by Eco-Mn Ox1 . [a] Entry Alcohol Catalyst Product Yield [%] [b] 1 Eco-Mn Ox1 100 2 Eco-Mn Ox1 100 [c] 3 Eco-Mn Ox1 [a] Reaction conditions: meso-hydrobenzoin (0.2 mmol), Eco-Mn Ox2 (10 mol% Mn), 1 mL of solvent, O 2 balloon, 1 h. [b] Conversion was determined by GC-MS after calibration, using dodecane as internal standard.

Selective aerobic oxidative cleavage of activated 1,2-diols catalyzed by Eco-Mn Ox2 catalyst

less wastes were generated compared to conventional systems, NaIO 4 or Pb(OAc) 4 respectively. Other substrates were tested and complete conversions were obtained on activated benzyl and allyl diols, with excellent selectivities towards corresponding aldehydes or ketones (98-99 %) and only 10 mol% of Mn, (Table 8). Among the diols studied, the superoxidized products, as carboxylic acids, were not detected. [36] Eco-Mn Ox3 catalyst is an unusual polymetallic complex of Mn(II) and Fe(III). Eco-Mn Ox3 was incorporated in Montmorillonite K10 giving a supported catalyst that was used to catalyze the epoxidation of alkenes with a mixture of H 2 O 2 (30 % w/w)/ NaHCO 3 (0.2 M) as a green terminal oxidative reagent.

Switchable alkene epoxidation/oxidative cleavage catalyzed by Eco-Mn Ox3 catalyst

The supported Eco-Mn Ox3 catalyst presented a high efficiency for the epoxidation of styrene using a much lower Mn content than previously described heterogeneous Mn catalysts (Table 9, entry 1). [84] On the contrary, Montmorillonite K10 alone and Eco-Mn LA1 did not result in the formation of the desired product (Table 9, entries 4 and 5). Inspired by the work of Richardson et al., [80] the addition of water into the reaction was tested and resulted in a quantitative conversion of styrene with a reduced loading of Eco-Mn Ox3 catalyst (Mn 0.31 mol%, Fe 1.82 mol%, Table 9, entry 3) and a reduced quantity of the ecounfriendly DMF.

The solvent classically used in this reaction is DMF, which is not recommended in green chemistry. An alternative solvent was investigated (Table 10), but DMF remains the best solvent for this reaction; all the other tested solvents led to conversions under 20 %. Even using 1,2-propylene carbonate, which has been described as an environmentally friendly alternative to DMF, [81] only 4 % conversion was obtained (Table 10, entry 2).

Eco-Mn Ox3 can be recycled by simple filtration up to four times with less than 10 % loss of Mn and no loss for Fe. The conversion and the yield remained unchanged after the fourth run, indicating a high stability of the Eco-Mn Ox3 catalyst (Table 11).

The scope of the method was extended to various alkenes, including complex natural products. Yields of epoxidation were mostly high (up to 99 %) and sometimes higher than those described with conventional Mn-catalysts as Mn(II)-zeolite (Table 12). [82] The use of norbornene confirmed the effectiveness of the method with a yield of 99 % of epoxy-norbornene (Table 12, entry 1). The reactions on (R)-(+)-limonene and citral (Table 12, entries 2 and 5) indicated that the electron rich double bonds are selectively epoxidized. Moderate activity was noted in the Table 8. Scope of the aerobic oxidative cleavage of 1,2-diols catalyzed by EcoMn Ox2 . [a] Entry Substituent (R) Table 9. Epoxidation of styrene -comparison of Eco-Mn OX3 with conventional catalysts. [a] Entry Catalyst Amount of Mn [mol %] Amount of Fe Table 10. Eco-Mn Ox3 catalyzed epoxidation of styrene -solvent optimization. [a] Entry Solvent Conversion case of (R)-(+)-pulegone, where the C=C double bond is strongly deactivated by conjugation with a carbonyl group (Table 12, entry 6).

Conversion [%] [b] Selectivity [%] [b,c]
[mol %] Conversion [%] [b] Yield [%] [b] Selectivity [%] [b,c]
[%] [b] Yield [%] [b] Selectivity [%] [b,c]
The method proved to be effective even on voluminous natural products, such as α-pinene, affording 75 % yield of αpinene epoxide, with complete selectivity (Table 12, entry 3). The Mn(II)-zeolite catalysts previously described in the literature were much less effective, indicating the advantage of the Eco-Mn Ox3 catalyst. [82] In agreement with the literature, the linalool epoxide rearranges as soon as it is formed, [83] to give the hydroxypyrane with 63 % yield (Table 12, entry 4).

Fair to good yields of epoxides with complex and bulky structures such as nopol (74 %, Table 12, entry 9) and βcaryophyllene (42 %, Table 12, entry 10) were obtained with excellent selectivity. However, the method proved to be ineffective with terminal aliphatic alkenes, such as n-octene, as already observed with other Mn(II) catalysts (Table 12, entry 11). [82,84] Indene, cyclooctene and cyclohexene epoxides were obtained in much higher yields than other catalysts based on Mn (II)-zeolite (Table 12, entries 12-14). [82] During the styrene epoxidation, a small amount of benzaldehyde was formed, suggesting that it might be possible to selectively obtain an aldehyde from an alkene by adjusting the reaction conditions. Indeed, by reproducing the optimal conditions of the epoxidation (4 h at 0 °C) and then heating the reaction medium to 80 °C styrene was quantitatively transformed into benzaldehyde (Table 13, entry 1).

The mechanism of the oxidative cleavage is firstly based on the formation of an epoxide, which is then open giving the corresponding diol. Then the diol undergoes an oxidative cleavage to form the corresponding aldehyde. This mechanism seems to be specific to Eco-Mn Ox3 . Indeed, a mixture of commercial salts MnCl 2 .4H 2 O/FeCl 3 .6H 2 O prepared in the same conditions as for the Eco-Mn Ox3 catalyst, leads to benzaldehyde with only 24 % yield, accompanied by the corresponding 1,2-diol, with 71 % yield (Table 13, entry 2). This observation suggests that the limiting step is not the formation of the 1,2diol but rather its subsequent cleavage.

Other alkenes were tested to determine the scope and limitations of the reaction. The presence of electron-withdrawing groups on the aromatic ring seemed to be unfavorable, since only 11 % yield of aldehyde was obtained from 3chlorostyrene (Table 14, entry 1). The corresponding epoxide, however, being formed with a yield of 68 %, suggests that in this case the limiting step is the opening of the epoxide. The trans-anethole carrying both a methyl-enriched C=C double bond and a methoxy-group on the aromatic ring, gave a better yield of p-anisaldehyde (51 % yield, Table 14, entry 2).

Reaction with cinnamyl acetate gave after heating, 53 % of the desired product, benzaldehyde, was obtained and 19 % of cinnamaldehyde was also observed (Table 14, entry 2).

In conclusion, Eco-Mn Ox3 catalyst allows a controlled access to aldehydes by oxidative cleavage of various alkenes. The developed process is therefore an interesting alternative not only to conventional epoxidation reagents, but also to oxidative cleavage process of styrene-derived alkenes, which generally involves the use of toxic and dangerous reagents.

Tandem sequential oxidation catalyzed by Eco-Mn LA and

Eco-Mn Ox catalysts [34] Tandem sequential oxidation can be described as a multi-step process in which the substrate is transformed by two mechanistically distinct processes, one of which is an oxidation through the action of species present at the beginning of the reaction. [85] Tandem processes reduce the number of operations, by allowing the preparation and in-situ reaction of reactive intermediates, thus avoiding their isolation and often preventing safety problems. Tandem processes are thus in line with the principles of green chemistry, [START_REF] Anastas | Green Chemistry: Theory and Practice[END_REF] and transition elements are frequently involved in tandem oxidation processes, as they often combine different chemical properties acting on different stages of the sequential reactions. [START_REF] Davi | [END_REF][88][89][90] Among these elements, manganese is the main topic of numerous publications devoted to tandem oxidation, mainly Mn(III) [91][92][93][94] and Mn(IV). [95][96][97][98] For instance, manganese has recently attracted wide interest for multicomponent reactions based on pincer-type manganese complexes. [99][100][101][102][103][104][105][106] Eco-Mn LA and Eco-Mn Ox catalysts possess a polymetallic composition which combines metal transition elements with Lewis or Brønsted acidity properties (Mn, Fe, Al) and oxidative properties (Mn, Fe). Eco-Mn LA and Eco-Mn Ox catalysts were studied in sequential tandem oxidation as a "Janus catalyst", which is an eco-material with two different but concomitant catalytic properties. [107,108] The ability to combine these two activities in a single material offers great benefits from a sustainable chemistry perspective, with reduced waste and process simplification.

Table 11. Recycling and leaching studies of the supported Eco-Mn Ox3 catalyst for the epoxidation reaction of styrene over 4 successive reactions. [a] Recycling Conversion

[%] [b] Yield [%] [b] Selectivity [%] [b,c] Post-run Mn content wt % [d] Post-run Fe content wt % [d] 4 times Stable 97-98

Tandem carbonyl-ene cyclisation and aromatization of citronellal to p-cymene catalyzed by Eco-Mn LA catalyst

The conversion of natural products into aromatic derivatives is of great importance. The p-cymene is used as a building block in many industrial applications such as the production of pesticides, fungicides, flavor and fragrance components and pharmaceuticals. [START_REF] Fiege | Ullmann's Encyclopedia of Industrial Chemistry[END_REF][START_REF] Roberge | [END_REF] It can also be used as a ligand of hydrogenation catalysts such as [(η 6 -cymene) RuCl 2 ] 2 . [111] Currently, p-cymene is mainly produced by Friedel-Crafts alkylation of toluene with 2-propanol using acid catalysts such as AlCl 3 and HCl. [START_REF] Eggersdorfer | Ullmann's Encyclopedia of Industrial Chemistry[END_REF] This reaction is carried out at high temperatures (� 200 °C) and produces undesired isomers along with polyalkylation products. The yield of p-cymene generally does not exceed 50 %. [START_REF] Barman | [END_REF] In addition, the use of large quantities of acid catalysts and hazardous solvents causes many environmental and safety issues, such as the handling of reagents and solvents, waste disposal and corrosion problems.

Depending of the loading of Eco-Mn LA1 catalyst (5 mol% or 15 mol%), the synthesis of p-cymene can be performed from (+)-citronellal, a natural and abundant terpene, or from (À )isopulegol under similar reaction conditions (Scheme 10).

Eco-Mn LA1 acted as a Janus catalyst in the transformation of (+)-citronellal into p-cymene, by promoting the carbonyl-ene reaction (Lewis acidity property) followed by the aromatization (oxidation). However, the catalysis of the last step was surprising, since Eco-Mn LA1 is supposed to lack any oxidizing properties. Indeed, Eco-Mn LA1 catalyst has Mn(II) species, which have no oxidative properties. The aromatization observed here might be due to the presence of oxidizing species, such as Mn (III) or Mn(IV), that might have been formed by oxidation of Mn (II) with O 2 present in the air. Previous studies have shown that the oxidation of Mn(II) to Mn(III) or Mn(IV) by atmospheric O 2 is a very slow process, but can be greatly accelerated with traces of Fe(III). [114,115] As Eco-Mn LA contains 0.90 wt% of Fe(III), the formation of such traces through this process is possible. Since Fe(III) is also an oxidizing agent, it is also possible that this species participates along with Mn(III) or Mn(IV) to the aromatization step.

[%] [b] Yield [%] [b] Selectivity [%] [b,c]

One pot synthesis of substituted pyridines catalyzed by Eco-Mn LA catalyst

The couple dihydropyridine/pyridine (or pyridinium) is the synthetic analog of NADPH/NADP + , a cofactor involved in natural redox systems. The couple dihydropyridine/pyridine is a useful combination to conduct traditional chemical reductions or oxidations in many examples. [START_REF] Dugas | Bioorganic Chemistry: A Chemical Approach to Enzyme Action[END_REF] The mechanism is based on a hydride transfer from dihydropyridine towards an electrophilic center (carbonyl compounds, imines, unsaturated carbonyl compounds), in which the driving force is the developing aromaticity of the pyridine cycle. [START_REF] Dugas | Bioorganic Chemistry: A Chemical Approach to Enzyme Action[END_REF] The preparation of 1,4-dihydropyridine is based on Hantzsch reaction between aldehydes, β-keto esters, and amines. The synthesis of pyridines can be performed by aromatization of dihydropyridines with various reagents such as HNO 3 , [START_REF] García | [END_REF] (NH 4 ) 2 Ce(NO 3 ) 6 , [118] Cu(NO 3 ) 2 , [119] Bi(NO 3 ) • 5H 2 O, [120] Mn(OAc) 3 , [121] Zr(NO 3 ) 4 , [122] Fe(NO 3 ) 3 , [123] PCC, [124] Pd/C, [125] I 2 /MeOH, [126] o-iodoxybenzoic acid (IBX), [127] SeO 2 , [128] KBrO 3 /SnCl 4 • 5H 2 O, [129] KMnO 4 , [130] MnO 2 under microwave irradiation or sonication [119] and DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone). [131] Despite these numerous works, the development of effective and green methods are still required.

In this context, Eco-Mn LA1 catalyst has proved to be an elegant solution in the direct synthesis of pyridines, obtained by in-situ oxidation of 1,4-dihydropyridines (DHP) resulting from the Hantzsch reaction. This reaction constitutes a second example of tandem sequential reaction.

The catalytic potential of Eco-Mn LA1 was studied under microwave irradiation after dispersion of the catalyst and reagents in silica gel. Surprisingly, none of the 1,4-dihydropyridines was observed, only the corresponding pyridine was obtained. Interestingly, the reaction is compatible with aromatic aldehydes as well as enolizable aldehydes, as phenyl acetaldehyde (69-96 %) (Scheme 11).

Oxidative iodination of ketones catalyzed by Eco-Mn Ox1 catalyst

The α-iodinated ketones are widely used and studied. Indeed, they have a high reactivity and can be used as intermediates for the synthesis of useful high added-value compounds, including pharmacologically active heterocycles. [132] The synthesis of αiodoketones frequently involves the formation of electrophilic derivatives of iodine, [START_REF] De Kimpe | α-Haloketones, α-Haloaldehydes and α-Haloimines[END_REF] using I 2 in combination with various oxidizing reagents, such as SeO 2 , [START_REF] Bekaert | [END_REF] Ce(IV) ammonium nitrate [135] or m-iodosobenzoic acid. [136] Despite their efficiency, these methods use toxic or unstable oxidizing reagents that are incompatible with the requirements of the REACH regulation. Moreover, since I 2 is a rather aggressive compound, its handling is problematic, especially on a large scale. For these reasons, various groups have proposed new methodologies involving the in-situ formation of electrophilic iodine by oxidation of safe iodide species such as NaI, KI or NH 4 I. Selected examples of this strategy include the use of NaI/H 2 O 2 /H 2 SO 4 , [137] KI/air/H 2 SO 4 / NaNO 2 [138] or NH 4 I/Oxone®. [139] Although these methods represent significant improvements, they still use corrosive acids (H 2 SO 4 ) or highly toxic compounds (NaNO 2 ).

The potential of Eco-Mn Ox1 has been studied in oxidative iodination of ketones, as an alternative and safe oxidative reagent, with NaI as the source of iodine (Scheme 12).

Thanks to its Lewis and Brønsted character, Eco-Mn Ox1 is able to promote the enolization of the ketones. At the same time, the oxidizing properties of Eco-Mn Ox1 allowed the in-situ formation of iodine, immediately consumed by reaction with the enolized ketone and thus forming the α-iodoketone.

In the case of the β-dicarbonyl compounds, 2 equivalents of Eco-Mn Ox1 at room temperature leads to a quantitative reaction after 12 h. Under the same conditions, cyclohexanone was directly iodinated to give 2-iodocyclohexanone with a yield of 64 %, thus confirming the activity of the system in the case of less easily enolizable ketones. Eco-Mn Ox1 is therefore a valuable ecological alternative to more harmful or unstable oxidizing reagents, avoiding the use of strong acids and toxic species.

Summary and outlook

Mn-accumulating plants of the Grevillea genus, that are used for the rehabilitation of metal contaminated mining sites, produce a biomass enriched in Mn and valuable for synthetic chemical applications. Using simple transformation procedures, the plant-based biomass led to the first bio-sourced Mncatalysts, Eco-Mn catalysts. The ICP-MS, XRD and XAS analyses revealed that the Eco-Mn catalysts contained original polymetallic complexes, which are linked to their unique reactivity. Eco-Mn LA catalysts showed a marked Lewis and Brønsted acidity, which makes them very competitive to other classical catalysts, for the preparation of enol ethers and the cyclisation of (+)-citronellal carbonyl-ene to isopulegol. Eco-Mn LA catalysts were also used in reductive catalysis, for reductive aminations of ketones when associated with the Hantzsch ester.

Three types of eco-catalysts with oxidative properties, Eco-Mn Ox , were prepared using Eco-Mn LA1 as a starting material. Eco-Mn OX catalysts were used as green and efficient oxidizing reagents for the controlled oxidation of allyl and benzyl alcohols to aldehydes as well as for the oxidative cleavage of diols and a functionalized polyol with very good yields.

Both Eco-Mn LA and Eco-Mn OX also behaved as Janus-type catalysts by promoting sequential tandem oxidation reactions, thanks to their Lewis acid character and oxidative properties.

The new concept of Ecocatalysis allowed the preparation of a wide range of efficient and ecological Eco-Mn catalysts with flexible reactivity, while respecting the principles of Green Chemistry. Eco-Mn allowed to revisit two major mechanisms of organic synthesis and to develop green substitution reagents for oxidants prohibited by REACH regulation. Ecocatalysts pave the way for sustainable chemistry able to combine efficiency, speed, Nature-origin and eco-responsibility.

  Tomasz K. Olszewski obtained his PhD degree in 2006 from Wroclaw University of Science and Technology (WUST), Poland (with Prof. Bogdan Boduszek) and from University of Montpellier, France (under the guidance of Prof. Claude Grison). After postdoctoral period and work in industry he returned to academia in 2012 and obtained habilitation in 2016. Currently, he holds position of Associate Professor at WUST. His research interests are focused on synthesis and applications of organophosphorus compounds and recently catalysis. Camille Bihanic was graduated in Chemistry at Ecole Normale Supérieure of Lyon (France). She first joined the Laboratory of Bio-inspired Chemistry and Ecological Innovations (France) for her master internship in 2017 and is currently pursuing her PhD studies in the laboratory, under the supervision of Dr. C. Grison. Her doctoral research focus on the preparation of Mn-based ecocatalysts from accumulating plant species, used in ecological rehabilitation of New Caledonian mining sites. Kenza Richards received her Green Chemistry Engineering degree from the ENSIACET and her Master's degree from the Université Paul Sabatier (Toulouse). She started her PhD in 2018 in Montpellier at the Laboratory of Bio-Inspired Chemistry and Ecological Innovations under the supervision of Pr. Claude Grison. Her research interests are focused on the ecological rehabilitation of degraded sites especially the metal decontamination of mining effluents through the mobilization of biomass, and the valorization of the latest by eco-catalysis.

Figure 2 .

 2 Figure 2. New Caledonian Mn-accumulators: a) Grevillea exul ssp exul; b) Grevillea exul ssp rubiginosa; c) Grevillea meisneri and d) Grevillea gillivrayi.

Figure 3 .Scheme 3 .

 33 Figure 3. Crystal of two mixed metal chlorides: a) KMnCl 3 and b) K 3 NaMnCl 6 from Eco-Mn LA identified by XRD analyses.

Scheme 4 .Scheme 5 .Scheme 6 .

 456 Scheme 4. Experimental conditions for the preparation of Eco-Mn Ox1 catalysts.

  Scheme 7. Conversion rates and selectivity of products from the rearrangement of ethylene acetal of 2-bromopropiophenone with commercial manganese or iron chlorides and with Eco-Mn. a Reaction conditions: 2bromopropiophenone (0.29 mmol), catalyst (4 mol%), water (0.29 mmol), 1,2-dichlorobenzene (3.3 mL), 180 °C, 20 h. b Determined by GC-MS analysis. c Ratio of yield to conversion as a percentage.

  Reaction conditions: 3-methyl-2-butanone (1 mmol), aniline (1 mmol), HEH (1.1 mmol), neat, 110 °C, 4 h. [b] Eco-Mn LA and "reconstituted" Eco-Mn LA loadings calculated in Mn mol %. [c] Yields were determined by GC-MS after calibration, using biphenyl as an internal standard. [d] In all cases, less than 5 % of residual imine were detected. [e] "Reconstituted" Eco-Mn LA prepared by mixing commercial chlorides of K, Mg, Ca, Al, Mn, Fe and Ni, in the same proportions than in Eco-Mn LA , according to its ICP-MS analysis. [f] 0.83 μL of 12 M HCl (aq) were added (1 mol% of HCl).

  Reaction conditions: ketone (1 mmol), amine (1 mmol), HEH (1.1 mmol), Eco-Mn LA (1 mol%) (calculated in Mn mol %), neat, 110 °C, 4 h. [b] Yields were determined by GC-MS after calibration, using biphenyl as an internal standard.

  Reaction conditions: alcohol (10 mmol), catalyst (38.9 mmol Mn), EtOAc (20 mL), 75 °C, 3 h. [b] Yields were determined by GC-MS after calibration, using biphenyl as internal standard. [c] > 95 % trans-citral was obtained. [d] Eco-Mn Ox1 (60 mmol).

  Reaction conditions: substrate (0.2 mmol), EcoMn Ox2 (10 mol% Mn), 1butanol (1 mL), O 2 balloon, 100 °C. [b] Conversion, yield and selectivity were determined by GC-MS after calibration, using dodecane as internal standard; [c] Ratio of yield to conversion as a percentage.

  .[d] [a] Reaction conditions: styrene (0.25 mmol), H 2 O 2 (30 wt%) (0.8 mmol), NaHCO 3 (0.2 M) (0.05 mmol), catalysts (*72 mg; **39 mg; *** 39 mg + 10 vol %H 2 O,), DMF (2 mL), 0 °C, 4 h. [b] Conversion and yield were determined by GC-MS after calibration, using biphenyl as an internal standard. [c] Ratio of yield to conversion as a percentage. [d] Not determined.

  carbonate acetonitrile, dichloromethane, water, isopropanol, 4-methyl-2-pentanol, glycerol, ethanol, THF, acetone, 2-butanone < 20 < 20 63-100 [a] Reaction conditions: styrene (0.25 mmol), H 2 O 2 (30 wt%) (0.8 mmol), NaHCO 3 (0.2 M) (0.05 mmol), supported Eco-Mn Ox3 catalyst (72 mg, Mn 0.58 mol%, Fe 3.35 mol%), solvent (2 mL), 0 °C, 4 h. [b] Conversion and yield were determined by GC-MS after calibration, using biphenyl as internal standard. [c] Ratio of yield to conversion as a percentage.

  Reaction conditions: styrene (0.25 mmol), H 2 O 2 (30 wt%) (0.8 mmol), NaHCO 3 (0.2 M) (0.05 mmol), recycled supported Eco-Mn Ox3 catalyst (39 mg, Mn 0.31 mol%, Fe 1.82 mol%), DMF/water 7 : 3 (2 mL), 0 °C, 4 h. [b] Conversion and yield were determined by GC-MS after calibration, using biphenyl as internal standard. [c] Ratio of yield to conversion as a percentage. [d] Determined by ICP-MS after EDTA extraction of a sample of supported Eco-Mn Ox3 catalyst following the reaction.

  Reaction conditions: alkene (0.25 mmol), H 2 O 2 (30 wt%) (0.8 mmol), NaHCO 3 (0.2 M) (0.05 mmol), supported Eco-Mn Ox3 catalyst (39 mg, Mn 0.31 mol%, Fe 1.82 mol%), DMF/water (2 mL), 0 °C, 4 h. [b] Conversion and yield were determined by GC-MS after calibration, using biphenyl as internal standard. Synthesized products were identified by comparison of their spectroscopic data with those previously published. [c] Ratio of yield to conversion as a percentage. [d] Not determined.

  Reaction conditions: alkene (0.25 mmol), H 2 O 2 (30 wt%) (3.0 mmol), NaHCO 3 (0.2 M) (0.05 mmol), catalysts (39 mg, Mn 0.31 mol%, Fe 1.82 mol%), DMF/water 7 : 3 (2 mL), 0 °C, 4 h, then 80 °C, 24 h. [b] Conversion and yield were determined by GC-MS after calibration, using biphenyl as internal standard. Synthesized products were identified by comparison of their spectroscopic data with those previously published. [c] Ratio of yield to conversion as a percentage. [d] 1-phenyl-1,2-ethanediol was obtained as side-product with 71 % yield.

Scheme 10 .

 10 Scheme 10. Conversion of (+)-citronellal or (À )-isopulegol into p-cymene catalyzed by Eco-Mn LA1 .

Table 1 . Composition of leaves of Grevillea and derived Eco-Mn LA catalysts determined by ICP-MS analysis.

 1

		Physiological metals [wt %]			Accumulated
								metals [wt %]
		Na	Mg	Al	K	Ca	Fe	Mn	Ni
	Leaves Grevillea	0.12 0.24 0.02 0.44 0.24	0.05 0.40	0.01
	exul ssp. rubigi-
	nosa
	Eco-Mn LA1 Gre-	0.81 5.44 0.29 3.79 7.86	0.72 6.46	0.20
	villea exul ssp.
	rubiginosa HCl
	12 M
	Leaves Grevillea	0.05 0.18 0	0.71 0.86	0.12 0.21	0.01
	exul ssp. exul
	Eco-Mn LA2 Gre-	0.58 3.27 0.17 9.87 9.98	2.77 3.99	0.26
	villea exul ssp.
	exul. HCl 12 M
	Leaves Grevillea	0.12 0.31 0.05 0.27 1.15	0.24 0.36	0.06
	gillivrayi
	Eco-Mn LA3 Gre-	0.61 4.96 0.22 3.11 9.99	2.16 5.51	0.09
	villea gillivrayi.
	HCl 12 M
	Leaves Grevillea	0.14 0.12 0	0.27 0.80	0.06 0.67	0.03
	meisneri
	Eco-Mn LA4 Gre-	0.84 2.01 1.16 3.93 12.54 0.
	villea meisneri
	HCl 12 M

37 11.71 0.04

Table 2 .

 2 ICP-AES analysis of Eco-Mn Ox compared to the starting Eco-Mn LA1 .

		Physiological metals [wt %]	Accumulated metals [wt %]
		Mg	Ca	Al	Fe	Mn	Ni
	Eco-Mn LA1	5.26	10.44 0.24 0.90 6.72	0.04
	Eco-Mn Ox1	0.71	2.85	0.78 6.79 48.22	0.35
	Eco-Mn Ox2	28.70 28.20 0	0.30 14.10	0.30
	Eco-Mn Ox3

Table 7 .

 7 Solvent screening for EcoMn Ox2 -catalysed oxidative cleavage of meso-hydrobenzoin.

	Entry Catalyst	Solvent	T	Conversion
				[°C]	[%] [b]
	1 [a]	EcoMn OX2	acetonitrile, ethyl acetate,	78	35-61
		(10 mol%)	toluene, water, ethanol, acetic
			acid
	2 [a]	EcoMn OX2	1-butanol	100 98
		(10 mol%)

Table 12 .

 12 Substrate scope of the epoxidation catalyzed by supported Eco-Mn Ox3 catalyst.[a]

	Entry	Substrate	Product	Solvent water content	Conversion	Yield	Selectivity
				[vol%]	[%] [b]	[%] [b]	[%] [b,c]
	1			0	100	99	99
	2			5	50	43	86/14 (endocyclic/exocyclic epoxide)
	3			0	75	75	100
	4			0	65	63	97/3 (internal/terminal epoxide)
	5			30	80	72	90/10 (mono/di epoxide)
	6			30	49	27	55
	7			15	81	78	96
	8			5	74	50	68/16/16 (mono epoxide 2 i/mono epoxide C 5 /di epoxide)
	9			30	77	74	96
	10			0	46	42	91/9 (endocyclic/exocyclic epoxide)
	11			0	0	0	n.d. [d]

Table 13 .

 13 Oxidative cleavage of alkenes -comparison of Eco-Mn Ox3 catalyst with mixed salts.

Table 14 .

 14 Substrate scope of oxidative cleavage catalyzed by Eco-Mn Ox3 catalyst.[a]

	Entry	Substrate	Product	Conversion

Acknowledgements

The authors would like to thank the French National Center for Scientific Research (CNRS) for financial support. Both North and South province of New Caledonia are acknowledged for permissions to harvest New Caledonian Mn-hyperaccumulating plants. In the same way, KNS, SLN and Dr. Cyril Poullain are acknowledged for the cultivation and harvest of plants in New Caledonia. Sci-GuidEdit is acknowledged for writing assistance and proofreading the article.

Conflict of Interest

The authors declare no conflict of interest.


Simon Khelissa

Adem Gharsallaoui

Alexandre Fadel

Alexandre Barras

Charafeddine Jama

Fouzia Jbilou

Nour-Eddine Eddine Chihib
email: nour-eddine.chihib@univ-lille.fr

Microencapsulation of benzalkonium chloride enhanced its antibacterial and antibiofilm

Keywords: Benzalkonium chloride, Listeria monocytogenes, Escherichia coli, microencapsulation, antibiofilm activity

come   L'archive ouverte pluridisciplinaire

Introduction

Spray-drying microencapsulation is the most common method for turning a fluid solution into dry particles due to its economical and easy operation [START_REF] Abrahão | Microencapsulation of bioactive compounds from espresso spent coffee by spray drying[END_REF]. Spray-drying is commonly used for microencapsulation in food and pharmaceutical industries. Applying spraydrying for the encapsulation may impose stresses on the encapsulated active molecules through exposure to high temperature, shearing, and dehydration. However, good control of the operating parameters during the drying process makes it possible to reduce the residence time of the capsules in the drying chamber and to protect the biological activity. Our previous researches have proved the effectiveness of spray-drying microencapsulation of different active compounds with pectin [START_REF] Ben Amara | Using complex coacervation for lysozyme encapsulation by spray-drying[END_REF], 2017).

Quaternary ammonium compounds (QACs) are the major class of cationic surfactants used as disinfectants in domestic, industrial, and medical applications [START_REF] Zhang | Quaternary ammonium compounds (QACs): a review on occurrence, fate and toxicity in the environment[END_REF]. Their structure consists of a positively charged nitrogen atom and four covalently bonded alkyl groups. There is at least one hydrophobic hydrocarbon chain in these four alkyl groups, and other alkyl groups are mostly short-chain moieties such as methyl or benzyl groups. The annual worldwide consumption of QACs was reported as 500,000 tons in 2004 [START_REF] Chen | Impacts of quaternary ammonium compounds on membrane bioreactor performance: Acute and chronic responses of microorganisms[END_REF], the production of QACs per year exceeds 344,000 tons in US [START_REF] Phan | Decision of the board of appeal of the european chemicals agency[END_REF], and it is expected to continuously increase in the future. QACs are generally considered to be biodegradable in an activated sludge system. However, this degradation varies depending on the QAC concentration, chemical structure, complexation with anionic surfactants, and microbial acclimation [START_REF] Zhang | Quaternary ammonium compounds (QACs): a review on occurrence, fate and toxicity in the environment[END_REF]. Benzalkonium chloride (BAC) is a broad-spectrum antimicrobial widely used to disinfect environmental surfaces in clinical and food industrial settings [START_REF] Kim | Widely used benzalkonium chloride disinfectants can promote antibiotic resistance[END_REF]. BAC has a benzyl group, two methyl groups and an alkyl chain (C12-C18). BAC of various carbo chain lengths have diverse applications ranging from surface disinfection in hospitals, food production, agriculture or dairy industry, to their use in eye drops Journal of Applied Microbiology and hair shampoos [START_REF] Kampf | Adaptive microbial response to low-level benzalkonium chloride exposure[END_REF][START_REF] Mulder | Quaternary ammonium compounds in soil: implications for antibiotic resistance development[END_REF]. On the other hand, BAC can also cause genotoxic effects in mammalian and plant cells at environmentally relevant concentrations [START_REF] Ferk | Benzalkonium chloride (BAC) and dimethyldioctadecyl-ammonium bromide (DDAB), two common quaternary ammonium compounds, cause genotoxic effects in mammalian and plant cells at environmentally relevant concentrations[END_REF]. The extensive use of QACs in domestic and industrial applications has outcompeted its biodegradation in the environment, which results in its enrichment in wastewaters and aquatic environment. When the concentration of some QACs excesses certain level in the environment, they become ecotoxic [START_REF] Elersek | Ecotoxicity of disinfectant benzalkonium chloride and its mixture with antineoplastic drug 5-fluorouracil towards alga Pseudokirchneriella subcapitata[END_REF]. Therefore, there is a growing concern on the presence of QACs in the environment, and the reduction of usage of QACs should be a good study direction.

Pectin is a water soluble polysaccharide widely used in food manufacturing for its thickening and gelling properties. Native pectins are high methoxyl pectin in which the majority of carboxylic acid group are esterified by methanol and consequently non ionizable: therefore, in the present work, LM pectin having a higher proportion of free ionized carboxylic acid groups was considered in order to favor electrostatic interactions with positively charged BAC.

In this study, we use an electrostatic anchoring strategy to fix BAC inside microcapsules containing pectin. The purpose is then to evaluate the effects of pectin complexation and spraydrying microencapsulation on the antibiofilm activity of BAC. The cytotoxicity of both free and microencapsulated BAC was evaluated. The overall goal was to improve the effectiveness of this disinfectant through its microencapsulation while reducing the used amounts and resulting cytotoxicity.

Materials and methods

Materials: bacteria and chemicals

The target strains used in this study were: Listeria monocytogenes ATCC 35152 and Escherichia coli CIP 54127. Strains were maintained at -80°C in Tryptone Soy Broth (TSB, Biokar Diagnistics, France) supplemented with 20% (v:v) glycerol. Pectin was from (Cargill, Journal of Applied Microbiology Baupte, France). The degree of esterification was from 22% to 28% and degree of acetylation was from 20% to 23%. Maltodextrins DE 19 (dextrose equivalent value of 19) were obtained from (Roquette-freres SA, Lestrem, France). Benzalkonium chloride (BAC), analytical grade imidazole (C3H4N2), acetic acid, sodium hydroxide (NaOH), hydrochloric acid (HCl), and ethanol were purchased from (Sigma-Aldrich, St Quentin Fallavier, France). Distilled water was used for the preparation of all solutions.

Stock solution preparation

Imidazole-acetate buffer solutions (5 m mol l -1 ) were prepared by dispersing weighed amounts of imidazole and acetic acid into distilled water. The stock solutions were prepared by dispersing BAC, pectin or maltodextrins powders into imidazole-acetate buffer stirring at room temperature until total hydration, and then the pH was adjusted to 7 by using HCl (0.1 or 1.0 mol l -1 ) or NaOH (0.1 or 1.0 mol l -1 ). The concentration of each stock solution (w:w) was: BAC 1%, pectin 3%, and maltodextrins 50%. The pH of each solution was adjusted again to 7 before analysis.

Complexes formation and spray-drying

The suspensions of complexes were prepared by mixing the stock solutions and imidazoleacetate buffer to the desired concentration. Stock maltodextrins solutions were added to the prepared suspensions, to obtain a final concentration (w:w) of 20% maltodextrins DE 19. Four complex pectin/BAC ratios of 1, 2, 2.5 and 3 were prepared. For complexed BACmicrocapsules study, only the ratio 3 was selected (20% maltodextrins DE 19, 0.4% BAC, 1.2% pectin). The mixtures were stirred for 30 min and then spray-dried using a laboratory scale device equipped with a 0.5 nm nozzle atomizer (Mini Spray-Dryer Büchi B-290, Switzerland).

The operational conditions of the drying process were as follow: feed flow rate: 0.5 l h -1 , inlet air temperature: 180 ± 2°C, and outlet air temperature: 80 ± 5°C. After spray-drying, the powders were collected in sealed containers and stored at 4°C until analysis. Reconstituted Journal of Applied Microbiology suspensions were prepared by dispersing weighted amounts (the same dry matter as before spray-drying) of spray-dried powders in imidazole-acetate buffer solutions (5 m mol l -1 , pH 7) and stirring for 1 h.

Zeta potential measurement

The electric charge of free molecules or complexes was determined using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). The zeta-potential (ZP) of BAC and pectin was measured in imidazole-acetate buffer at different pH values. If necessary, the samples were diluted with corresponded pH imidazole-acetate buffer. Each test was repeated at least three times. The mean ZP values (±SD: standard deviation) were obtained from the instrument.

Particle size measurement

Particle size distribution of complexes were assessed by a laser diffraction instrument (Mastersizer 3000, Malvern Instruments, Malvern, UK) with a value of 1.33 for water phase.

To measure the particle size distribution of spray-dried microcapsules, the powder was slowly added into the measurement chamber containing ethanol (refractive index: 1.36) to avoid the dissolution of maltodextrins during the measurement. The spray-dried powders were continuously stirred throughout the measurement to ensure that the samples were homogeneous. The volume mean particle diameter (D43) was calculated by the software from the three injections of three separate samples with five readings per sample.

Moisture content

The moisture content of spray-dried microcapsules was measured by drying in an oven at 160°C under air circulation for 24 h. Spray-dried microcapsules (∼1 g) were placed in caps that were weighed before and after oven drying. Moisture content values were calculated as a fraction of the initial microcapsules weight lost during drying and were reported on a wet basis.

Bacterial susceptibility testing

Journal of Applied Microbiology L. monocytogenes and E. coli, were cultured overnight in TSB at 37°C. Cells were pelleted by centrifugation (5000 g, 5 min, 20°C) then washed with Potassium Phosphate Buffer (PPB; 100 m mol l -1 , pH 7). Bacterial suspensions were diluted in TS broth to 10 6 CFU ml -1 . The Minimum inhibitory concentrations (MICs) of free-BAC, uncomplexed BAC-microcapsules (UBM) and complexed BAC-microcapsules (CBM) were determined by measuring kinetically, the development of turbidity by vertical photometry using a Bioscreen C (Labsystems, Helsinki, Finland), [START_REF] Khelissa | Comparative study of growth temperature impact on the susceptibility of biofilm-detached and planktonic Staphylococcus aureus cells to benzalkonium chloride[END_REF]. Briefly, 100 μl of bacterial suspension were added to the plate wells containing serial 2-fold dilutions from microcapsules or free BAC stock solution prepared in TSB. A growth control (bacteria with microcapsule-free TSB), and a sterility control (with only microcapsule-free TSB) were added to each plate. Plates were incubated at 37°C under continuous shaking in the Bioscreen C which measured the optic density (OD) at 600 nm every 2 h during 24 h. The MIC was defined as the lowest concentration preventing the bacterial growth, as measured by optical density. The experiment was repeated three times and mean OD 600 nm values were plotted versus time.

Biofilm installation and disinfection assay

All bacterial strains were precultured in TSB (5 ml) for 24 h at 37°C. Cultures were prepared by inoculating 50 ml of TSB with 10 4 CFU ml -1 from the preculture and incubated at 37°C overnight. Cells were and harvested by centrifugation (5000 g, 5 min, 20°C) and washed twice with PPB. Bacterial suspensions were adjusted to 10 8 CFU ml -1 in PPB (OD 600 nm 0.110 ± 0.005). Stainless steel slides (50 x 25 mm) were covered with 3 ml of the cell suspension adjusted to 10 7 CFU ml -1 and allowed to stand at 20 °C for 1 h. Afterwards, slides were rinsed in PPB, to remove loosely attached cells. Each slide was covered with 3 ml of TSB and incubated statically at 37°C for 24 h. After 24 h of incubation, slides covered with biofilm were rinsed with PPB then placed horizontally in 20 ml of the tested solution (at the MIC) and incubated for 10 min. Afterwards, slides were withdrawn from the tested solution and immersed Journal of Applied Microbiology in 20 ml of neutralizing solution [START_REF] Toté | Inhibitory effect of biocides on the viable masses and matrices of Staphylococcus aureus and Pseudomonas aeruginosa biofilms[END_REF]) containing 1 g of 1 mm diameter glass beads in 100 ml sterile pot to stop the antibacterial action. In order to detach the biofilm cells were from the stainless steel surface, pots were vortexed for 30 s followed by a sonication for 5 min (37 kHz, 20 °C) (Elmasonic S60H, Elma, Germany). Thereafter, pots were vortexed again for 30 s and serial dilutions were realized in TS. Samples of 100 μl were spread onto TSA plates and incubated at 37°C for 24 h. The number of viable and culturable cells was counted on plates and results were expressed in log CFU cm -2 . For the control assays, the disinfectant solution was replaced by the TS. The cell viability percentage in treated biofilms was determined after staining with the LIVE/DEAD® BacLight Bacterial Viability kit (Invitrogen Molecular Probes, USA). Briefly, 10 7 CFU ml -1 of detached cells were filtered through a 0.2 μm-pore-size black polycarbonate filters (Millipore, France) and stained for 10 min in the dark. Stained cells were washed once with 1 ml of distillated water, and filters were air dried in the dark then observed under epifluorescence microscopic (Olympus BX43, Germany) for enumeration. The green cells (viable) and red ones (dead) were counted over 50 microscopic fields. The percentage of viable cells was calculated as follows: (Number of green cells x 100) / total number of both red and cells. Results represent the average of three independent experiments and in each case, two slides were used.

Scanning electron microscopy (SEM)

The morphology of biofilm bacterial cells treated for 10 min with free BAC or CBM at MICs was assessed by SEM and compared to negative control where cells were treated with Tryptone Salt (TS). Biofilm treatment cells were recovered according to [START_REF] Abdallah | Effect of growth temperature, surface type and incubation time on the resistance of Staphylococcus aureus biofilms to disinfectants[END_REF] and diluted (10-fold) Tryptone Salt (TS). Samples of 1 mL volume from the diluted biofilm cell suspensions were filtered through a 0.2-μm-pore-size polycarbonate membrane filter (Schleicher & Schuell, Dassel, Germany) then fixed overnight with 2% glutaraldehyde, cacodylate buffer 0.1 mol l -1 , pH 7, at 4°C. Fixed cell samples were dehydrated in an ascending Journal of Applied Microbiology ethanol series (50%, 70%, 95%, and 100% (×2) (v:v) ethanol), for 10 min at each concentration and dried. Fixed bacterial samples were carbon coated and examined at an accelerating voltage of 3 kV.

Epifluorescence microscopy analysis

Biofilms were treated with free and encapsulated BAC and for the control biofilm were treated with TS. Biofilms were stained with LIVE/DEAD BacLight kit (Invitrogen Molecular Probes, USA), according to the manufacturer instruction for 15 min. Biofilms were washed by genteelly dipping the stainless steel coupons in distillated water, air dried in the dark then observed under epifluorescence microscopic (Olympus BX43, Germany). The green cells were considered viable and red ones were defined as non-viable.

Cytotoxicity assay

The HeLa cell line derived from cervical carcinoma from a 31 year old female (ATCC® CCL-2™, ECACC, Sigma Aldrich, Saint-Quentin Fallavier, France) were cultured and maintained in Dulbecco's Modified Eagle's medium (DMEM, Gibco®) supplemented with 10% fetal bovine serum (FBS, Gibco®) and 1% penicillin-streptomycin (Gibco®) in a humidified incubator at 37 C and 5% CO2. Cells were seeded at a density of 10 4 cells per well in a 96-well plate and grown for 24 h before assay. The culture medium was replaced with a fresh medium that contains the compounds from 12.5 to 400 µg ml -1 . After 24 h, the old medium was removed and cells were washed with PBS. The cell viability was evaluated using resazurin cell viability method. Briefly, 100 l of the resazurin solution (11 µg ml -1 ) in complete medium were added to each well and the plate was incubated for 4 h in the humidified incubator. The fluorescence emission of each well was measured at 593 nm (20-nm bandwidth) with an excitation at 554 nm (18-nm bandwidth) using a Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek Instruments SAS, France). Each condition was replicated three times and the mean fluorescence value of non-exposed cells (treated with TSB) was taken as 100% cellular viability.

Journal of Applied Microbiology

Statistical analysis

All experiments were carried out at least three times. Statistical study was performed with IBM SPSS 19 statistics software following the one-way ANOVA. Results are considered significantly different when P < 0.05.

Results

BAC-LM pectin interactions and properties of the formed complexes

Figure 1

To choose optimum pH for the complexation, the zeta-potential (ZP) of BAC and pectin as function of pH was measured as shown in Figure 1. At the pH range from 3 to 8, pectin was negatively charged. In detail, the absolute value of ZP for pectin obviously increased with increasing the pH from 3 to 5, which was due to the deprotonation of carboxyl groups. With continual increase of pH over 5, the changes in ZP value became unobvious, which indicated the end of deprotonation of carboxyl groups. The zeta-potential of BAC was overall stable at around +20 mV whatever the pH value. At pectin/BAC mass ratio 1.5, the complexes of BAC and pectin became electrical neutral at pH 7 (Figure 2). The ZP of the complexes reached a plateau after the pectin/BAC mass ratio reached 3, which could be indicated that positively charged BAC groups were fully occupied by pectin (Figure 2). Therefore, this ratio was chosen for the following experiments as it allowed to have small complexes having a compact structure (Figure 3). Nevertheless, at ratio 4, the high pectin amount resulted in the dissociation of pectin/BAC complexes. This could be due to the bridging of BAC molecules which significantly (P < 0.05) increased the size of complexes (Figure 3). The spray-dried BAC microcapsules were white color with low moisture content of 3.70% and 5.15% for UBM and CBM, respectively. Pectin is a polyanionic biopolymer which has a high affinity with water molecules, which explains the highest water content for CBM. On the other hand, for the 2 types of capsules, these water contents should make it possible to have good storage capacities. The particle size of spray-dried microcapsules with and without pectin showed no significant difference (P > 0.05) in their particle size distribution (Figure 4

Assessment of bacterial susceptibility to free and microencapsulated BAC

Table 1. Minimal inhibitory concentration (in mg l -1 ) of free and encapsulated BAC against E. coli and L. monocytogenes.

Bacterial strains free BAC CBM UBM

E. coli 12.5 3.12 6.25

L. monocytogenes 6.25 1.56 3.12 Abbreviations: BAC, benzalkonium chloride; CBM: complexed BAC-microcapsules; UBM:

uncomplexed BAC-microcapsules.

The MIC of CBM and UBM microcapsules were determined and compared to those of free BAC against L. monocytogenes ATCC 35152 and E. coli CIP 54127. The obtained results are

shown in Table 1. The MICs of the encapsulated BAC were significantly (P < 0.05) lower than those of free BAC against all studied strains. In addition, the MICs of CBM were 2-fold (P < 0.05) lower than those of UBM whatever the studied strain (Table 1). L. monocytogenes was 2fold more susceptible to all studied BAC forms than E. coli (Table 1).

Antibiofilm assays

Journal of Applied Microbiology The antibiofilm efficacy of CBM and UBM used at their MICs was investigated on L.

monocytogenes and E. coli biofilms and compared to that of free BAC, respectively. The results obtained were expressed as log CFU cm -2 reductions in treated biofilms, compared to untreated controls (Table 2). The treatment with free BAC resulted in log reduction of the initial biomasses of E. coli and L. monocytogenes biofilms by 1.8 and 2.5 log CFU cm -2 (P < 0.05), respectively (Table 2). Similar results were observed after treatment with UBM (Table 2). The treatment with CBM resulted in a significant log reduction of ca 3.1 and 3.6 log CFU cm -2 (P < 0.05), for E. coli and L. monocytogenes, respectively. CBM showed higher antibiofilm effect as compared to UBM (P < 0.05). Furthermore, the percentage of viable cells after staining with the SYTO9 and propidium iodide (PI), showed that untreated E. coli and L. monocytogenes biofilms were composed mainly of viable cells (respectively of ca 93 and 96%, data not shown).

The results presented in Table 2 also suggest that the treatment with free BAC reduced the viability of E. coli and L. monocytogenes biofilms to 69 and 52% respectively (P < 0.05). After treatment with CBM and UBM the remaining E. coli viable cells percentage was of ca 57 and Journal of Applied Microbiology with CBM and UBM were of 37 and 48% respectively (Table 2). Upon antibacterial treatment the viability percentage results were in agreement with the cellular biomass reduction (Table 2). In the free and microencapsulated BAC-treated biofilms the decrease of viable cells indicates the increase of percentage of membrane-compromised stained with PI (Table 2).

Impact of free and encapsulated BAC treatment on biofilm cells morphology

Control free-BAC CMB

Escherichia coli Listeria monocytogenes

To investigate the morphological modifications of E. coli and L. monocytogenes biofilm cells after the treatment with either free-BAC or BAC microcapsules. Bacterial samples were analyzed by SEM. As the effect of both free BAC and UBM on treated cells morphology was similar, UBM-treated cells micrographs were not shown (Figure 5). The negative control (cells Journal of Applied Microbiology treated with TS) showed intact cells which exhibited a normal and smooth lining (Figure 5).

However, whatever the studied bacteria strain, the exposure to both free-BAC and CBM solutions (concentration adjusted to the MIC) for 15 min led to significant structural collapse (Figure 5). Cells seemed irreversibly lysed, deformed and showed holes in their cell walls (Figure 5).

Cell Viability and biofilm architecture

Control free-BAC CMB

Escherichia coli

Listeria monocytogenes

Figure 6

Figure 6 showed epifluorescence microscopy images of 24 h E. coli and L. monocytogenes biofilms stained with SYTO9 and PI after treatment with free and encapsulated BAC. The control (biofilm treated with TS) showed a thick and covering viable biomass mainly stained with SYTO9. The treatment with BAC and CBM resulted in a significant removal of biofilm Journal of Applied Microbiology biomass from the SS coupon. Furthermore, the density of remaining biofilm cells stained with PI after antibacterial treatment increased significantly. The results demonstrated a higher level of cell dispersion after treatment with CBM as a very thin cell layer mostly stained with PI was observed (Figure 6). According to our results, there was a correlation between the cellular membrane integrity and the remaining biofilm cell biomass after treatment with both free BAC and CBM which highlights the possible modes of action of these antimicrobials.

Cytotoxicity of free and microencapsulated BAC

The viability of HeLa cells was assessed after 24 h contact with free and micro-encapsulated BAC concentrations ranging from 12.5 to 400 µg ml -1 (Figure 7). The TSB was used as a negative control. Two additional controls consisting of microcapsules formulated without BAC (empty UBM and CBM). Figure 7 showed that TSB had no negative effect on HeLa cells viability. When used at concentrations between 12.5 and 100 µg ml -1 , both empty UBM and CBM had no cytotoxic effect on HeLa cells which showed a viability percentage of ca 100 % after being in contact with empty microcapsules for 24 h. However, at concentrations of 200 and 400 µg ml -1 the viability of HeLa cells was reduced by 10 and 20 % respectively, whatever Journal of Applied Microbiology the studied empty microcapsules (Figure 7). Contact with BAC at 12.5 µg ml -1 was not toxic to HeLa cells which remained 100 % viable (Figure 7). Our findings, underlined that exposure to BAC concentrations greater than, or equal to 25 µg ml -1 was significantly cytotoxic (P < 0.05) and led to 100 % HeLa cells viability reduction (Figure 7). Furthermore, CBM and UBM demonstrated reduced cytotoxicity towards HeLa cell, with a minimum needed concentration of 200 µl ml -1 to completely kill the HeLa cells. Nevertheless, CBM were significantly more cytotoxic then UBM (P < 0.05) (Figure 7). These results confirm the ability of microencapsulation to enhance the antibacterial effect of BAC while reducing its health hazard even at doses that would otherwise be completely toxic.

Discussion

BAC is different from biomacromolecules like pectin having a complex molecular structure.

The antimicrobial used in this study has a simpler molecular structure which consists of quaternary cationic nitrogen atom linked with a benzyl group, two methyl groups and an alkyl chain (C16). The molecular structure of BAC could induce, in aqueous phase, the formation of micelles by inside hydrophobic long alkyl chains that can be surrounded by positively charged nitrogen atoms. The structure of BAC micelles with positively charged nitrogen atoms on the surface would not change with the change of pH [START_REF] Ilari | Effect of the surfactant benzalkonium chloride in the sorption of paraquat and cadmium on montmorillonite[END_REF]. Considering that pectin and BAC carried opposite charges at the tested pH range, the most common pH for several applications, pH 7, was chosen for the formation of electrostatic complexes in this study. Pectin is net negatively charged at pH 7, and can combine sufficiently with positively charged BAC via electrostatic attractive interactions. Therefore, the ZP of pectin/BAC complexes gradually decreased with increasing the amount of added pectin (Figure 2). Furthermore, our results showed that CBM and UBM microcapsules had low moisture content witch was slightly higher for those containing pectin. This could be due to the water holding capacity of pectin. In this context, [START_REF] Botrel | Properties of spray-dried fish oil with different carbohydrates as carriers[END_REF] reported that the differences in the Journal of Applied Microbiology moisture content presented between particles were due to the affinities of the materials to water and moisture diffusivities through the wall matrix. This moisture content is fitted in the ideal moisture content range as 3-5% [START_REF] Crouter | The effect of moisture on the flowability of pharmaceutical excipients[END_REF].

The size distribution of spray-dried microcapsules with and without pectin showed no significant difference (P > 0.05). This result was expected since the same drying conditions were used for all the feed solutions (temperature, pulverization pressure). However, [START_REF] Sansone | Maltodextrin/pectin microparticles by spray drying as carrier for nutraceutical extracts[END_REF] have shown that the presence of pectin affects the maltodextrin/pectin particle size significantly. To assess the susceptibility of L. monocytogenes and E. coli to the different form of BAC, the MIC of CBM and UBM microcapsules were determined and compared to those of free BAC. Our results showed that the encapsulation of BAC reduced the its MIC significantly (P < 0.05). In addition, the MICs of CBM were 2-fold (P < 0.05) lower than those of UBM whatever the studied strain. We demonstrated that treatment with CBM used at the MIC reduced significantly the viability and cultivability of L. monocytogenes and E. coli biofilm cells. The antibiofilm effect of CMB was significantly greater than those of free BAC and UBM whatever the studied strain. These results are consistent with those obtained by [START_REF] Wang | Effect of drying and interfacial membrane composition on the antimicrobial activity of emulsified citral[END_REF] for citral microcapsules which showed an increase of the antibacterial effect of this essential oil against several food pathogens when pectin was added to emulsified citral. This could be due to the presence of a proper concentration of BAC in close of bacterial cells that could result from a controlled release of BAC encapsulated in presence of pectin.

It has been reported that the antibacterial activity of many antimicrobial agents including BAC results in the disruption of the cell membrane integrity through the insertion of the antimicrobial agent in the cell membrane, leading to the collapse of the membrane and to the leakage of cytoplasmic pool or penetration of the compound into the cell, affecting internal homeostasis [START_REF] Mclandsborough | Biofilms: at the interface between biophysics and microbiology[END_REF][START_REF] Khelissa | Comparative study of growth temperature impact on the susceptibility of biofilm-detached and planktonic Staphylococcus aureus cells to benzalkonium chloride[END_REF][START_REF] Khelissa | Actively detached Pseudomonas aeruginosa biofilm cell susceptibility to benzalkonium chloride and associated resistance mechanism[END_REF]. Thus, we observed the morphology of bacterial cells after antibacterial treatment with SEM. SEM micrographs showed the Journal of Applied Microbiology structural collapse and the irreversible lysis of cell walls. These observations are consistent with our previously reported studies regarding the damage made by BAC to bacterial cells membrane integrity [START_REF] Khelissa | Comparative study of growth temperature impact on the susceptibility of biofilm-detached and planktonic Staphylococcus aureus cells to benzalkonium chloride[END_REF][START_REF] Khelissa | Actively detached Pseudomonas aeruginosa biofilm cell susceptibility to benzalkonium chloride and associated resistance mechanism[END_REF]. Like any other QAC, BAC is toxic for mammalian cells.

Our findings, underlined that exposure to BAC concentrations greater than, or equal to 25 µg ml -1 killed HeLa cells up to 100 %. Our results, are consistent with those reported by [START_REF] Deutschle | In vitro genotoxicity and cytotoxicity of benzalkonium chloride[END_REF], who reported that BAC used at low concentration (0.01%) nearly kill all human respiratory epithelial BEAS-2B cell line after 2 h of exposure. These results highlighted the health and environmental hazards associated to the BAC even at very low concentrations. In addition, cytotoxicity assays showed apoptotic effects for lower concentrations and necrotic effects for higher concentrations of BAC [START_REF] Deutschle | In vitro genotoxicity and cytotoxicity of benzalkonium chloride[END_REF].

Exposure of HeLa cells to high concentrations of empty microcapsules (formulated without BAC) resulted in up to 20% reduction of initial viability. This could be explained by the highdose-toxicity effect over 200 µg ml -1 . It has been demonstrated that pectin-derived acidic oligosaccharides in mammalian assays were classified as cytotoxic only at highly concentrations [START_REF] Garthoff | Safety evaluation of pectin-derived acidic oligosaccharides (pAOS): Genotoxicity and sub-chronic studies[END_REF][START_REF] Fiume | Safety assessment of microbial polysaccharide gums as used in cosmetics[END_REF]. Our results showed that the microencapsulation of BAC significantly reduced its cytotoxicity toward HeLa cells. The significant reduction in BAC toxicity is an important step in advancing the developed UBM and CBM towards industrial application.

In summary, BAC was successfully encapsulated by electrostatic complexation with pectin using spray-drying technique. We studied the effect of complexation and spray-drying on the antimicrobial action of BAC. The results showed that the complexation of BAC with pectin would control its release kinetics from microcapsules, and therefore improve its effectiveness against bacteria contaminating food. The increase of the pectin amount influenced the particle size of BAC/pectin complexes before spray-drying. Microbiological tests demonstrated that complexed BAC microcapsules (CBM) had the highest antimicrobial activity than Journal of Applied Microbiology uncomplexed ones (UBM). Exposure of E. coli and L. monocytogenes biofilms MICs of free and microencapsulated BAC resulted in the modification of overall biofilm cell viability, cell morphology, and cell biomass dispersal. In addition, both CBM and UBM were more efficient than free BAC when they are applied against several food pathogen biofilms. The application of such a BAC microcapsule-based delivery system can improve the surface disinfection procedures and reduce the generated chemical wastes that seems to be a good strategy to maintain hygiene in concerned sectors.

Figures Captions

Figure 1. Zeta-potential of benzalkonium chloride (BAC) -----and pectin -in imidazole acetate buffer at different pH.

Figure 2. Zeta-potential of BAC/pectin complexes as a function of pectin concentration at pH 7 (the concentration of BAC is constant at 1 g l -1 ).

  Figure 2

For

  Figure 3

  mean diameters (D43) were ranged between 17.3 μm for spray-dried BAC without pectin and 16 μm for spray-dried BAC with pectin microcapsules (Figure4 b). These particle distribution properties should make it possible to have fluid powders with good properties of use whatever the type of microcapsules.

Figure 3 .

 3 Figure 3. Particle size distribution of BAC/pectin complexes as a function of pectin/BAC ratio.

Figure 4 .

 4 Figure 4. Particle size distribution and corresponding average size of spray-dried

Figure 5 .

 5 Figure 5. Scanning electron micrographs of Escherichia coli and Listeria monocytogenes,

Figure 6 .

 6 Figure 6. Fluorescence microscopy images of Escherichia coli and Listeria monocytogenes,

Figure 7 .

 7 Figure7. HeLa cells viability percentage after 24 h exposure to concentrations (between 12.5 and 400 µg ml -1 ) of free BAC ; pectin-complexed BAC-microcapsules (CBM) ; Spray

Table 2 .

 2 The reduction (in log CFU cm -²) and viability percentage of E. coli and L. monocytogenes biofilm biomass after treatments with free or microencapsulated BAC.

	Bacteria	Viable cells %		Biomass log reduction
		BAC	CBM	UBM	BAC	CBM	UBM
	E. coli	69 ± 2	57 ± 3	65 ± 2	1.8 ± 0.2	3.1 ± 0.1	2.1 ± 0.1
	L. monocytogenes	52 ± 3	37 ± 2	48 ± 3	2.5 ± 0.3	3.6 ± 0.2	2.3 ± 0.1
	Abbreviations: BAC: benzalkonium chloride; CBM: complexed BAC-microcapsules; UBM:
	uncomplexed BAC-microcapsules. Data presented as means (±SD) of three independent
	repeats.

Journal of Applied Microbiology

Acknowledgments

This work was carried out within the framework of ALIBIOTECH program (Agroalimentaire et Biotechnologie). The authors also thank the Haut de France region and FEDER (Fonds Européen de Développement Régional) for their financial support.

Conflict of Interest